玉米多态性和基因分型的方法 Corn polymorphisms and gene typing methods

并入的序列表 Incorporated Sequence Listing

两份序列表拷贝（拷贝l和拷贝2)和一份计算机可读形式（CRF) 的序列表，都在CD-ROM上，每份CD-ROM中都包含名为"pa_ 0 0 35 8B. rpt" 的文件，该文件大小为10.8MB (在MS-DOS中测定），所有序列表都创建于2006年8月10日，通过引用并入本文。 Two copies of the sequence table (copy copy l and 2) and a computer-readable form (CRF) of sequence table, in the CD-ROM, CD-ROM each are included named "pa_ 0 0 35 8B. rpt "The file size is 10.8MB (measured in MS-DOS), all sequences tables are created in August 10, 2006, incorporated herein by reference.

并入的表格 Incorporated in the form

CD-ROM中的两份表格拷贝（拷贝l和拷贝2)(每份CD-ROM中包含名为"Table 1. txt，，的3MB(在MS-DOS中测定）的文件和名为"Table 3.doc"的2. 2MB的文件）都创建于2006年8月11日，通过引用并入本文。 CD-ROM copies of two forms (copy l and 2 copies) (each CD-ROM contains the named "Table 1. txt ,, the 3MB (measured in MS-DOS) file and named" Table 3.doc "of 2. 2MB file) are created on August 11, 2006, incorporated herein by reference.

发明领域 Field of the Invention

本发明公开了玉米多态性，涉及所述多态性的核酸分子以及使用所述多态性和分子的方法，例如，在基因分型中。 The present invention discloses a maize polymorphism, the polymorphic nucleic acid molecules and methods of using the polymorphism and molecules involved, e.g., in genotyping.

背景技术 BACKGROUND

多态性用作遗传标记，用于农业领域中的基因分型应用，例如， 在植物遗传研究和商业育种中。 Polymorphism used as genetic markers for the agricultural sector in genotyping applications, for example in plant genetic research and commercial breeding. 参见例如美国专利5,385, 835; 5， 437， 697 ; 5， 385， 835; 5， 492， 547; 5， 746， 023; 5， 962， 764; 5， 981， 832和6， 100， 030,所有这些专利的公开内容通过引用并入本文。 See, e.g., U.S. Patent No. 5,385, 835; 5, 437, 697; 5, 385, 835; 5, 492, 547; 5, 746, 023; 5, 962, 764; 5, 981, 832 and 6, 100, 030, All disclosures of these patents are incorporated herein by reference. DM的高度保守性与极少发生的稳定的多态性相结合提供了遗传标记，这两者都是可预测的并且都可以辨别不同的基因型。 DM is highly conserved and polymorphic stability rare combination provides genetic markers, both of which are predictable and can distinguish different genotypes. 现有的遗传标记种类之一是指示遗传变异的多种多态性，包括限制性片段长度多态性（RFLPs )，扩增片段长度多态性（AFLPs )，简单序列重复（SSRs )， One kind of conventional genetic markers is more polymorphisms indicative of genetic variation, including restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs),

6)和插入/缺失多态性（Indels)。 6) and insertion / deletion polymorphism (Indels). 用于植物物种的遗传标记的数量是有限的，因此额外的遗传标记的发现将促进基因分型应用，包括标记性状关联研究，基因定位，基因发现，标记辅助选择和标记辅助育种。 The number of plant species for genetic markers are limited, so find additional genetic markers will facilitate genotyping applications including studies related trait markers, gene mapping, gene discovery, marker-assisted selection and marker-assisted breeding. 发展中的技术使得一些遗传标记更适于快速的，大规模用途。 Developing technology makes some genetic markers more suitable for rapid, large-scale applications. 例如，SNP检测技术表明SNP是优选的遗传标记。 For example, SNP detection technology show SNP genetic markers are preferred.

发明概述 Summary of the Invention

本发明提供了大量的玉米遗传标记。 The present invention provides a number of maize genetic markers. 这些遗传标记包括玉米DNA 基因座，其用于至少两种玉米品种之间的基因分型应用。 These genetic markers including maize DNA loci for genotyping applications between at least two maize varieties. 本发明的多态性玉米基因座包含至少20个连续的核苷酸，其包括多态性或与多态性相邻，所述多态性是本文（例如，表1中）鉴定的多态性。 Maize locus polymorphism present invention comprises at least 20 consecutive nucleotides, which include polymorphisms or adjacent to a polymorphism, the polymorphism (e.g., Table 1) polymorphisms identified herein sex. 更具体地，本发明的多态性玉米基因座具有这样的核苷酸序列，其与包括多态性或者与多态性相邻的玉米DNA片段的任一条链中的相同核苷酸数量的序列至少90%，优选至少95°/。 More specifically, the present invention is polymorphic loci of maize has a nucleotide sequence with any one chain comprises a polymorphism or a polymorphism of DNA fragments adjacent to corn in the number of identical nucleotides sequence at least 90%, preferably at least 95 ° /. 同一。 The same. 如在表l中所示，SEQIDN0: 1至SEQIDN0: 10373的核酸序列包含一个或多个多态性，例如，单核苷酸多态性（SNPs)和插入/缺失多态性（Indels)。 As shown in Table l, SEQIDN0: 1 to SEQIDN0: 10373 nucleic acid sequence that contains one or more polymorphisms, such as single nucleotide polymorphisms (SNPs) and insertion / deletion polymorphism (Indels).

在本发明的一个方面中，在一个或多个DNA序列的数据组中提供了多态性玉米基因座，即数据组包含高达限定数量的不同序列的多态性基因座。 In one aspect of the present invention, in the data set of one or more DNA sequences are provided in the polymorphic locus of maize, i.e. the data set contains a sequence of up to a limited number of different polymorphic loci. 数据组中的限定数量的多态性基因座可以少至2个或高达1000个以上或更多，例如，5、 10、 25、 40、 75、 100或500个基因座。 Data set limited number of polymorphic locus may be as few as two or as high as 1000 or more, or more, e.g., 5, 10, 25, 40, 75, 100 or 500 loci. 此类数据组用于大规模的或包括大量的植物的基因分型应用。 This data set is used to include a large number of large-scale plant or genotyping applications. 在本发明的一个有用的方面中，多态性玉米基因座的数据组记录在计算机可读介质上。 In one useful aspect of the present invention, the polymorphic loci maize data set recorded on a computer readable medium.

在本发明的另一个方面中，将本发明的基因座中的多态性定位在玉米基因组上，例如，作为包含两个或多个多态性的图谱位置的玉米基因组的遗传图镨，如表1中所示，更优选如表3中所示。 In another aspect of the present invention, the locus of the polymorphism in the present invention positioned on the maize genome, for example, as a map that contains two or more polymorphic positions of the maize genome genetic map praseodymium, e.g. shown in Table 1, and more preferably as shown in Table 3. 此类遗传图谱在图1中说明。 Such genetic map is illustrated in Figure 1. 遗传图谱数据还可以记录在计算机可读介质上。 Genetic map data can also be recorded on a computer readable medium. 本发明的优选实施方案提供高密度的多态性遗传图镨，例如，至少150 个或更多，比如说至少500个或1000个，跨越玉米基因组图傳的多态 A preferred embodiment of the present invention to provide a high-density genetic map polymorphism praseodymium, e.g., at least 150 or more, for example at least 500 or 1000, pass across the maize genome mapping polymorphic

7性。 7 sex. 特别地，有用的遗传图语包含在连锁群上平均距离不多于IO厘摩(cM)的多态性。 In particular, useful in the genetic map language linkage group containing an average of not more than IO centimorgan (cM) polymorphism.

本发明还提供了用于鉴定多态性的核酸分子，此类分子优选是寡核苷酸，其用作用于扩增玉米基因组片段（例如，多态性基因座）的PCR引物，和用于在玉米DM中鉴定特定多态性的存在或缺失的测定的杂交探针。 The present invention also provides a nucleic acid molecule for the identification of polymorphisms, such molecules preferably is an oligonucleotide, which is used for amplification of fragments of the maize genome (e.g., polymorphic locus) of the PCR primers, and for Determination of specific identification of the presence or absence of polymorphism in maize DM hybridization probes.

通常，用作PCR引物的核酸分子是成对提供的，用于扩增包含至少一个多态性的玉米DNA片段，其中各个分子包含至少15个核苷酸碱基。 Typically, the nucleic acid molecule used for PCR primers are provided in pairs for the amplification of corn comprising at least one polymorphic DNA fragment, wherein each molecule comprises at least 15 nucleotide bases. 引物分子之一的核苷酸序列与多态性基因座中的玉米DNA片段的一条链中的相同连续核苷酸数量的序列优选至少90%同一。 The nucleotide sequence of one strand of one molecule and the primer polymorphic locus in maize DNA fragment the same as in the number of contiguous nucleotide sequence is preferably at least 90% identical. 优选地， 引物可以在高严紧条件下与多态性基因座DNA的链杂交。 Preferably, the primer can hybridize to polymorphic locus strand DNA under high stringency conditions. 优选地，成对地提供和使用此类引物，所述引物在多态性基因座中的玉米DNA片段中的至少一个多态性的侧翼。 Preferably, provided in pairs and the use of such primers, the primer polymorphic locus in maize DNA fragments flanking the at least one polymorphism.

用作杂交探针的核酸分子，用于检测玉米DM中的多态性，其可以针对多种测定进行设计。 Used as nucleic acid hybridization probes for detecting a polymorphism in maize DM, which may be designed for a variety of assay. 为测定目的（其中探针意欲于与包括多态性的片段杂交），此类分子可以包含至少12个核苷酸碱基和可检测的标记。 To determine the object (wherein the probe is intended to hybridize with a fragment comprising the polymorphism), such molecules may comprise at least 12 nucleotide bases and a detectable label. 核苷酸碱基的序列与本发明的多态性基因座中的玉米DNA片段的任一条链中相同连续核苷酸数量的序列优选至少90%同一，更优选至少95%同一。 Any one polymorphic locus strand the nucleotide sequence of the present invention in maize DNA fragments of the same number of contiguous nucleotide sequence is preferably at least 90% identical, more preferably at least 95% identical. 本发明优选的方面中，可检测的标记是位于所述分子的一个末端的染料。 A preferred aspect of this invention, the detectable marker is located at one end of the molecule of the dye. 在更优选的方面中，所述分子在其末端包含染料和染料淬灭剂。 In a more preferred aspect, the dye molecule comprising at its distal end and a quencher dye. 就SNP测定而言，成对提供此类分子是有用的，例如， 其中各个分子在5，端具有不同的荧光染料并且除单核苷酸多态性以 On SNP assays, to provide a pair of such molecules are useful, for example, wherein each molecule at 5, ends with different fluorescent dyes and in addition to a single nucleotide polymorphism

外具有相同的核苷酸序列。 Having the same nucleotide sequence outside.

为了测定目的（其中分子设计为杂交于多态性邻近，所述多态性通过单碱基延伸检测，例如，标记的双脱氧核苷酸的单碱基延伸）， 此类分子可以在序列中包含至少15个，更优选至少16个或17个核苷酸碱基，所述序列与多态性玉米DM片段的任一条链中的相同连续核苷酸数量的序列至少90%同一，优选至少95%同一。 To determine the object (wherein the molecule is designed to hybridize to adjacent polymorphism, single base extension of the polymorphisms detected by, e.g., labeled dideoxy nucleotides single base extension), in the sequence of such molecules can be comprising at least 15, more preferably at least 16 or 17 nucleotide bases, and any one strand of the sequence polymorphism in maize DM fragment of the same number of contiguous nucleotide sequence of at least 90% identical, preferably at least 95% of the same.

本发明的另一方面是杂交探针和玉米基因组DM片段的复合物。 Another aspect of the invention is a complex of a hybridization probe and DM maize genomic fragments.

8本发明另一方面提供了寡核苷酸组，其包含用于PCR扩增多态性玉米DM片段的核酸分子引物对，和用于检测所述片段中的多态性的至少一个检测核酸分子。 8 another aspect the invention provides a set of oligonucleotides comprising a nucleic acid molecule primers for PCR amplification of polymorphic fragments of maize DM and for the detection of a polymorphism in the fragment at least one detector nucleic acid molecules. 此类组以至少2组或高达1000组或更多，例如，高达5、 10、 25、 40、 75、 100或500组的引物对和杂交探针的集合形式提供。 Such a group has at least 2 groups or groups of up to 1000 or more, e.g., up to 5, 10, 25, 40, 75, 100 or 500 and the group of primer pairs hybridized to form a set of probes.

本发明的另一方面提供了用于确定多态性的方法，就真核基因组中的基因分型应用而言，所述多态性作为标记很可能是有用的。 Another aspect the present invention provides a method for determining the polymorphism, it eukaryotic genome genotyping applications, the polymorphic markers as may be useful. 此类方法包括通过分离来自一个物种的至少两个品种的基因组DM片段的重复序列，基于核苷酸序列的大小将分离的基因组DNA片段分开，并且比较级分中的片段的序列以确定多态性来构建约化表示文库 Such methods include separation by repeating sequences from two species of at least one species of DM genomic fragments, genomic DNA fragments based on the nucleotide sequence of the isolated separately, and the comparison sequence fragment fraction to determine the polymorphic to build a representation of about library

(reduced representation 1 ibraries )。 (Reduced representation 1 ibraries). 更具体地,鉴定基因组DNA 中的多态性的方法包括用甲基化敏感的核酸内切酶消化来自一个真核物种的至少两个品种的总基因组DNA,以提供经消化的DM片段的库 More particularly, the identification of genomic DNA polymorphism method comprises cleaved with a methylation-sensitive nucleic acid digestion of total genomic DNA from one of at least two species of eukaryotic species, to provide a digested fragment library DM

(pool)。 (Pool). 对于由更低百分比的5-曱基化胞嘧啶表征的DNA区域，片段的平均核苷酸长度更小。 For the lower percentage 5- 曱 base cytosine characterized DNA region, the average length of the nucleotide fragment smaller. 此类片段是可分离的，例如，通过凝胶电泳，基于核苷酸长度。 Such fragments are separable, e.g., by gel electrophoresis, based on the nucleotide length. 具有少于平均核苷酸长度的DNA级分分离自经消化的DNA库。 Having less than the average length of the DNA nucleotide fractions isolated from the digested DNA library. 比较级分中的DNA序列以鉴定多态性。 Comparison of the fraction to identify DNA sequence polymorphisms. 与编码序列相比，重复序列更可能包含5-甲基化胞嘧啶，例如在-CG-和-CNG序列片段中。 Compared with the coding sequence, repeat sequences are more likely to contain 5-methyl cytosine, e.g. -CG- and -CNG sequence fragment. 在该方法优选的一个方面中，用甲基化敏感的核酸内切酶消化来自一种作物的至少两种不同的近交品种的基因组DM,所述核酸内 In a preferred aspect of the method, the use of methylation-sensitive endonuclease digestion of genomic DM at least two different inbred varieties derived from one crop, the nucleic acid within

切酶选自由Aci I， Apa I， Age I, Bsr FI, BssH II， Eag I， Eae I， Hha I， HinPl I， Hpa II, Msp I， MspM II， Nar I, Not I， Pst I， Pvu I， Sac II， Sma I, Stu I和Xho I组成的组，以提供一皮物理分离（例如，通过凝胶电泳）的经消化的MA库。 Enzyme selected from Aci I, Apa I, Age I, Bsr FI, BssH II, Eag I, Eae I, Hha I, HinPl I, Hpa II, Msp I, MspM II, Nar I, Not I, Pst I, Pvu I, Sac II, Sma I, Stu I and Xho I the group consisting of, in order to provide a physical separation of the skin (e.g., by gel electrophoresis) of the digested library MA. 相当大小的DM级分获得自各个所述品种的经消化的DM。 DM sizable fraction obtained by the digestion of DM from each of the species. 将来自相当级分的DM分子插入到载体中以构建基因组DNA克隆的约化表示文库，对其进行测序和比较，以鉴定多态性。 The DM molecules from considerable fraction into a vector to construct a genomic DNA library cloned from about representation, be sequenced and compared in order to identify polymorphisms.

在可选择的方法中，通过用核酸内切酶消化来自一个真核物种的至少两个品种的总基因组DM以提供经消化的DNA片段的库，从而鉴定基因组DNA中的多态性。 In an alternative method, the DM digestion of total genomic eukaryotic species from one species with at least two endonucleases to provide the digested DNA fragment library, thereby identifying a genomic DNA polymorphism. 将经消化的DNA片段在基底上的阵列中进行分离并且与一种或多种经标记的具有重复序列元件的寡核苷酸接触，所述重复序列元件表征该物种中的MA。 The digested DNA fragments were separated in an array on the substrate and in contact with one or more labeled oligonucleotide having a sequence element is repeated, the repeated sequence elements characterizing the species of MA. 杂交鉴定由重复序列表征的DNA片段。 Blotting characterized by repeated sequences of DNA fragments. 针对多态性，比较不与重复序列寡核苷酸杂交的DNA 片段的序列。 For polymorphisms, less oligonucleotide sequences that hybridize with the repeat sequence DNA fragment nucleotides. 此类方法提供了来自植物的约化表示的基因组DNA的片段，所述植物具有这样的基因组DM，其包含具有相对较高水平的甲基化胞嘧啶的DNA区和具有相对较低水平的曱基化胞嘧啶的DNA区。 Such methods provide a fragment of genomic DNA from some of the plant represented by the plant has a genome of DM, which comprises a relatively high level of methylated cytosine in a DNA region having a relatively low level of 曱DNA base cytosine area. 本发明的约化表示片段包含来自具有相对较低水平的甲基化胞嘧啶的DNA区的基因组DNA,并且在由所述片段的核酸大小表征的级分中提供，例如，在500至3000bp范围内。 Reduction of the invention showing the genomic DNA fragment containing a relatively low level from the methylated cytosine of DNA region, and is characterized by the size of the nucleic acid fragment fraction, e.g., in the range 500 to 3000bp inside.

本发明还提供了使用本发明的基因座和多态性的方法，例如，在基因分型和相关的应用中。 The present invention also provides the use of the present invention and polymorphism locus methods, e.g., in genotyping and related applications. 本发明一方面提供了通过比较至少两种玉米品系中的DNA序列找出玉米DNA中的多态性的方法，其中通过使用多态性玉米DNA基因座的片段选择序列。 The present invention provides in one aspect by comparing at least two strains of maize corn DNA sequence to identify DNA polymorphisms, wherein by using a locus polymorphic fragments of maize DNA sequence of choice. 用于比较的DNA序列优选与多态性基因座的序列至少80%同一。 The DNA sequence for comparison with the polymorphic locus sequence preferably is at least 80% identical. 更优选地，所述序列与多态性基因座相连锁。 More preferably, the sequence polymorphism loci with linkage.

本发明还提供通过测定来自至少一种玉米品系的组织的DNA或mRM以鉴定与本发明的多态性基因座连锁的核酸多态性的存在的基因分型的方法。 The present invention also provides a nucleic acid polymorphism genotyping methods by measuring the presence of at least one tissue from maize lines with DNA or mRM polymorphic loci identified with the present invention is linked. 在本发明优选方面中，基因分型使用在图l的遗传图谱中鉴定的多态性，如通过表3所扩增。 In a preferred aspect of the present invention, genotyping identified using genetic map in Figure l of the polymorphisms, e.g., by amplification in Table 3. 在本发明的另一个优选的方面，基因分型包括针对至少两个玉米品系鉴定一个或多个表型性状以及确定性状和多态性之间的关联（association)，例如鉴定和选择性状互补的林系用于育种以提高杂种优势。 In another preferred aspect of the invention, genotyping, including at least two maize lines for the identification of one or more phenotypic traits and determine the association between traits and polymorphism (association), such as the identification and selection of complementary traits Forest Department for heterosis breeding to improve. 用于此类基因分型的测定可以使用足够的核酸分子以鉴定至少2个和高达5000个或更多的不同的多态性的存在，例如，其中不同多态性的数量为5、 10、 25、 40、 75、 100、 500、 1000、 2000、 3000或4000个。 Genotyping for such measurement may be used to identify nucleic acid molecules sufficient at least two and up to 5000 or more different polymorphism exists, for example, where the number of different polymorphic 5, 10, 25, 40, 75, 100, 500, 1000, 2000, 3000 or 4000.

本发明还提供了通过确定分离自一种或多种玉米植物的核酸分子的核酸序列中多态性的存在研究玉米等位基因的方法，其中所述多态性与本发明的多态性基因座连锁。 The present invention also provides a self-determined by one or more of the nucleic acid sequence of the nucleic acid molecule of maize plants in the presence of polymorphism alleles Maize separation, wherein the gene polymorphism and polymorphism of the present invention Block chain.

10本发明还提供了通过鉴定基因组序列中已定位的多态性的存在定位玉米基因组序列的方法，其中已定位的多态性与本发明的多态性连锁，例如在本发明的遗传图谱中已定位的多态性。 10 The invention also provides a polymorphism exists in Maize genomic sequences identified by methods genomic sequence has been positioned, wherein the linkage has been positioned polymorphism polymorphism of the present invention, for example, in the genetic map of the present invention positioned polymorphism.

本发明还提供了通过选择具有借助连锁不平衡而与目的性状相关联的多态性的玉米品系来进行玉米育种的方法，其中所述多态性与本发明的多态性基因座相连锁。 The present invention also provides a selection polymorphism by means of linkage disequilibrium maize lines and associated with the trait for corn breeding, wherein the polymorphic loci polymorphism and linkage of the present invention.

本发明还提供了通过鉴定表征玉米植物的一种或多种不同的表型性状的组将玉米植物中的表型性状与基因型相关联的方法。 The present invention also provides a characterization of a corn plant or more different phenotypic traits through the identification group of maize plants phenotypic traits associated with the genotype. 测定来自 Determined from

至少两种具有等位基因DNA的玉米植物的组织中的DM或mRM，以鉴定不同多态性的组的存在或缺失。 At least two tissues of maize plants with the alleles in DNA of DM or mRM, to identify the presence or absence of different polymorphic group. 鉴定多态性的组和表型性状的组之间的关联，其中多态性的组包括至少一个，更优选至少10个，与本发明的多态性基因座连锁的多态性，例如，与已定位的多态性（例如， 如表3中所鉴定的）相连锁的至少IO个多态性。 The group identified the association between polymorphism and phenotypic trait group, which includes at least one set of polymorphisms, more preferably at least 10, and polymorphic loci linked polymorphisms of the present invention, for example, and the localized polymorphism (e.g., as identified in Table 3) with at least IO linked polymorphisms. 在更优选的方面中， 在玉米植物的分离群体中（所述的玉米植物具有染色体基因座中的等位基因DNA)性状与基因型相关联，所述基因型对目的性状赋予表型影响，并且其中多态性定位于此类基因座中，并且其中多态性之间以及多态性和性状之间的关联程度使得能够确定多态性和性状基因座的线性次序。 In a more preferred aspect, in a segregating population of maize plants (maize plants having said chromosome locus allele DNA) traits associated with the genotypes, the genotype effect on phenotype conferred trait, and positioned in such a polymorphic loci, and in which the degree of association between the polymorphism and polymorphism traits enabling the determination of polymorphism and trait loci linear order. 在此类方法中，至少5个多态性与基因座连锁，允许所述基因座的不均衡定位。 In such methods, at least five loci polymorphisms and linkage, allowing uneven positioning of the locus.

本发明还提供了通过鉴定至少一种多态性与目的性状的连锁，其中所述多态性与本发明的多态性基因座连锁，鉴定包含所述基因座的基因组克隆和鉴定与所述基因座连锁的基因而鉴定与目的性状相关联的基因的方法。 The present invention also provides through the identification of at least one polymorphism and the trait chain, wherein the polymorphisms and polymorphic locus chain present invention, the identification of the locus containing genomic clones and the identification and Locus method of gene identification and gene associated with the trait. 在本发明的一个优选的方面，此类关联用于标记辅助育种和/或标记辅助选择。 In a preferred aspect of the invention, such association for marker-assisted breeding and / or marker-assisted selection.

本发明进一步提供了用于改进杂交玉米中的杂种优势的方法。 The present invention further provides a method for improving the hybrid maize heterosis. 在此类方法中，开发大量与本发明的多态性基因座连锁的多态性和两个以上玉米近交系中的性状之间的关联。 In such methods, the development is associated with a large number of polymorphic loci linked polymorphisms present invention and two or more inbred lines of maize between traits. 选择具有互补的杂种优势组的两种此类近交系用于育种，预测所述互补的杂种优势组提高杂种优势。 Such a choice of two inbred lines with complementary heterosis breeding group for predicting the complementary set of heterosis improve heterosis.

本发明还提供了通过查询（interrogating)玉米遗传图谱中小于 The present invention also provides a query (interrogating) maize genetic map in less than

ii10cM的平均密度上的SNP的集合篩选性状的方法。 SNP screening method for collection of traits on the average density ii10cM. 与本发明的多态性基因座连锁的SNP的存在或缺失与此类性状相关联。 Polymorphic loci with the invention of the chain of the SNP and the presence or absence of these traits are associated. 在本发明的另一个方面，所述多态性用于鉴定单倍型，所述单倍型是由连锁不平衡的至少两种多态性表征的基因组DNA的等位基因的片段并且其中所述多态性在长度不大于IO厘摩的基因组窗口（window)中，例如，不大于8厘摩或者更小的窗口，例如在比如说1至5厘摩范围内。 In a further aspect of the invention, for the identification of the polymorphism haplotypes, the haplotype is a fragment from allele in linkage disequilibrium with at least two polymorphic characterization of genomic DNA and wherein length polymorphism described in no more than IO centiMorgans genome window (window), for example, no more than 8% or less of the window Morocco, for example, for instance within 1-5 centiMorgan range. 本发明尤其有用的方法使用此类多态性，以鉴定各玉米染色体中一系列相邻的基因组窗口中的大量单倍型，例如，提供用此类窗口的基本完全的基因组覆盖。 The method of the present invention is particularly useful in the use of such polymorphisms to identify each chromosome of a series of adjacent corn genome in a large number of window haplotype, e.g., to provide essentially complete genome is covered with such a window. 用足够大量的和不同的玉米育种群体，能够在各个窗口中鉴定大量的单倍型，因此提供能够与一种或多种性状相关联的等位基因DNA以允许聚焦（focused)的标记辅助育种。 With a sufficiently large number of different maize breeding population, able to identify a large number of haplotypes in each window, and therefore capable of providing a variety of traits or alleles associated with DNA to allow the focus (focused) marker-assisted breeding . 因此，本发明的玉米 Accordingly, the present invention is corn

分析的一个方面进一步包括针对所述玉米植物群体表征一种或多种性状以及将所述性状与所述等位基因的SNP或Indel多态性进行关联的步骤，优选地，进行组织（organized to)以定义单倍型。 A further aspect of the analysis for the maize plant comprises the step of one or more groups characterizing trait and the trait and the allele or SNP Indel polymorphisms associated, preferably organized (organized to ) to define haplotypes. 此类性状包括产量，倒伏，成熟期，植林高度和疾病抗性，例如，对玉米孢嚢线虫，玉米锈病，褐色茎腐病，摔死症（sudden death syndrome )等等的抗性。 Such traits including yield, lodging, maturity, plant height and disease resistance forests, for example, corn spore Nang nematodes, corn rust, brown stem rot, which killed disorder (sudden death syndrome), etc. resistance. 为了促进育种，计算各个性状的值或性状组合的值是有用的，例如，多重性状指数（multiple trait index)。 In order to promote the breeding, the value calculated for each trait or a combination of traits are useful, for example, multiple trait index (multiple trait index). 分配给多重性状指数中的不同性状的权重可以根据育种的目标而改变。 Right index assigned to multiple traits of different traits of heavy breeding goals can change. 例如，如果产量是关键目标，产量值的权重可以为50至80%，成熟期，倒伏，植林高度或疾病抗性在多重性状指数中的权重可以为较低的百分比。 For example, if the output is a key goal, the right weight yield value can be 50 to 80%, maturity, lodging, disease resistance afforestation or right height in multiple trait index may be to a lower weight percentage.

本发明的另一个方面提供了基因分型的方法，其进一步包括针对至少两种玉米品系鉴定一种或多种表型性状和确定所述性状和多态性之间的关联。 Another aspect of the invention provides a method of genotyping, further comprising at least two corn lines for identifying one or more phenotypic traits and determine the association between the trait and polymorphism.

本发明的另一个方面关注于经选择的多态性玉米DNA序列组在玉米育种中的用途，例如，通过基于其位于多态性基因座的基因型选择玉米品系，所述玉米品系具有经选择的多态性玉米DNA序列的组中的序列。 Another aspect of the present invention is concerned with a selected group of polymorphic DNA sequences in maize corn breeding purposes, e.g., by selecting based on maize lines located polymorphic locus genotype, the corn line having a selected Corn DNA sequence polymorphisms in the sequence group.

本发明的另一个方面提供对玉米植物进行育种的方法，包括步骤： Provides a corn plant breeding methods to another aspect of the invention, comprising the steps of:

12(a) 针对至少两种玉米植物的育种群体，鉴定至少两个高达IO厘摩的基因组窗口中的至少两个单倍型的性状值； 12 (a) for at least two corn plant breeding populations, identify at least two property values ​​up to IO centiMorgans genome window at least two haplotypes;

(b) 在所述的育种群体中对两种玉米植物进行育种，以产生后代种子群体； (B) in the breeding population of two maize plant breeding to produce progeny seed population;

(c) 鉴定所述后代种子中的各个所述窗口中的多态性等位基因的状态，以确定所述单倍型的存在；和 (C) identifying the status of each of said progeny seed of the polymorphic alleles of the window, to determine the presence of said haplotype; and

(d ) 选择具有针对所述后代种子中的确定的单倍型而鉴定的较高性状值的后代种子。 (D) selecting progeny seed having a higher value for the trait in progeny seeds identified haplotype and identified.

在育种方法的方面中，针对基本上覆盖各个染色体全部的各个相邻的基因组窗口中的至少两个单倍型鉴定性状值。 In terms of the breeding process, for substantially covering at least two haplotype identification character value of each of all the chromosomes of the genome of the respective adjacent window. 在该方法的另一个有用的方面，针对各个染色体中高达IO厘摩的基因組窗口中的单倍型 In another useful aspect of the method, for each chromosome of up to IO centimorgan genome haplotype window

就较高的产量性状值选择后代种子。 On higher value selection progeny seed yield traits. 在本发明的另一方面，育种方法关注于增加的产量，其中性状值针对产量性状，其中针对各个窗口中的单倍型对性状值进行分级，并且其中选择这样的后代种子，其在窗口中的产量性状值高于所述窗口中的平均产量性状值。 In another aspect of the present invention, breeding methods focus on increasing the yield, which the property value for yield traits, wherein for each window in the haplotype of trait values ​​were graded, and wherein the selection of such progeny seed, which in the window The yield is higher than the average yield traits trait value of the window. 在育种方法的某些方面中，用表1中鉴定的多态性定义单倍型或单倍型定义为在DM 序列的组中，所述DNA序列的组包括SEQ IDN0: 1至SEQ ID NO: 10373 的所有DNA序列，或定义为与这些多态性之一连锁不平衡。 In some aspects of breeding methods, polymorphism is defined in Table 1 identified haplotype or haplotype is defined as the DM group sequence, the DNA sequence of the group consisting of SEQ IDN0: 1 to SEQ ID NO : All DNA sequences 10373 or defined as polymorphism in linkage disequilibrium with one of these.

由标记鉴定表征的本发明的方法可以一使用寡核苷酸引物和寡核苷酸检测子（detector )进行。 Characterized by the method of the present invention, markers may be one using oligonucleotide primers and oligonucleotide detection sub (detector) carried out. 因此，本发明的另一个方面关注于此类 Accordingly, another aspect of the present invention is concerned with such

寡核苷酸，例如与标记起作用的寡核苷酸组。 Oligonucleotides, such as oligonucleotides labeled with functioning. 更具体地，本发明提供分离的核酸分子对，其包含用于扩增玉米DNA以鉴定DNA中多态性的存在的寡核苷酸引物，例如包含至少12个连续核苷酸的寡核苷酸，其与多态性玉米DNA基因座的对应链中相同核苷酸数量的DM片段末端至少90%同一，所述多态性玉米DNA基因座具有与本文中公开的多态性玉米DNA序列的子集中的序列（或其互补物）至少90%同一的序列。 More particularly, the present invention provides an isolated nucleic acid molecule of comprising corn DNA for amplification of polymorphic DNA to identify the presence of oligonucleotide primers, e.g., comprising at least 12 contiguous nucleotides of the oligonucleotide acid, corn with polymorphic DNA loci in the same chain of nucleotides corresponding number of DM-terminal fragment of at least 90% identical, the maize DNA loci polymorphism DNA sequence polymorphism corn disclosed herein The subset of sequences (or the complement thereof) at least 90% identical sequence. 更优选的，此类寡核苷酸对包含至少15个连续的核苷酸，或更多，例如，至少20个连续的核苷酸。 Containing at least 15 consecutive nucleotides, or more, e.g., at least 20 contiguous nucleotides and more preferably, such oligonucleotides. 更具体地，当打算将与SNP的杂交用于鉴定玉米DM中的多态性的标记测定时，该组将包含四个寡核苷酸，例如， 一对分离的核酸分子，其用于扩增可以与多态性侧翼的DNA杂交的DM,以及一对检测子核酸分子，其用于检测扩增的DM的片段中单核苷酸多态性中的各个核普酸。 More specifically, when it is intended to be used to identify the SNP hybrid maize DM polymorphism markers measured, this group will contain four oligonucleotides, e.g., the pair of isolated nucleic acid molecule, which is used to expand by hybridizing with DNA flanking polymorphisms DM, and a pair of detecting the nucleic acid molecule, for the detection of the amplified fragments DM single nucleotide polymorphisms in each core Pu acid. 在本发明的优选的方面，此类检测子核酸分子包含至少12个核苷酸碱基和可检测的标记，或至少15 个核苷酸碱基，并且检测子核酸分子的序列除所述核苷酸多态性（例如SNP或Indel)外与含有多态性的多态性玉米DNA基因座的片段的任一链中的相同连续核苷酸数量的序列同一且至少95%同一。 In a preferred aspect of the invention, such testing promoter nucleic acid molecule comprises at least 12 nucleotide bases and a detectable label, or at least 15 nucleotide bases, and detects the sub-sequence of the nucleic acid molecule in addition to nuclear nucleotide polymorphisms (e.g., SNP or Indel) outer with either strand of the polymorphic locus of maize DNA fragment containing the polymorphism of the same number of contiguous nucleotides and at least 95% sequence identity to the same.

附图简介 Brief Introduction

图1是玉米的遗传图谱，其展示本发明的已定位的多态性的密度。 Figure 1 is a genetic map of maize, which demonstrate the present invention have been positioned polymorphism density.

图2是是基因分型测定的对偶图（allelograra)图示结果。 2 yes yes genotyping assay dual graph (allelograra) illustrates the results. 定义：本文中使用的一些术语定义如下： Definition: Some terms used herein is defined as follows:

"等位基因"表示位于特定基因座的可选择的序列；等位基因的长度可以小到1个核苷酸碱基，但是通常更大。 "Allele" indicates an alternative sequence at a particular locus; allele lengths as small as 1 nucleotide base, but usually greater. 等位基因序列可以是氨基酸序列或核酸序列。 Allele sequence may be an amino acid sequence or nucleic acid sequence. "基因座"是一种短序列，其通常是独特的并且通常通过参照点在基因组中的特定位点发现，例如短DM序列， 其是基因，或基因的部分或基因间区域。 "Locus" is a short sequence, which is typically unique and generally by a reference point in the genome specific sites found, such as a short sequence of DM, which is an intermediate portion or gene, or gene regions. 本发明的基因座可以是基因组中特定位点外的独特的PCR产物。 Locus present invention may be genomic locus specific PCR product unique outside. 本发明的基因座包含一个或多个多态性，即在一些个体中存在的可选的等位基因。 Locus present invention comprises one or more polymorphisms, i.e., in some alternative alleles present in an individual.

"基因型"指位于个体生物体中的一个或多个基因座的等位基因组成的详述。 "Genotype" refers to an individual organism DETAILED DESCRIPTION located one or more loci thereof. 就二倍体生物来说，在各个基因座有两个等位基因；当等位基因相同时，二倍体基因型被称为纯合的，而当等位基因不同时， 被称为杂合的。 On diploid organisms, two at each locus alleles; when the same allele, homozygous diploid genotype is called, and when the alleles are different, known as hybrid bonded. "单倍型"表示基因组DNA的等位基因片段，其倾向于作为一个单位进行遗传；此类单倍型可以由两种或多种多态性表征并且可以由不大于IO厘摩的大小定义，例如不大于8厘摩。 "Haplotype" means the allele of genomic DNA, which tend as a genetic unit; haplotype may be, and such can be defined by two or more polymorphisms characterized by not more than the size of the IO centimorgan , such as no more than 8% friction. 随着更高的精度，来自更高的密度的多态性，单倍型可以由l-5厘摩范围内的 With higher accuracy, higher density polymorphism from haplotype l-5 can be made within the scope centiMorgan

14基因组窗口表征。 14 genome characterization window.

"共有序列"指构建的DNA序列，其鉴定位于基因座的等位基因中的SNP和Indel多态性。 "Consensus sequence" refers to DNA sequences constructed, which is located in loci identified in SNP and Indel polymorphisms. 共有序列可以基于位于基因座的DNA的任一条链并且表述基因座中各个SNP的任一的核苷酸碱基和基因座中所有Indel的核苷酸碱基。 Consensus sequence may be based on either strand of DNA located locus and expression of nucleotide bases and locus of each SNP locus of any one of the nucleotide bases of all Indel. 因此，尽管共有序列可能不是实际的DNA序列的拷贝，共有序列用于精确设计针对基因座中的实际的多态性的引物和探针。 Thus, although a consensus sequence may not copy the actual DNA sequence, a consensus sequence for the precise design for the actual locus polymorphic primers and probes.

"表型"指细胞或生物体的可检测的特征，所述特征是基因表达的表现。 "Phenotype" refers to a cell or organism detectable feature, the feature is the performance of gene expression.

"标记"指多态性序列。 "Label" refers to a sequence polymorphism. "多态性"是序列中个体间的变异，尤其在DNA序列中。 "Polymorphism" is a variation among individuals in sequence, particularly in DNA sequence. 有用的多态性包括单核苷酸多态性（SNPs) , DNA 序列中的插入或缺失（Indel)和DM序列的简单序列重复（SSRs)。 Useful simple sequence polymorphisms include single nucleotide polymorphisms (SNPs), DNA sequence insertion or deletion (Indel) and DM sequence repeat (SSRs).

"标记测定"指通过使用特定方法检测位于特定基因座的多态性的方法，例如表型分型（例如种子颜色，花的颜色，或其他直观地可检测的性状），限制性片段长度多态性（RFLP),单碱基延伸，电泳， 序列比对，等位基因特异性寡核苷酸杂交（ASO) , RAPID等。 "Determination mark" means by using a specific method for detecting a polymorphism at a particular locus methods, e.g., phenotyping (e.g., seed color, flower color, or other visually detectable trait), restriction fragment length Normality (RFLP), single base extension, electrophoresis, sequence alignment, allele-specific oligonucleotide hybridization (ASO), RAPID and so on. 优选的标记测定包括如美国专利6,013,431中公开的单碱基延伸和如美国专利5, 538, 848中公开的等位基因分型，其中核酸内切酶活性释放来自杂交探针的报告染料，两者公开的内容均通过引用并入本文。 Preferred marker assays include single base as described in U.S. Patent No. 6,013,431 and as disclosed in U.S. Patent No. 5 extends, 538, 848 in the allele-disclosed, wherein the endonuclease activity of the hybridization probe releases the reporter dye from the two those disclosures are incorporated herein by reference.

"连锁"涉及杂交中产生配子的类型的相对频率。 "Chain" refers to the relative frequency of the type of produce gametes hybridization. 例如，如果基因座A具有基因"A"或"a"而基因座B具有基因"B"或"b，，，并且具有AABB的亲本I和具有aabb的亲本B之间的杂交产生四种可能的配子，其中基因分离为AB、 Ab、 aB和ab。空期望值（null expectation)为独立均等地分离到四种可能的基因型中的每一种，即没有连锁，配子的1/4将是每一种基因型。配子分离为不同于l/4的基因型归因于连锁。 For example, if you have the gene locus A "A" or "a" and has a gene locus B "B" or "b ,,, and I have AABB parent and have cross between parental B aabb produce four possible gametes, wherein the gene is separated into AB, Ab, aB and ab. Empty expected value (null expectation) independent uniformly separated into four possible genotypes in each, i.e. no chain, gametes would be 1/4 each genotype. Genotype gametes is separated into different than l / 4 is attributed to the chain.

"连锁不平衡"在一代的许多个体的群体中配子类型的相对频率的背景下进行定义。 "Linkage disequilibrium" in many individuals in the population at the generation of the relative frequency of gamete types of background defined. 如果等位基因A的频率是p, a的频率是p， ， B 的频率是q而b的频率是q，，则基因型AB预期的频率（没有连锁不 If the frequency of allele A is p, a frequency is p,, the frequency B is q and b is the frequency of the genotype AB q ,, is the expected frequency (with no linkage does

15平衡）是PQ， Ab是pq， ， aB是p， q，而ab是p， q，。 15 balance) is PQ, Ab is pq,, aB is p, q, and ab is p, q ,. 预期频率的任何偏差都叫做连锁不平衡。 Any deviation from the expected frequency is called linkage disequilibrium. 当在连锁不平衡中时两个基因座称为"基因连锁"。 When in linkage disequilibrium with two loci called "genetic linkage."

"数量性状基因座（QTL)"指这样的基因座，其在一定程度上数字化地控制其代表的性状，所述性状通常连续分布。 "Quantitative trait locus (QTL)" refers to the locus, which to a certain extent, digitally controlled traits of their representatives, the trait is usually a continuous distribution.

本发明的核酸分子或其片段在某些情况下能够与其他核酸分子杂交。 Nucleic acid molecule of the invention or fragments thereof, in some cases capable of hybridizing with other nucleic acid molecules. 如本文所用的，如果两种分子能够形成反平行，双链核酸结构，这两种核酸分子称为能够相互杂交。 As used herein, if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure, known as the two nucleic acid molecules capable of hybridizing to each other. 一种核酸分子称为另一种核酸分子的"互补物，，，如果它们表现为"完全互补，，，即一条序列中的每个核苷酸与另一条序列中的其碱基配对伴倡核苷酸互补。 A nucleic acid molecule referred to another nucleic acid molecule "complement ,,, if they behave as" fully complementary ,,, i.e. a sequence in each nucleotide sequence and the other one of its base pairing with initiative nucleotides complementary. 两种分子称为"最低限度互补"，如果它们能够以足够的稳定性相互杂交，以在至少常规的"低严紧条件"下允许它们保持相互退火。 Two molecules called "minimum complementary" if they can hybridize to each other with sufficient stability to at least conventional "low stringency conditions" permit them to remain annealed to each other. 类似地，分子称为"互补"，如果它们能够以足够的稳定性相互杂交，以在常规的"高严紧"条件下允许它们保持互相退火。 Similarly, molecules called "complementary" if they can hybridize to each other with sufficient stability to under conventional "high stringency" conditions permit them to remain annealed to each other. 与其它核酸分子杂交的核酸分子，例如，至少在低严紧条件下，被称为其他核酸分子的"可杂交同源物（hybridizable cognate )"。 And other nucleic acid molecules hybridizing to a nucleic acid molecule, e.g., at least under low stringency conditions, other nucleic acid molecules are called "hybridizable homolog (hybridizable cognate)". 常规的严紧条件由Sambrook等人，Molecular Cloning. A Laboratory Manual, 2nd Ed. ， ColdSpring Harbor Press, Cold Spring Harbor, New York (1989) 和Haymes等人，Nucleic Acid Hybridization, A Practical Approach,IRL Press, Washington, DC (1985)描述，其分别通过引用并入本文。 Conventional stringency conditions are described by Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Ed., ColdSpring Harbor Press, Cold Spring Harbor, New York (1989) and Haymes, et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington , DC (1985) described, which are incorporated herein by reference. 因此，允许偏离完全互补，只要此类偏离不完全排除分子形成双链结构的能力。 Thus, allowing deviation from a perfectly complementary, as long as these deviations do not completely rule out the ability of the molecules to form a double-stranded structure. 因此，为了使核酸分子用作引物或探针，其只要在序列中足够互补从在特定的溶液和所用的盐浓度下能够形成稳定的双链结构。 Therefore, in order to make the nucleic acid molecule is used as a primer or probe, which is sufficient as long as the sequence complementary to the solution and in a particular salt concentration can be used to form a stable double-stranded structure.

促进DNA杂交的合适的严紧条件，例如，6. Qx氯化钠/柠檬酸钠(SSC)，在约45。 Suitable stringency conditions that promote DNA hybridization, for example, 6. Qx sodium chloride / sodium citrate (SSC), at about 45. C，然后在50。 C, and then at 50. C用2. OxSSC冲洗，是本领域技术人员已知的或者可以在Current Protocols inMolecular Biology, JohnWiley & Sons, NY (1989)， 6. 3. 1-6. 3. 6 (通过引用并入本文）中找到。 C with 2. OxSSC flushing, are known to those skilled in the art or can be in Current Protocols inMolecular Biology, JohnWiley & Sons, NY (1989), 6. 3. 1-6. 3. 6 (incorporated herein by reference) found. 例如，冲洗步骤中盐浓度可以从50。 For example, the salt concentration in the washing step can be from 50. C约2. OxSSC的低严紧性至50。 C about 2. OxSSC low stringency to 50. C约0. 2xSSC的高严紧性中选择。 C about 0. 2xSSC high stringency of selection. 另外，冲洗步骤中的温度可以从室温，约22。 Further, the temperature in the wash step can be from room temperature, about 22. C的低严紧条件增加到约65。 Low stringency conditions increased to about 65 C. C的高严紧条件。 C in high stringency conditions. 温度和盐都可以改变，或者温度或盐浓度可以保持恒定而另一个量发生改变。 Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other volume change.

在优选的实施方案中，本发明的核酸分子可以特异性与具有如SEQ ID NO: 1至SEQ ID NO: 10373中列出的核酸序列的玉米DNA片段的一条链杂交，在中等严紧条件下，例如，约2. OxSSC和约65°C,更优选在高严紧条件下，例如0. 2xSSC和约65°C。 In a preferred embodiment, the nucleic acid molecule of the invention can be specifically having as SEQ ID NO: 1 to SEQ ID NO: one strand of the nucleic acid sequences listed 10373 maize DNA fragments, under medium stringency conditions, e.g., from about 2. OxSSC about 65 ° C, more preferably at high stringency conditions, e.g., 0. 2xSSC and about 65 ° C.

本文所用的"序列同一性，，涉及两个最佳比对的多核苷酸或肽序列贯穿組分（例如核苷酸或氨基酸）的比对的窗口不变的程度。对测试序列和参照序列的比对的片段来说，"同一性分数"是由两个比对的序列共有的同一组分的数目除以参照序列片段中组分的总数目，即完整的参照序列或较小的定义的参照序列的部分。"百分比同一性"是同一性分数的100倍。 "Sequence identity as used herein ,, degree polynucleotide or peptide sequences involving two optimally aligned through component (e.g., nucleotides or amino acids, respectively) of the same window. Test and reference sequences are than the pair fragment, the "identity fraction" is the total number of components in a sequence fragment from the same number of sequences common to the two component alignment divided by the reference, i.e., a complete reference sequence or a smaller defined part of the reference sequence. "percent identity" is 100 times the identity scores.

优选的实施方案的详述 DETAILED DESCRIPTION A preferred embodiment of the

A. 核酸分子一基因座，引物和探针 A. nucleic acid molecule a locus primers and probes

本发明的玉米基因座包含DM序列，所述DM序列包含至少20个连续的核苷酸并且包括或与表1中鉴定的一个或多个多态性相邻。 Maize locus of the invention comprises a sequence DM, the DM sequence comprises at least 20 contiguous nucleotides and comprising one or more polymorphic or adjacent identified in Table 1. 此类玉米基因座具有核酸序列，所述核酸序列与玉米DNA片段的任一条链中的相同核苷酸数量的序列具有至少90%序列同一性，更优选地至少95%,或对一些等位基因来说更优选地至少98%,以及在很多情况下至少99°/。 Such maize locus having a nucleic acid sequence, said nucleic acid sequence according to any one strand of the DNA fragment and corn are the same as the number of the nucleotide sequence having at least 90% sequence identity, more preferably at least 95%, or for some allelic genes, more preferably at least 98%, and in many cases at least 99 ° /. 序列同一性，所述玉米DNA片段包括或与多态性相邻。 Sequence identity, including the corn DNA fragment polymorphisms or adjacent. 可以在由SEQ ID NO: 1至SEQ ID NO: 10373组成的组中的序列中发现此类玉米DM片段的一条链的核苷酸序列。 Can by SEQ ID NO: a nucleotide sequence of one strand of a fragment of such corn DM discovery group consisting of the sequence of 10373: 1 to SEQ ID NO. 借助多态性的特别的本质，应理解，对至少一些等位基因来说，本身与多态性没有同一性。 With particular nature of the polymorphism, to be understood that, for at least some of the alleles, the identity of itself with no polymorphism. 因此，对多态性序列以外的序列，可以确定序列同一性。 Therefore, other than the polymorphic sequence sequences, sequence identity can be determined. 更具体地，各个基因座中的多态性在表l中进行鉴定。 More specifically, each locus polymorphism identified in Table l.

对许多基因分型应用来说，使用来自一个以上基因座的多态性作为标记是有用的。 For many genotyping applications, the use of more than one locus from polymorphism as a marker is useful. 因此，本发明的一个方面提供不同基因座的集合。 Accordingly, one aspect of the present invention provides a collection of different loci.

17在此类集合中基因座的数目可以改变，但将是限定的数量，例如，少 17 The number of loci can be varied in such a collection, but the number will be limited, e.g., less

至2或5或10或25个基因座或更多，例如高达40或75或100或更多个基因座。 To 2 or 5 or 10 or 25 or more loci, e.g., up to 40 or 75 or 100 or more loci.

本发明的另一个方面提供核酸分子，其能够与本发明的多态性玉米基因座杂交。 Another aspect of the invention provides a nucleic acid molecule, which is capable of polymorphic loci present invention maize hybrid. 在本发明的一些实施方案中（例如，其提供PCR引物），此类分子包含至少15个核苷酸碱基。 In some embodiments of the present invention (e.g., which provides PCR primer), such molecules comprise at least 15 nucleotide bases. 用作引物的分子能够在高严紧条件下与本发明的多态性基因座中的DM片段的一条链杂交。 Molecules can be used as primers under high stringency conditions to a polymorphic locus in the present invention, a fragment of a strand DM. 成对提供用于扩增DNA的引物，即正向引物和反向引物。 Provided in pairs for the amplification of DNA primers, i.e., forward primer and reverse primer. 一条引物可以与基因座中的DNA的一条链互补并且另一条引物可以与基因座中的DNA的另一条链互补，即引物的序列与一条链中的相同核苷酸数量的序列优选至少90°/。 A primer may locus a DNA strand complementary to and the other primer may other strand complementary locus DNA, i.e., the primer sequence with one strand of the same nucleotide number sequence is preferably at least 90 ° /. ，更优选至少95%同一。 , More preferably at least 95% identical. 应理解，此类引物可以与远离多态性的基因座中的序列杂交，例如，离多态性至少5、 10、 20、 50个或高达约IOO个的核苷酸碱基。 It should be understood, such primers may hybridize to sequences locus away from the polymorphism, e.g., from the polymorphism at least 5, 10, 20, 50, or up to about IOO a nucleotide bases. 本发明的引物设计依赖于本领域已知的因素，例如避免或重复序列。 Primer design of the present invention relies on factors known in the art, such as avoiding or repeats.

本发明的核酸分子的另一个方面是用于多态性测定的杂交探针。 Another aspect of the nucleic acid molecule of the present invention is a hybridization probe polymorphism assay. 本发明的一个方面中，此类探针是包含至少12个核苷酸碱基和可检测的标记的寡核苷酸。 One aspect of the present invention, such a probe is an oligonucleotide comprising at least 12 nucleotide bases and a detectable marker. 此类分子的目的是用于杂交，例如在高严紧条件下，与包括或与在多态性基因座的扩增部分中的目的多态性相邻的核苷酸碱基片段中的DNA的一条链。 The purpose of such molecules is used for hybridization, for example, under high stringency conditions, with a nucleotide fragment comprising a nucleotide polymorphisms adjacent object or part in the amplification of the polymorphic locus in a DNA a chain. 此类寡核苷酸优选与多态性基因座中的玉米DNA的一条链中的相同核苷酸数量的片段的序列至少90%，更优选至少95%同一。 Such oligonucleotides are preferably polymorphic locus in maize DNA strand in the sequence at least 90%, more preferably at least 95% identical to the number of segments of identical nucleotides. 本发明的优选的方面中，杂交探针进一步包含荧光标记和淬灭子，例如用于使用杂交探针的测定类型（已知如Taqman邻'J定，可从AB Biosystems获得）。 A preferred aspect of the present invention, the hybridization probe further comprises a fluorescent label and a quencher promoter, e.g., for determination of the type using hybridization probes (e.g. Taqman known o 'J set, available from AB Biosystems).

对于其中设计分子用于杂交至多态性邻近且通过单碱基延伸（例如，经标记的双脱氧核苷酸）检测的测定来说，此类分子可以在序列中包含至少15个，更优选至少16或17个核苷酸碱基，所述序列与多态性玉米DNA片段的任一条链中的相同连续核苷酸数量的序列至少90%,优选至少95%同一。 For design molecules which hybridize to polymorphic and adjacent to a single base extension (e.g., by a dideoxynucleotide labeled) is detected by measuring, in a sequence of such molecules can comprise at least 15, more preferably at least either strand 16 or 17 nucleotide bases, said sequence polymorphism in maize DNA fragments of the same number of contiguous nucleotide sequence of at least 90%, preferably at least 95% identical. 用于单碱基延伸测定的寡核苷酸可获自Orchid Bioystems。 Oligonucleotides used for single-base extension assay available from Orchid Bioystems. 此类引物和探针分子通常以用于基因分型测定中的两条引物和一种或多种探针的组提供。 Such primers and probe molecules typically measured for genotyping of two primers and one or more probes of group provides. 此外，经常希望针对大量多态性进行大量基因分型测定。 In addition, it is often desirable for a lot of a lot of polymorphism genotyping determination. 因此，本发明还提供核酸分子集合，例如，以表征大量多态性的组提供。 Accordingly, the present invention also provides a nucleic acid molecule set, e.g., to characterize a large number of polymorphic group provides.

B.识别多态性 B. identify polymorphisms

基因组中的多态性可以通过比较来自不同林系的cDNA序列进行确定。 Genome polymorphism can be determined by comparing the cDNA sequences from different forest department. 虽然通过比较cDNA序列检测多态性相对更方便，但cDNA序列的评价没有提供关于相应的基因组DNA中的内含子位置的信息。 Although relatively easier detection by comparing the cDNA sequence polymorphism, but the evaluation does not provide the cDNA sequence information on the corresponding genomic DNA intron positions. 此外，非编码序列中的多态性不能从cDNA鉴定。 In addition, non-coding sequence polymorphisms can not be identified from the cDNA. 这是一个缺点，例如，当使用cDM来源生的多态性作为标记用于基因組DNA的基因分型时。 This is a disadvantage, for example, when using a source of raw cDM polymorphism as a marker for genotyping genomic DNA. 可以设计更有效的基因分型测定，如果多态性的范围包括存在于非编码独特序列中的那些多态性。 Can design more effective genotyping determination, if the polymorphism is present in a range including the non-coding sequence of those unique polymorphisms.

对于鉴定和检测多态性来说，基因组DNA序列比cDM更有用。 For identification and detection polymorphism, the genomic DNA sequence ratio cDM more useful. 基因组中的多态性可以通过比较来自不同林系的基因组DNA序列而确定。 Genome polymorphism can be determined by comparing the genomic DNA sequences from different forest department. 然而，较高级的真核细胞的基因组DNA通常含有大量重复序列和转化子。 However, the genomic DNA of higher eukaryotic cells typically contain a large number of repeats and transformants. 基因组DNA可以是更有效的序列，如果通过减去或除去重复序列富集（enrich)编码/独特部分。 Genomic DNA sequences may be more effective, if by subtracting or removing the repeat enrichment (enrich) encoding / unique part.

很多策略可以用于富集编码/独特序列。 Many strategies can be used to enrich the coding / unique sequence. 这些例子包括使用对胞嗜啶曱基化敏感的酶，使用切割重复序列的McrBC核酸内切酶，印记(printing)基因组文库的微阵列，所述微阵列接着与重复序列探针杂交。 Examples include the use of cell tropism piperidin 曱 yl-sensitive enzyme, endonuclease McrBC cleavage repeat sequences using a nucleic acid microarray imprint (printing) a genomic library, the microarray and then repeat sequence probe hybridization.

a. 曱基化胞嘧啶敏感的酶： . a 曱 sensitive enzyme cytosine base:

较高级的真核细胞的DNA倾向于非常高度曱基化，然而其不是均匀甲基化。 Higher eukaryotic cells tend to be very highly Yue DNA glycosylation, methylation, however, not uniform. 事实上，重复序列比编码序列更高度甲基化。 In fact, the sequence is repeated more than the coding sequence of highly methylated. 因此，编码/独特序列可以通过利用曱基化模式中的这一区别来进行富集。 Thus, the coding / unique sequence can be enriched by using 曱 glycosylation pattern of this difference. 参见美国专利6， 017, 704，定位和评估CG岛中的DNA甲基化模式的方法。 See U.S. Patent No. 6, 017, 704, CG island location and methods to assess the DNA methylation patterns. 一些限制性核酸内切酶对其识别位点中甲基化胞嗜啶残基的存在敏 Some restriction enzyme recognition sites for its nucleic acid methylation cell tropism pyridine residues exist Min

19感。 19 sense. 此类甲基化敏感的限制性核酸内切酶不能在其识別位点切割，如 Enzymes such methylation-sensitive restriction endonuclease recognition sites which can not be cut, e.g.

果5'-CG-3'重叠或5'-CNG-3'重叠中的胞嘧啶残基被甲基化。 If the 5'-CG-3 'overlap or 5'-CNG-3' overlapping the cytosine residues are methylated. 甲基化敏感的限制性核酸内切酶包括4碱基切割酶（cutter): Aci I, Hha I， HinPl I, HpaII和Msp I， 6碱基切割酶：Apa I， Age I， Bsr FI， BssH II， Eag I， Eae I， MspM II， Nar I， Pst I， Pvu I， Sac II， Sma I， Stu I和Xho I以及8碱基切割酶：Not I。 Endo methylation-sensitive restriction endonuclease cleavage enzyme include four bases (cutter): Aci I, Hha I, HinPl I, HpaII and Msp I, 6 bp cutting enzymes: Apa I, Age I, Bsr FI, BssH II, Eag I, Eae I, MspM II, Nar I, Pst I, Pvu I, Sac II, Sma I, Stu I and Xho I and 8-base cutting enzymes: Not I. 例如，当C残基被甲基化，Pst I在CTGCAG位点进行的DNA切割被抑制。 For example, when a C residue is methylated, Pst I at CTGCAG loci DNA cleavage is suppressed. 为了富集编码/独特序列，玉米文库可以从用Pst I (或其他甲基化敏感的酶）消化的并通过琼脂糖凝胶电泳大小分离的基因组DNA构建。 To enriched coding / unique sequence, can be constructed from a library of maize with Pst I (or other methylation-sensitive enzyme) and digested genomic DNA size fractionated by agarose gel electrophoresis. 高度甲基化的基因组区域（即，具有大量重复序列的区域）具有更多的被甲基化的Pst I位点。 Highly methylated genomic region (ie, the region has a large number of repeat sequences) with more to be methylated Pst I sites. 因此，在Pst I消化期间重复DNA中的大部分Pst I位点不能被切割，并且重复序列倾向于主要由高分子量，未切割的MA组成。 Thus, during the Pst I digested DNA repeated Most Pst I site can not be cut, and repeats tend mainly high molecular weight, uncut MA composition. 反之，未被高度甲基化的基因组区域（即包含大量编码/独特序列的区域）包含大量未甲基化的Pst I位点，在消化期间这些位点被切割， 产生相对较小的片段。 Conversely, not hypermethylated genomic region (i.e., contains a large region encoding / unique sequence) contains a large number of unmethylated Pst I site during the digestion of these sites are cut, produces a relatively smaller fragments. 当经消化的DNA经过琼脂糖电泳时，来自高度甲基化的，非编码DNA区的相对较大的片段与来源于编码/独特序列的相对较小的片段分离。 When subjected to agarose electrophoresis of the digested DNA,, a relatively large non-coding DNA fragment from the region hypermethylation and from the encoding / unique sequence of relatively small fragment was isolated. 编码区-富集的DNA片段（通常在500 - 3000bp 之间）可以从凝胶中切除，纯化并连接入经Pst I消化的载体中，例如pUC18。 Coding region - enriched DNA fragment (typically 500 - Between 3000bp) can be excised from the gel, purified and ligated into the vector digested by Pst I, for example, pUC18. 连接产物通过电穿孔转化到大量合适的细菌宿主中，例如DH10B,以产生对编码/独特序列富集的克隆文库。 Ligation products were transformed by electroporation into a large number of suitable bacterial host, e.g., DH10B, to generate the coding / unique sequence enriched clone library. 可以对单独的克隆测序以提供插入的编码区MA序列。 You can separate clones were sequenced to provide coding region sequence inserted MA.

为了减少任意特定文库的序列复杂度，500至10000bp范围内的DNA可以进一步通过从凝胶递增地切除片段进行大小分离。 In order to reduce the complexity of any particular sequence library, DNA within the range 500 to 10000bp by further fragment excised from the gel incrementally size fractionated. 有用的大小分离的片段范围包括500-600 bp, 600-700 bp, 700-800 bp, 800-900 bp, 900—1100 bp, 1100-1500 bp， 1500-2000 bp， 2000-2500 bp和2500-3000bp。 Useful fragment size range, including the separation of 500-600 bp, 600-700 bp, 700-800 bp, 800-900 bp, 900-1100 bp, 1100-1500 bp, 1500-2000 bp, 2000-2500 bp and 2500- 3000bp. 一系列大小分离的约化表示文库通过将来自各个大小级分的纯化的DNA分别连接入载体而构建。 About the size of a series of separate representation library from purified DNA by various size fractions were ligated into the vector constructed. 对来自各个文库的克隆的小量样品（例如约400个克隆）进行测序以确定各个特定文库中存在的重复序列的分数。 On a small sample (e.g., approximately 400 clones) clones from each library were sequenced to determine the presence of scores of each particular library repeats. 从多种不同玉米品系制备的约化表示文库的比较表明许多级分含有少于10%重复序列和一些级分含有多于20% 重复序列。 From a variety of different maize lines produced about representation comparison suggests that many library fractions containing less than 10% repeat and some fractions containing more than 20% of the repeat sequence. 优选的约化表示文库包含少于20%重复序列，更优选少于15%重复序列和更优选少于10%重复序列。 The preferred representation of library comprises less than about 20% of repeat sequences, and more preferably less than 15% of the repeat sequence and more preferably less than 10% of the repeat sequence. 通过确定遍及所有大小分离的约化表示文库的重复序列的分数，可以选择具有最小重复序列分数的文库用于深度测序（通常10000-20000个克隆）。 By determining the size of the separation throughout all about representation fraction repeats library, you can choose to repeat the sequence with the smallest fraction of a library for deep sequencing (usually 10000-20000 clones). 因为获得序列的目的是为了检测多态性，对两种玉米品系的代表相同大小级分的等价文库测序。 Because the purpose is to obtain sequence polymorphisms, on behalf of the same size as the two maize lines library sequencing equivalent fractions. 对多态性检测而言，使用约化表示文库的另一个优点是其增加了从两种玉米品系回收等价序列的可能性。 Polymorphism detection, another advantage of using a representation about the library is that it increases the likelihood of recovery from two maize lines equivalent sequence. 只有等价序列可获自两种品系，多态性才能被检测。 Only available from two strains of the equivalent sequence polymorphism can be detected.

b. McrBC核酸内切酶 endonuclease b. McrBC nucleic acid

可选择的用于富集编码区DNA序列的方法使用McrBC限制性核酸内切酶(endonuclease restriction )。 Alternative method for the enrichment of the DNA sequence of the coding region provided with McrBC restriction endonuclease (endonuclease restriction). 作为对来自谨菌体/病毒的侵入的外源DNA的防御，大肠杆菌包含核酸内切酶，例如，McrBC核酸内切酶，其切割含有甲基化胞嘧啶的DNA。 As I would like to bacterial invasion from / virus defense exogenous DNA, E. coli endonuclease containing, for example, within McrBC endonuclease, which cut containing methyl cytosine DNA. 可以使用这一特征富集非高度甲基化的基因组区域的DM，例如，假定的编码区DNA。 This feature may be used non-enriched DM hypermethylated genomic region, for example, assume that the coding region DNA. 约化表示文库可以用通过物理剪切或用限制性酶消化切割的基因组DM片段构建。 Representation about the library can be used by physical shearing or by restriction enzyme digestion of genomic fragments cut DM building. 将DNA片段转化到大肠杆菌宿主中，所述大肠杆菌宿主包含McrBC 核酸内切酶，例如，大肠杆菌菌林JM107或DH5a。 The DNA fragment was transformed into E. coli host, the E. coli host containing the endonuclease McrBC, e.g., Escherichia coli JM107 or forest DH5a. 当用包含甲基化DNA 区域的DNA片段转化细菌宿主时，McrBC核酸内切酶将切割插入的DM 并且质粒不增殖。 When the DNA fragment containing the methylated DNA region transformed bacterial host, the endonuclease McrBC cutting insert of plasmid DM and not proliferate. 当用未甲基化的DNA片段转化细菌宿主时，质粒增殖，并且菌落将在琼脂平板上生长使得克隆可被测序。 When using the unmethylated DNA fragment transformed bacterial hosts, plasmid proliferation and colony clones can be sequenced so that growth on agar plates. 对来自用这种方式产生的文库的克隆的小量样品取样，并且确定重复序列的分数。 Of clones from the library generated in this way with a small sample of the sample, and determining the fraction of repeat sequences. McrBC核酸内切酶还可以与曱基化胞嘧啶敏感的核酸内切酶一起使用，以进一步减少文库中不适于测序的重复序列的分数，例如，包含15%以上的重复序列的序列。 The endonuclease McrBC may be glycosylated with 曱 cytosine nucleic acid-sensitive endonucleases used together, to further reduce repeats sequenced library suitable fraction, e.g., comprising a sequence of more than 15% of the repeat sequence.

c. 微阵列约化表示文库 c. microarray representation about library

另一种用于富集编码/独特序列的方法是构建约化表示文库（使用甲基化敏感或非-甲基化敏感的酶），在尼龙膜上印记文库的微阵列， 并且与由已知存在于文库中的重复元件制备的探针杂交。 Another method of enrichment coding / unique sequence is used to construct the library of about representation (using methylation sensitive and non - methylation-sensitive enzyme), the nylon membrane imprint library microarray, and by the already known to exist in the library of repetitive elements prepared probe hybridization. 鉴定重复序列元件，并且通过仅挑选阴性克隆重排列文库。 Identification of repeat sequence elements, and rearrange the library by selecting only negative clones. 上述过程通过随机挑 The above process by randomly pick

选来自约化表示文库的克隆到384孔平板中并且培养它们来进行。 Choose from a library of clones about representation to 384-well plates and cultured them for. 微阵列可以通过以确定模式在支持物上（例如玻璃支持物或尼龙膜）印记克隆DM制备，所述印记克隆DM来自384孔平板的集合。 Microarray to determine the mode can be on the support (e.g., a glass support or a nylon membrane) Stamp clone DM prepared, set the mark from clone DM 384-well plate. 包含数千个不同克隆（例如高达约25000个克隆或更多）的微阵列的制备是本领域公知的。 Preparation containing thousands of different clones (e.g., up to about 25,000 clones or more) of the microarray is well known in the art. 参见例如美国专利5， 807， 522，用于制备以高密度点印的多核苷酸的微阵列的方法。 See, e.g., U.S. Patent No. 5, 807, 522, a method for preparing a polynucleotide microarray with high density of the printed dot. 可以对来自约化表示文库的克隆的小量样品（如约400个克隆）进行测序以鉴定重复序列元件。 Can a small sample (such as about 400 clones) from some representation library clones were sequenced to identify repeat elements. 恢复(retrieve)包含重复序列的克隆，并且将克隆用于制备放射性探针， 所述探针在尼龙阵列上进行杂交。 Restore (retrieve) clones containing repetitive sequences, and the clone used to prepare radioactive probes, the probes were hybridized on nylon array. 放射性同位素标记元素包括32P， 33P， 35S, 1251等等，尤其优选"P。通过检测放射性分析杂交的阵列，例如， 使用Fuji Phosphoimager™ Imaging Screen。在合适的曝光时间后， 阵列影像被读取为数字文件，其代表来自各个阵列元件的杂交强度， 所述杂交强度与标记的重复序列的数量成比例。该放射性影像鉴定阵列上所有对应于重复序列克隆的克隆，还鉴定384孔平板和各个重复序列克隆的孔的位置。利用这些信息，可以从原始平板挑选出所有的非重复序列克隆并且将其重新置于一组新的平板上，这些平板不包含重复序列克隆。该方法可以用于将约化表示文库中的重复序列分数从约25%降低到约1-2%。 Radiolabelled elements include 32P, 33P, 35S, 1251, etc., particularly preferably "P. array hybridization analysis by detecting radioactivity, e.g., using Fuji Phosphoimager ™ Imaging Screen. After proper exposure time, the image is read as an array proportional to the number of digital documents, with representatives from hybridization intensity of each element of the array, the intensity of the hybridization with the labeled repetitive sequences. The radioactivity on the image to identify all of the clones corresponding to the array repeat clones, also identified 384-well plates and each repeat position sequences cloned hole. Using this information, the tablet can be selected from all of the original non-repeat is cloned and placed it back on a new set of plates, these plates do not contain repetitive sequences cloned. The method may be used to about representation library repeats fraction from about 25% to about 1-2%.

C.检测多态性 C. polymorphisms

DNA序列中的多态性可以通过多种本领域公知的有效方法检测， 包括在美国专利5,468,613和5，217，863; 5，210，015; 5, 876,930; 6, 030, 787 6,004, 744; 6,013,431; 5, 595, 890; 5， 762， 876; 5,945,283; 5， 468, 613; 6,090, 558; 5， 800， 944和5， 616， 464中公开的方法，所有的专利通过引用并入本文。 DNA sequence polymorphisms can be detected by a variety of effective methods well known in the art, including in U.S. Patents 5,468,613 and 5,217,863; 5,210,015; 5, 876,930; 6, 030, 787 6,004, 744; 6,013,431; 5, 595, 890; 5, 762, 876; 5,945,283; 5, 468, 613; 6,090, 558; 5, 800, 944 and 5, 616, 464 in the disclosed method, all of the patents are incorporated herein by reference . 例如，DNA序列中的多态性可以通过与等位基因-特异性寡核苷酸（ASO)探针杂交来检测，如在 For example, DNA sequence polymorphisms by allele - specific oligonucleotide (ASO) hybridization probes to detect, such as in

22美国专利5, 468,613和5， 217， 863中所公开。 22 US patents 5, 468,613 and 5, 217, 863 disclosed. 设计ASO探针的核苷酸序列以形成完全匹配的杂交或在可变核苷酸残基的位点包含错配碱基对。 ASO probes designed nucleotide sequence to form a hybrid or exact match at the site contains the variable nucleotide residues mismatched base pairs. 匹配的杂交和错配的杂交之间的区别基于在杂交或冲洗过程中使用的条件下杂交的热稳定性上的区别，通过变性梯度电泳或在错配位点的化学切割分析杂交稳定性上的区别。 Difference on the thermal stability of hybridized under hybridization and difference mismatch hybridization based on matching between the hybridization or washing conditions used in the process, or chemically by denaturing gradient electrophoresis in the mismatch site cleavage analysis of the hybridization stability distinction.

美国专利5， 468, 613公开了等位基因特异性寡核苷酸杂交，其中在核酸序列中的单个或多个核苷酸的变异可以通过如下过程在核酸中检测：扩增含有核苷酸变异的序列，将其点印在膜上并用标记的序列-特异性寡核苷酸探针处理。 U.S. Patent No. 5, 468, 613 discloses allele specific oligonucleotide hybridization, in which a single mutation in the nucleic acid sequence or a plurality of nucleotides in a nucleic acid can be detected in the following process: the amplification comprises a nucleotide sequence variation, which was printed on the film and the point marked with a sequence - specific oligonucleotide probes processing.

DNA核苷酸序列重复（例如微卫星，简单序列重复（SSRs)和短串联重复（STRs))中的长度变异，可以通过如美国专利6， 090,558 中公开的质傳法检测。 DNA nucleotide sequence repeats (eg microsatellites, simple sequence repeats (SSRs) and short tandem repeats (STRs)) of length variation, by United States Patent 6, 090,558 discloses a mass transfer assay. 使用质谱法的优点包括分析速度（每个样品数秒）和直接质量测量准确度的显著增加。 The advantage of using mass spectrometry, including analysis speed (seconds per sample) and a significant increase in direct mass measurement accuracy.

靶核酸序列还可以通过如美国专利5， 800， 944中公开的探针连接方法检测，其中扩增目的序列并且与探针杂交，然后进行连接以检测探针的标记的部分。 Target nucleic acid sequences are also possible as described in U.S. Patent No. 5, 800, 944 in the probe connection method disclosed in the detection, wherein the amplified target sequence and hybridized with the probe, and then connected to the portion labeled detection probe.

靶核酸序列还可以通过如美国专利5, 616, 464中公开的探针连接方法检测，该方法使用至少一对具有与靼核酸序列的相邻部分同源的序列和具有侧链的探针，所述侧链在所述探针与所述靶核酸序列配对后非共价性结合以形成茎（stem)。 Target nucleic acid sequences are also possible as described in U.S. Patent No. 5, 616, 464 in the probe connection method disclosed in the detection, the method using a sequence homologous to adjacent portions of at least one pair having pedaled nucleic acid sequence and a probe having a side chain, After the side chain of the probe and the target nucleic acid sequence non-covalently binding pair to form the stem (stem). 至少一条侧链具有光敏基团，其可以与茎的另一条侧链成员形成共价交联。 At least one side chain having a photosensitive group, which can be covalently cross-linked with another member of the side chain of the stem formed.

a. 引物碱基延伸测定 a. primer extension measurement bases

优选的用于检测SNP和Indel的方法是标记碱基延伸方法，如在美国专利6， 004， 744; 6,013,431 ; 5， 595:890; 5， 762， 876; 和5， 945， 283中所公开。 A preferred method for detecting SNP and Indel is labeled base extension method, as described in U.S. Patent No. 6, 004, 744; 6,013,431; 5, 595: 890; 5, 762, 876; and 5, 945, 283 disclosed . 此类方法基于引物延伸和可检测的核苷三磷酸的掺入。 Such methods based on primer extension and detection of nucleotide triphosphate incorporation. 引物设计为退火到与可变核苷酸紧邻的序列，在少至一个标记的核苷三磷酸掺入后，可以检测可变核苷酸。 Primers are designed to anneal immediately adjacent to the variable nucleotide sequence, after as few as one labeled nucleoside triphosphate incorporation, variable nucleotide can be detected. 该方法使用三条合成的寡核苷酸。 This method uses three synthetic oligonucleotides. 两条寡核苷酸用作PCR引物并且与玉米基因组DNA基因 Two oligonucleotides used as PCR primers and maize genomic DNA gene

23座的序列互补，所述序列位于包含待测定的多态性的区域的侧翼。 Sequence 23 complementary to, sequences flanking the polymorphism to be assayed contains a region. 将 Will

玉米基因组DNA用作模板，在PCR中使用引物寡核苷酸以产生足够拷贝的包含多态性的基因座区域，以区别等位基因。 Maize genomic DNA was used as template, using the oligonucleotide primers to generate sufficient copy locus region containing polymorphism in PCR to distinguish between alleles. 扩增包含多态性的玉米基因组区域后，PCR产物与第三条寡核苷酸（称为延伸引物）混合，所述第三条寡核苷酸设计为在DM聚合酶和两种差别标记的双脱氧核苷酸三磷酸存在下与扩增的紧邻多态性的DNA杂交。 After amplification of the maize genome comprising polymorphic region, PCR product with the third oligonucleotide (called extension primer) were mixed, and the third oligonucleotide is designed to mark the difference between DM polymerase and two dideoxynucleotide triphosphates under the presence of the amplified DNA polymorphism adjacent to hybridize. 如果多态性存在于模板上，在单碱基链延伸中，经标记的双脱氧核苷酸三磷酸之一可以加入到引物。 If the polymorphism is present on the template, in a single base chain extension, the one labeled dideoxynucleotide triphosphates may be added to by the primers. 通过检测两种差别标记中哪一种加入到延伸引物中，推断等位基因的存在。 By detecting the difference between the two markers which was added to the extension primer, the inferred allele. 纯合样品使得两种经标记的碱基中只有一种被掺入并且因此两种标记中只有一种被检测到。 Homozygous samples such that two kinds of labeled bases being incorporated and thus only one of the two labels only one is detected. 杂合样品存在两种等位基因，因此指示两种标记的掺入（掺入到延伸引物的不同分子） 并且因此将检测到两种标记。 Heterozygous samples there are two alleles, thus indicating two markers incorporated (incorporated into the extension primer is different molecules) and thus both labels will be detected.

为了设计用于通过单碱基延伸检测玉米多态性的引物，基因座序列开始是隐藏的，以防止针对与已知玉米重复元件（例如转座子）匹配的或具有非常低的序列复杂度（二或三-核苷酸重复序列）的位点设计三条引物中任一条。 In order to design for the detection of single-base extension primers corn polymorphism, locus sequence starts is hidden to prevent against the known corn repeat element (e.g., transposons) match or have a very low sequence complexity (two or three - nucleotide repeats) site design either a three primers. 针对此类重复元件设计的引物导致由多基因座的扩增或延伸引物针对多位点的退火引起的低特异性的测定。 For such repetitive elements designed primers resulting in a low specificity determined by multi-locus amplification or extension primer for annealing multilocus caused.

PCR引物优选地设计为（a)具有最佳的退火温度，就PCR而言， 在55-60。 PCR primers are preferably designed to (a) has the best annealing temperature, the PCR, in 55-60. C范围内，（b)具有18-25个碱基的长度，和（c)产生大小在75-200个碱基对范围的具有待测定的多态性的产物，所述待测定的多态性离各个引物的3，末端至少25个碱基。 Within the C range, (b) have a length 18-25 bases, and (c) generating size 75-200 bp polymorphic product range to be measured of, said polymorphism to be measured of from 3, the end of at least 25 bases of each primer. 必须选择延伸引物以含有最小的引物自我互补或引物间互补，否则PCR反应的效率和/或特异性将降低。 Extension primer must be selected to contain a minimum of self-complementary primer or a primer complementary to room, otherwise the efficiency of the PCR reaction and / or specificity will decrease.

延伸引物设计为退火到紧邻多态性，以便退火的延伸引物的3， 末端紧邻多态性位点。 Extension primer is designed to anneal to immediately adjacent polymorphism, so as to extend the annealed primer 3, the end close to the polymorphic site. 延伸引物可以位于多态性的5，侧或3，侧；然而，如果设计为位于3，侧，则延伸引物的序列必须与紧邻多态性的序列的反向互补序列匹配。 Extension primer may be located polymorphism 5, side or 3, the side; however, if the design is located 3, side, then extending the primer sequence must match a sequence adjacent the polymorphism reverse complement sequence. 延伸引物必须不包含自我互补序列，自我互补序列可以自我退火，或标记的ddNTPs的摻入可以由延伸引物的自我启动（priming)引起，使得多态性指导的掺入的结果不明显。 DdNTPs must be incorporated to extend the primer does not contain self-complementary sequences, self annealed self-complementary sequences, or may be labeled (priming) caused by the self-extension primer to start, so that the result is not obvious incorporated polymorphism guidance. 如果 In case

24紧邻多态性位点的序列的性质使得不可能设计完全不自我互补的延伸引物，则可以通过用脱碱基位点取代延伸引物的一个或两个碱基，限 Properties 24 sequence adjacent to the polymorphic site of the design makes it impossible to completely self-complementary extension primer, it can be substituted with abasic sites extension primer one or two bases, limit

制自我退火的程度，只要脱碱基位点没有插入到最3，的三个位点。 The degree of self-annealing of the system, as long as the abasic site is not inserted into the most 3, the three sites.

通过多态性的性质确定选择用于反应中的包含物的标记的ddNTP，无论延伸引物是否位于与多态性的第一个碱基匹配的位点，如果延伸引物位于多态性的5，或3，。 Determined by the nature of the polymorphism ddNTP reactions selection marker for inclusion, whether the primer extension site is located and the first nucleotide polymorphism matching, if the extension primer located polymorphism 5, or 3 ,. 如果延伸引物位于多态性的5，， 则ddNTP是多态性本身的核苷酸。 If the extension primer located 5 ,, the ddNTP polymorphism is a nucleotide polymorphism itself. 例如，在AG多态性情况下，ddNTP 将是ddATP-标记（1 )和ddGTP-标记（2 )。 For example, in the case of AG polymorphism, ddNTP will be ddATP- mark (1) and ddGTP- mark (2). 如果延伸引物位于多态性位点的3，，则ddNTP是多态性中包含的碱基的互补碱基；在这个例子中，是ddTTP-标记（1 )和ddCTP-标记（2 )。 If the extension primer polymorphic site located 3 ,, the ddNTP is complementary to the nucleotide polymorphism bases contained; in this case, is ddTTP- mark (1) and ddCTP- mark (2). 标记可以选自多种化学部分，包括亲和或免疫学标记，荧光染料和质量标签。 Tag may be selected from a variety of chemical moieties, including affinity or immunological markers, fluorescent dyes and mass labels. 该方法的最普遍的实施方案中，使用亲和和免疫学标记，然后使用适当的检测试剂。 The most common embodiment of the method, the use of affinity and immunological markers, and then use the appropriate detection reagents. 在这个例子中，可以使用ddATP-FITC和ddGTP-生物素，然后和偶联到辣根过氧化物酶的抗-FITC-抗体（HRP-抗-FITC )和偶联到碱性磷酸酶的链霉亲和素（AP-链霉亲和素） 一起温育。 In this example, may be used ddGTP- ddATP-FITC and biotin, and then conjugated to horseradish peroxidase-conjugated anti -FITC- antibody (HRP- Anti -FITC) and a chain conjugated to alkaline phosphatase streptavidin (AP- streptavidin-biotin) were incubated together.

b. 标记的探针降解测定 b. probes labeled degradation determination

另一种用于检测多态性的优选的方法中，SNP和Indel可以通过在美国专利5'210，015; 5， 876， 930和6， 030， 787中公开的方法检测，其中寡核苷酸探针具有共价连接到探针的5，和3，末端的5，焚光报告染料和3，淬灭染料。 Another method is preferred for detecting polymorphisms in, SNP and Indel by in U.S. Patent No. 5'210,015; 5, 876, 930 and 6, 030, 787 in the detection method disclosed, wherein the oligonucleotide acid covalently linked to the probe having the probe 5, and 3, the end of 5, the burning of the optical reporter dye and 3, quencher dye. 当探针完整时，报告染料与淬灭染料接近抑制报告荧光，例如，通过Forster类型的能量转移。 When the probe is intact, the reporter dye and the quencher fluorescent dye close to suppress report, e.g., by Forster type energy transfer. PCR期间， 正向和反向引物与位于多态性侧翼的靶DNA的特异序列杂交。 During PCR, forward and reverse primers located in the flanking target DNA polymorphism specific sequences. 杂交探针与扩增的PCR产物内的包含多态性的序列杂交。 Contains a sequence polymorphism hybridization probe hybridized with PCR products amplified within. 在后续的PCR循环中，具有5， 一3，核酸外切酶活性的DNA聚合酶切割探针并且使报告染料和淬灭染料分离，导致报告荧光增加。 In the subsequent PCR cycles, having 5, a 3, exonuclease activity of DNA polymerase so that cleavage probe and reporter dye and the quencher dye, resulting in increased fluorescence report. 有用的测定可以获自AB Biosystems如Taqman⑧测定，其在单一反应中使用四种合成的寡核苷酸，该反应同时扩增玉米基因组DNA，辨别存在的等位基因，并直接提供用于辨别和检测的信号。 Useful assays can be obtained from AB Biosystems e.g. Taqman⑧ measured, using four synthetic oligonucleotides in a single reaction, the reaction simultaneously amplifying maize genomic DNA, to identify the presence of alleles, and directly provides for the identification and signal detection. 四种寡核苷酸的两种用作PCR引物并且 Two of the four oligonucleotides used as PCR primers and

25产生包含待检测的多态性的PCR产物。 25 PCR products containing polymorphic be detected. 其他两种寡核苷酸是等位基因特异性荧光共振能量转移（FRET)探针。 The other two allele-specific oligonucleotides are fluorescence resonance energy transfer (FRET) probe. FRET探针合并了紧密接近的荧光基团和淬灭分子，以便荧光基团的荧光被淬灭。 FRET probe incorporated in close proximity fluorophore and quencher molecules for fluorescence fluorophore is quenched. 通过FRET寡核苷酸的降解产生来自FRET探针的信号，以便荧光基团从淬灭剂的附近释放，并且因此当被合适的波长激发时能够发光。 Generated by FRET oligonucleotide degradation signal from FRET probe to fluorophore is released from the vicinity of the quencher, and therefore when it is appropriate excitation wavelength to emit light. 在该测定中，使用两个具有不同荧光报告染料的FRET探针，其中独特的染料结合到寡核苷酸中，所述寡核苷酸可以以高特异性退火到两个等位基因中的仅一个。 In this assay, using two FRET probes of different fluorescent reporter dyes, wherein the dye binding to a unique oligonucleotide, the oligonucleotide can be annealed to high specificity of the two alleles one only. 有用的报告染料包括6-羧基-4， 7， ， -四氯荧光素（TET ) , ( VIC ) 和6-羧基荧光素亚磷酰胺（FAM )。 Useful reporter dyes include 6-carboxy-4, 7,, - tetrachloro-fluorescein (TET), (VIC), and 6-carboxy-fluorescein phosphoramidite (FAM). 有用的淬灭剂是6-羧基-N， N， N， ， N' -四甲基罗丹明（TAMRA)。 Useful quencher is 6-carboxy--N, N, N,, N '- tetramethyl rhodamine (TAMRA). 另外，化学封闭各个FRET探针的3，端，以便它不能作为PCR引物。 Further, the chemical blocking each FRET probe 3, the end, so that it can not be used as PCR primers. 测定期间，玉米基因组DNA加入到包含两种PCR引物和两种FRET探针的緩冲液中。 During the measurement, maize genomic DNA was added to the buffer containing the two PCR primers and FRET probes of both. 还存在第三种荧光基团用作参比（passive reference),例如罗丹明X (R0X)，以帮助之后的相关荧光值的标准化（校正反应组装中的体积误差）。 There is a third fluorophore is used as the reference (passive reference), such as standardized rhodamine X (R0X), fluorescence correlation values ​​to aid later (assembly reaction volume correction error). 开始基因组DM 扩增。 DM began genome amplification. 在PCR的各个循环期间，FRET探针以等位基因特异方式退火到模板DM分子。 During each cycle of PCR, FRET probe annealing allele-specific manner to the template molecule DM. 当酶遇到退火的探针的5，末端时，退火的（但不是非退火的）FRET探针被TAQ DNA聚合酶降解，因此从探针的淬灭剂附近释放荧光基团。 When the enzyme encounters a probe annealed 5, end, annealing (but not non-annealed) FRET probes are degraded TAQ DNA polymerase, thus releasing the fluorophore from the quencher close to the probe. PCR反应后，使用荧光确定法检测两种荧光剂中每一种的荧光，和参比的荧光。 After the PCR reaction, using a fluorescent assay to determine each of the two fluorescent agent fluorescence, and the reference fluorescence. 两种染料的每一种的荧光的标准化强度与最初存在于样品中的各个等位基因的量成比例，并且因此可以推断样品的基因型。 Standardized strength of each of the two fluorescent dyes and amount of each allele originally present in the sample is proportional to, and therefore the genotype of the sample can be inferred.

为了设计用于测定的引物和探针，最初，基因座序列是隐藏的， 以防止针对与已知的玉米重复元件（例如，转座子）匹配的位点或具有非常低的序列复杂度（二-或三-核苷酸重复序列）的位点设计三条引物中的任一条。 In order to design for the determination of the primers and probes, initially, locus sequence is hidden to prevent against corn loci known repetitive elements (e.g., transposons) match or very low sequence complexity with ( two - or three - nucleotide repeats) site design three primers either one. 针对此类重复元件设计探针将导致由多个基因座的扩增或FRET探针针对多个位点的退火引起的低特异性的测定。 Such probes are designed for the repeat elements would cause low specificity is measured by a plurality of loci amplification or FRET probes for multiple sites annealing caused.

PCR引物设计为（a)具有大小在18-25个碱基范围的长度和与多态性基因座中的序列匹配，（b)具有57-60。 PCR primers were designed to (a) having a sequence matching the size range of 18-25 bases in length and the polymorphic locus, (b) having 57-60. C范围的计算的熔解温度， 例如，相应于52-55。 C calculated melting temperature range, e.g., corresponding to 52-55. C的最佳的PCR退火温度，（c)产生包括多态性 Optimal PCR annealing temperature of C, (c) comprises generating polymorphic

26位点并且具有大小在75-250个碱基对范围的长度的产物。 26 points and having a product size in the range of 75-250 base pairs in length. 优选PCR 引物定位于基因座，以便多态性位点离各个PCR引物的3，末端至少一个碱基。 Preferably PCR primers located in the locus, in order from the respective polymorphic loci PCR primer 3, the end of at least one base. PCR引物必须不包含大范围的自我互补或引物间互补的区域。 PCR primers must not contain a wide range of inter-self-complementary or complementary to the primer region.

FRET探针设计成跨越多态性位点的序列，优选多态性位于寡核苷酸的最3，端的2/3。 FRET probes are designed to span the polymorphic site sequence, preferably located at the oligonucleotide polymorphisms most 3, end 2/3. 在优选的实施方案中，FRET探针在其3，末端合并化学部分，当探针退火到模板DNA时，其结合到DNA小沟，因此， 增强探针-模板复合物的稳定性。 In a preferred embodiment, FRET probes in its 3, end consolidation chemical moieties when the probe is annealed to the template DNA, which binds to the DNA minor groove, thus, enhancing the probe - the stability of the template complex. 探针具有12-17个碱基范围的长度， 并且具有3， MGB，具有比PCR引物的熔解温度高5-7。 A probe having a length of 12-17 base range, and has a 3, MGB, having a high specific PCR primer melting temperature 5-7. C的计算的熔解温度。 C calculated melting temperature. 探针设计在美国专利5， 538， 848; 6, 084, 102和6， 127， 121中公开。 Probe design in U.S. Patent No. 5, 538, 848; 6, 084, 102 and 6, 127, 121 discloses.

D.多态性用于建立标记/性状关联的用途 D. polymorphic markers used to establish / trait association uses

本发明的基因座中的多态性可以用于标记/性状关联，其从群体的成员的基因型和表型的统计分析推断得到。 Locus of the present invention can be used for polymorphic markers / associated traits, from members of the community whose genotype and phenotype of the statistical analysis inferred. 这些成员可以是个体生物(例如，玉米），紧密相关的个体的家族，近交系，双单倍体或其他紧密相关的个体的组。 These members can be individual organism (e.g., corn), family of closely related individuals, inbred, double haploids or other closely related group of individuals. 此类玉米组称为"品系，，，表示遗传（descent) 品系。群体可以来自两种个体或两种品系（例如，定位群体）之间的单交或者其可以由具有多种遗传品系的个体组成。各个个体或品系由单个或平均性状表型和位于一个或多个标记基因座的基因型表征。 Such single cross maize group called "genetic strains ,,, represents (descent) strains. Two individuals or groups can come from two strains (eg, mapping population) or it can be made between individuals with a variety of genetic strains composition. characterizing the genotype of each individual or line of one or more marker loci by a single average trait or phenotype and located.

一些类型的统计分析可以用于从表型/基因型数据推断标记/性状的关联，但基本的设想是检测标记，即，多态性，对多态性而言，可选择的基因型具有显著不同的平均表型。 Some types of statistical analysis can be used to correlate the phenotype / genotype data to infer marks / traits, but the basic idea is to detect the mark, that is, polymorphism, polymorphism, the alternative genotype significantly different average phenotypes. 例如，如果给定的标记基因座A具有三种可选择的基因型（AA，Aa和aa)，并且如果这三种类型的个体具有显著不同的表型，则推断出基因座A与性状相关联。 For example, if a given marker locus genotype A has three selectable (AA, Aa and aa), and if these three types of individuals with significantly different phenotypes, the inferred locus A and traits Union. 表型上的区别的显著性可以通过数种标准统计检验（例如表型的标记基因型的线性回归或方差分析（AN0VA))进行检验。 Significant differences in phenotype can be produced by several types of standard statistical tests (e.g., the phenotype of the marker genotype of a linear regression or analysis of variance (AN0VA)) were tested. 商业上可获得的通常用于进行这类分析的统计软件包，包括SAS Enterprise Miner (SAS Institute Inc., Cary， NC) 和Splus (Insightful Corporation. Commercially available software packages such typically used for statistical analysis, including SAS Enterprise Miner (SAS Institute Inc., Cary, NC) and Splus (Insightful Corporation.

27Cambridge, MA)。 27Cambridge, MA). 当同时检验多种标记时，在所需的显著性水平进行<务正，例如Bonferonni 4交正，以表明关联。 When testing a variety of tags simultaneously when the required significance level <works are, e.g. Bonferonni 4 post is to show association.

通常，关联研究的目的并不是简单为了检测标记/性状关联，还为了评估直接影响性状（即QTL)的基因相对于标记位点的位置。 Generally, the purpose of the study is not simply related to the detection label / associated traits, but also in order to assess the direct impact of traits (ie QTL) gene marker loci with respect to the position. 在达到这一目的的简单方法中，在标记基因座差值量级之间，在可选的基因型或差异的显著性水平之间进行比较。 In a simple way to achieve this purpose, the difference in magnitude between the marker locus, between significance level selectable genotype or differences are compared. 性状基因推断为最接近这样的标记，其具有最相关的基因型差异。 Trait inference to the nearest such markers, which has the most relevant genotypic differences. 在更复杂的分析中，例如区间定位(interval mapping ) (Lander 和Botstein， Genetics 121:185-199 (1989)，针对QTL位于该位点的可能性检验遗传图镨上的许多位点中的每一个（比如说在lcM区间）。基因型/表型数据用于计算各个检验位点的L0D分值（似然比的对数）。当LOD分值超过临界阈值时，对QTL定位于遗传图谱上的该位置（其落在两个特异的标记基因座之间）来说存在显著的证据。 In a more sophisticated analysis, such as interval mapping (interval mapping) (Lander and Botstein, Genetics 121: 185-199 (1989), for the QTL located in many sites of the sites on the possibility of testing the genetic map of each praseodymium one (for example, in lcM interval). genotypic / phenotypic data used to calculate the various test sites L0D score (logarithm of the likelihood ratio). When LOD score exceeds a critical threshold for QTL mapping in the genetic map The position (which falls between two specific marker loci) is significant evidence exists.

a. 连锁不平衡定位和关联研究 a. linkage disequilibrium mapping and association studies

用于确定性状基因位点的另一种方法是分析群体中的性状-标记关联，所述群体中的个体在性状和标记基因座上都有区别。 Another method for determining the trait locus is to analyze population traits - mark associated with a group of individuals have differences in traits and marker loci. 一些标记等位基因可以与这一群体中的一些性状基因座等位基因相关联，由于群体遗传过程，例如独特起源的突变，建立者事件（founder event)， 随机漂变和群体结构。 Some marker alleles with this group of some trait loci associated allele, due to population genetic processes, such as the unique origin of the mutation, the founder of the event (founder event), random drift and population structure. 所述关联称为连锁不平衡。 The association is called linkage disequilibrium. 在连锁不平衡定位中，将个体的性状值与位于标记基因座的不同基因型比较。 In linkage disequilibrium location, the property value is compared with the individual marker loci located on different genotypes. 通常地， 显著的性状差异表明标记基因座和一个或多个性状基因座之间紧密接 Generally, a significant difference suggests a close connection between trait marker locus and one or more trait loci

近。 Closer. 如果标记密度适当高并且连锁不平衡只在染色体上非常紧密连锁的位点之间出现，那么可以非常精确地定位性状基因座。 If the marker density is high and appear appropriate linkage disequilibrium between loci on chromosomes only very closely linked, it can be very accurately positioned trait loci.

特定类型的连锁不平衡定位称为关联研究。 Locate a particular type of linkage disequilibrium called association studies. 该方法利用候选基因内的标记，所述基因被认为是功能性地涉及性状发生的基因，由于信息，例如生物化学，生理学，转录谱和模型生物中的反向遗传试验。 The method uses the markers within the candidate gene, the gene is considered to be functionally related to gene traits, since information, such as biochemistry, physiology, and transcriptional profiling model organisms reverse genetics tests. 在关联研究中，针对与性状变异的关联，检验候选基因内的标记。 In related studies, associated with the trait variation for inspection within the candidate gene markers. 如果研究群体中的连锁不平衡限制于非常紧密连锁的位点（即在基因内 If the study population linkage disequilibrium limited to sites very closely linked (ie, within the gene

28或在紧邻基因之间），则正关联提供了几乎确切的证据表明候选基因是性状基因。 28 or between adjacent genes), the positive association provides almost definitive evidence that the candidate gene trait.

b. 定位克隆（positional cloning)和转基因应用传统的连锁定位通常将性状基因定位于两个遗传标记之间的区间 b. positional cloning (positional cloning) and genetically modified application of traditional chain positioning trait usually located in the interval between the two genetic markers

(称为侧翼标记）。 (Called flanking markers). 当该区间相对小时（比如小于1Mb)，通过定位克隆方法精确鉴定性状基因是可行的。 When the range is relatively small (eg less than 1Mb), identified by positional cloning method precise trait is feasible. 需要高标记密度以充分减小区间长度。 High marker density required to sufficiently reduce the interval length. 该方法需要巨大的插入基因组克隆的文库（例如BAC文库）， 其中插入的是来自目的物种的基因组DM的片段（通常长度为100-150kb)。 This method requires a huge insert genomic clone library (e.g. BAC library), wherein the insert is a fragment from the genome of a species DM (usually a length of 100-150kb). 通过探针杂交或PCR篩选文库，以鉴定包含侧翼标记序列的克隆。 By probe hybridization or PCR screening of libraries to identify clones containing sequences flanking markers. 然后，通过物理定位方法，构建连接两个侧翼克隆的一系列部分重叠克隆（重叠群）。 Then, by physical mapping method, construct a series of connecting two portions flanking clones overlapping clones (contigs). 该方法包括指紋图镨，STS内容物作图和序列标签连接子方法学。 The method includes fingerprinting praseodymium, STS mapping and sequence tag contents linkers methodology. 一旦物理重叠群构建完成并测序，则针对所有的转录单位搜索序列。 Once the physical contigs built and sequencing, the search sequence for all transcription units. 可以通过比较突变体和野生型抹系之间的序列，通过另外的精密标度的遗传定位，和/或通过植物转化过程中的功能检测确定相应于性状基因的转录单位。 By comparing the mutant sequences and wild-type wipe system, through additional genetic positioning precision scale, and / or to determine the appropriate trait gene transcription unit by plant transformation process function tests. 用该方法鉴定的性状基因成为转基因产物开发的线索Uead)。 Trait loci identified by this method become transgene product development clue Uead). 类似地，通过与候选基因的关联研究鉴定的性状基因，成为转基因产物开发的线索。 Similarly, by identifying trait gene association studies with candidate genes, become transgene product development leads.

c. 标记辅助育种和标记辅助选择 c. marker-assisted breeding and marker-assisted selection

当性状基因定位于基因组标记附近时，这些标记可以用于选择性状的改良值，不需要在每个选择循环进行表型分析。 When the trait gene is located in the vicinity of the genome of tags that can be used to improve the value of select traits, phenotypic analysis is not required in each selection cycle. 在标记辅助育种和标记辅助选择中，性状基因和标记之间的关联最初通过遗传定位分析建立（如在A. l或A. 2中）。 In marker-assisted breeding and marker-assisted selection, the association between trait and marker analysis initially established through genetic mapping (as in A. l or A. 2 in). 在相同的方法中，确定哪些标记等位基因与有利的性状等位基因连锁。 In the same way, and to determine which marker allele and a favorable trait alleles chain. 然后，在群体中选择与有利的性状等位基因相关联的标记等位基因。 Then, select the marker alleles with favorable traits associated alleles in the population. 如果标记和性状基因之间有足够紧密的连锁，该方法将改善性状值。 If there is enough tight linkage between markers and trait genes, which will improve the property value. 需要的连锁程度依赖于选择的代数， 因为在每一代，都有可能通过重组破坏关联。 Chain extent required depends on the choice of algebra, because in every generation, are likely related damage through restructuring.

29用于开发新的近交系的杂交的预测 29 used to develop new inbred lines hybrid prediction

特定标记等位基因和有利的性状等位基因之间的关联还可以用于预测后代的哪些类型可以从给定的杂交中分离。 What types associated with a particular marker alleles and alleles between favorable traits can also be used to predict future generations can be isolated from a given hybridization. 这种预测可以允许选择合适的亲本以产生群体，从所述群体中，组装有利的性状等位基因 This prediction can allow parents to choose the right group to produce, from the group, assembled favorable traits allele

的新的组合以产生新的近交系。 The new combination to produce new inbred lines. 例如，如果A品系具有标记等位基因， 先前已知该标记等位基因与位于基因座1，20和31的有利的性状等位基因相关联，而B品系具有标记等位基因，该标记等位基因与位于基因座15， 27和29的有利的作用相关联，那么可以通过AxB杂交和选择在所有6个性状基因座具有有利的等位基因的后代开发新品系。 For example, if A strain having a marker alleles, with 1,20 and 31 located locus advantageous trait alleles associated with marker allele and B strains, the mark of the marker alleles and other previously known allele located locus beneficial effect 15, 27 and 29 associated, then the crossing and selection by AxB progeny develop new strains having favorable alleles at all loci 6 traits.

d. 杂种预测 d. hybrid forecast

通过在属于不同"杂种优势组"的两个优良近交系之间进行杂交， 产生商业玉米种子。 By belonging to different hybridization between "heterosis group" of two elite inbred lines, resulting in commercial maize seed. 这些组在遗传上足够不同，使得它们之间的杂交表现出高水平的杂种优势或杂种活力（即相对于亲本系增强的性能）。 These groups are genetically different enough so that hybridization between them exhibit a high level of heterosis or hybrid vigor (ie, the department enhanced performance relative to the parent). 通过分析好的杂种的标记构成，可以鉴定位于雄系和雌系中不同基因座的等位基因的组，所雄系和雌系良好结合以产生杂种优势。 By analyzing the mark constitutes good hybrids, can be identified and located the male line female lines of different loci of the group, a good combination of the male and female line system to produce heterosis. 理解了这些模式，并知道了不同近交系的标记构成，就可以允许预测不同品系对之间的杂种优势水平。 Understanding these patterns, and know the mark constitutes different inbred lines, you can allow the prediction of heterosis levels between different strains. 这种预测可减少对应杂种优势组的品系用于测试新的近交系的性能的可能性。 This prediction can be used to reduce the possibility of new strains of inbred heterosis performance tests corresponding group.

e. 遗传同一性(Identity by descent) e. the genetic identity (Identity by descent)

杂种优势的一种理论预测，用于产生杂种的雄系和雌系之间遗传同一性（IBD)的区域将降低杂种性能。 One theory of heterosis prediction, genetic identity between the male and female lines produced hybrid system (IBD) is used to reduce the area of ​​hybrid performance. 遗传同一性可以从不同品系中的标记等位基因模式推断。 Genetic identity can be inferred from the different strains of the marker allele model. 如果不可能偶然地独立发生，则位于一系列相邻基因座的同一的标记串可以认为是遗传同一的。 If you can not accidentally occur independently, are located in the same series of adjacent marker loci string can be considered genetically identical. 雄系和雌系中的标记指紋图镨分析可以鉴定IBD区域。 Male and female lines lines mark fingerprint analysis can identify IBD praseodymium area. 这些区域的认识可以告知杂种亲本的选择，因为在杂种中避免IBD可能提高性能。 Understanding of these areas can be informed parental choice of hybrids, because avoid IBD may improve performance in hybrids. 这一认识还可以告知育种程序，其中杂交可以设计为产生显示较少或没有IBD的近交系对（一个雄性和一个雌性）。 This understanding can also inform the breeding program, which can be designed to produce hybrids show little or no IBD inbred pair (one male and one female).

30近交系的指紋图谱是在一组标记基因座处的等位基因的纽合。 30 inbred fingerprint is a group of New York together in allele at the marker loci. 高密度指紋图语可以用于建立和描绘种质同一性，其用于种质所有权保护。 High-density fingerprints language can be used to establish and characterize germplasm identity, ownership protection for germplasm.

遗传标记用于加速转基因渗入到新的遗传背景（即渗入到不同范围的种质）。 Transgenic genetic markers for accelerating penetration to a new genetic background (i.e., penetrate to a different range of germplasm). 筒单基因渗入包括将转基因系与优良近交系杂交，然后将杂种与优良（循环的）亲代重复回交，并针对转基因的维持进行选择。 Single tube introgression including transgenic lines with excellent inbred lines, hybrids and then fine (cycle) repeated backcross parent, and for the maintenance of transgenic choose. 多个回交代后，通过重组和分离，起始转基因系的遗传背景逐渐被优良近交系的遗传背景取代。 After multiple backcross, through restructuring and separation, the genetic background of the initial gene transfer system is gradually being replaced by the genetic background of elite inbred lines. 该过程可以通过选择来源于循环亲代的标记等位基因进行加速。 This process can be accelerated by selecting from the cycle of parental marker alleles.

E.多态性测定用于定位DNA克隆文库的用途 E. Determination of use polymorphism DNA clone libraries for positioning

本发明的多态性和基因座用于与多态性连锁的QTL和基因的DM 序列的鉴定和定位。 Polymorphism loci present invention is used with the polymorphism linked QTL and DM sequence of the gene identification and positioning. 例如，可以用与性状连锁的多态性，查询BAC或YAC克隆文库，以发现包含特定QTL和与性状相关联的基因的克隆。 For example, you can use the traits linked polymorphic queries BAC or YAC clone libraries to discover and contains specific QTL cloning and genetic traits associated. 例如，通过与寡核普酸探针杂交鉴定大量的QTL和基因，例如数百或数千的巨大的多基因的序列，所述寡核苷酸探针可以与已定位的和/ 或连锁的多态性杂交。 For example, by oligonucleotide probe hybridization to identify a large number of genes and the QTL, e.g. huge multigene hundreds or thousands of sequences, said oligonucleotide probe may be positioned and / or chain polymorphism hybridization. 可以通过以高密度阵列形式提供克隆序列改善此类杂交篩选。 Can be provided in the form of high-density arrays of such hybrid screening to improve the cloned sequences. 更优选地，通过使用混合策略（pooling strategy) 增强篩选方法，以显著减少鉴定包含所述多态性的克隆所必需的杂交的数目。 More preferably, by using a mixed strategy (pooling strategy) enhanced screening method to significantly reduce the number of identifying comprises hybridization of the polymorphism of clones required. 当多态性被定位时，该筛选有效地定位克隆。 When polymorphism is located, the screening effectively positional cloning.

例如，如杲其中数千克隆排列在已定义的阵列中，例如，96孔板中，平板可以任意地在三维上排列，经排列的堆积（stack)的孔中分别包含独特的DNA克隆。 For example, as Gao wherein arranged in an array of thousands of clones have been defined, for example, 96-well plate, the plate can be arranged arbitrarily in three dimensions, by stacking (stack) of the pore array respectively include a unique DNA clone. 各个堆积中的孔可以表示为行，列和板的三维阵列中的离散单元。 Accumulation in each hole can be represented as lines, three-dimensional array of columns and plates discrete units. 本发明的一个方面中，堆积和堆积中的平板的数目约等于最小化的测定数。 One aspect of the invention, the number of stacking and stacking of the plate is approximately equal to minimize the number of measurement. 平板的堆积允许构建克隆的DM的库。 Cloning allows stacking plates of DM libraries.

对于三维排列的堆积，可以针对（a)各行中的所有单元，（b) 各列中的所有单元，和（c)各板中的所有单元建立克隆的DNA的库。 For three-dimensional arrangement of stacking can for (a) all the cells of each row, (b) all the cells in each column, and (c) in all cells of each plate to establish a library of cloned DNA. 用寡核苷酸探针杂交筛选的库将提供针对一列库， 一行库和一板库的正向指示，所述寡核苷酸探针与对克隆之一独特的多态性杂交，从而 Oligonucleotide probe hybridization screening with libraries will provide a positive indication for one column libraries, libraries, and a plate line of the library, the oligonucleotide probes for cloning the unique one polymorphism hybridization, thereby

31指示包含靶克隆的孔单元。 31 indicates clones containing the target hole unit.

在多重堆积的情况下，各个堆积中的所有克隆DNA的额外的库允许指示具有靶克隆的行-列-板坐标的堆积。 In the case of multiple stacked, each piled extra libraries allow all cloned DNA clones indicates a target row - column - stacking plate coordinates. 例如，4608个克隆组排列于48个96-孔板中。 For example, the 4608 clone group 48 are arranged in 96-well plates. 48个平板可以排列为8组，每组6个平板堆积，提供6 x 12 x 8三维阵列的单元，即，每个堆积包含6个8行和12列的堆积。 48 plates may be arranged as eight groups of six stacked plates, provide 6 x 12 x 8 three-dimensional array of elements, i.e., each containing six 8 stacked rows and 12 columns stacked. 对完整的克隆组来说，有36个库，即6个堆积库，8行库，12个列库和8个堆积库。 For a complete clone group, there are 36 libraries, namely six stacked library, 8 rows libraries, 12 libraries and eight columns stacked library. 因此，为了找到含有与各个已定位的多态性相关联或连锁的QTL或基因的克隆，需要最多36个杂交反应。 Therefore, in order to find containing polymorphisms or linked QTL associated with individual or gene cloning has been positioned, take up to 36 hybridization reaction.

一旦鉴定了克隆，从多态性基因座设计的寡核苷酸引物可以用于定位克隆连锁的QTL和/或基因。 Once identified, cloned, from polymorphic locus Designed oligonucleotide primers may be used to locate linked QTL cloning and / or genes.

F.计算机可读介质和数据库 F. computer readable medium and a database

本发明的核酸分子序列可以在多种介质中"提供，，，以方便使用，例如，数据库或计算机可读介质，其还可以包含描述性注释，所述注释的形式使得本领域技术人员能够研究或查询序列并获得有用的信息。在本发明的一个实施方案中，可以制备计算机可读介质，其包含核酸序列，其中本发明的基因座和核酸分子的序列的至少10%或更多，例如至少25%，或至少50%或更多。例如，此类数据库或计算机可读介 The nucleic acid molecule sequences of the invention can be in a variety of media ",,, provided to facilitate the use, for example, a database or a computer-readable medium, which may also include descriptive comment, the comment form enable those skilled in the art to study or query sequence and obtain useful information. In one embodiment of the present invention may be prepared a computer readable medium, comprising a nucleic acid sequence, wherein at least 10% of the locus and the nucleic acid molecule sequence of the present invention or more, e.g. at least 25%, or at least 50% or more. For example, such a database or a computer readable medium

的组。 Group. 此外，此类数据库或计算机可读介质可以包含已定位的或未定位的多态性或本发明和遗传图镨的图或表。 In addition, such a database or a computer-readable medium may contain positioning or localization of genetic polymorphism or invention and FIG praseodymium present charts or tables.

本文所用的"数据库"指可获取的收集的数据的任意表示，包括计算机文件，例如文本文件，数据库文件，电子数据表文件和图像文件，打印的表格（printed tabulat ion )和图示以及数字和图像数据组合的组合。 As used herein, "database" refers to any collection of available data representation, including computer files, such as text files, database files, spreadsheet files and image files, print form (printed tabulat ion) and shown as well as digital and combined-image data in combination. 在本发明的优选的方面，"数据库，，表示记忆系统，其可以存储计算机可搜索的信息。通常地，优选的数据库软件包括由DB2，Sybase和Oracle提供的数据库软件。 In a preferred aspect of the present invention, "Database, and represents memory system that can store a computer search. Typically database software, database software includes preferably provided by DB2, Sybase and Oracle.

本文所用的"计算机可读介质"指任何可以通过计算机直接读取和访问的介质。 As used herein, "computer readable medium" refers to anything that can be read and accessed directly by a computer medium. 此类介质包括，但不限于：磁存储器，例如软盘，硬 Such media include, but are not limited to: magnetic storage, such as floppy disks, hard

32盘，存储介质和磁带；光存储介质例如CD-ROM;电存储介质例如RAM和ROM;和这些种类的混合例如磁/光存储介质。 32, the storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and a mixture of these species such as magnetic / optical storage media. 本领域技术人员很容易知道任何目前已知的计算机可读介质如何可以用于创建包含其上记 Those skilled in the art will readily know how any of the presently known computer readable media may be used to create note thereon comprising

录有本发明的核苷酸序列的计算机可读介质的产品。 Recorded with the nucleotide sequence of the present invention is a computer-readable medium product.

本文所用的"记录的"、"记录有，，指在可获取的数据库或计算机可读介质中存储信息的方法的结果。例如，本领域技术人员可以很容易地采用任意目前已知的方法在计算机可读介质上记录信息，以产生包含本发明的已定位的多态性和其他核苷酸序列信息的介质。对本领域技术人员来说，可利用多种数据存储结构创建计算机可读介质，其中数据存储结构的选择通常基于所选的访问存储信息的方法。另外， Any known method, "record", ",, means recorded in a database or the results of a computer-readable medium can be obtained in the method of storing information. For example, those skilled in the art can easily adopt as used herein in computer-readable information recording medium, the medium to produce a nucleotide sequence polymorphisms and other information comprising the present invention have been positioned. skilled artisan, can utilize a variety of data structures to create a computer-readable storage medium, wherein the selection data storage structure is typically based on information stored in the selected access method. Further,

多态性和核苷酸序列信息。 Polymorphism and nucleotide sequence information.

计算机软件是公众可获得的，其使得本领域技术人员可以访问计 Computer software is publicly available which enable those skilled in the art can access count

算机可读介质中提供的序列信息。 The computer-readable medium provided sequence information. 以下实施例示范软件（其运行搜索运算法则，例如BLAST运算法则（Altschul等，J. Mol. Biol.215: 403-410 (1990)，通过引用并入本文）和Sybase系统中的BLAZE运算法则（Brutlag等人，Comp. Chem. 17:203-207 (1993)，通过引用并入本文））如何可以用于鉴定DNA序列，所述DNA序列以高同一性水平与本发明的基因座序列同源。 The following examples demonstrate the software (which run the search algorithms, such as BLAST algorithm (Altschul et al, J Mol Biol.215:.. 403-410 (1990), incorporated herein by reference) and Sybase system BLAZE algorithm ( . Brutlag et al., Comp Chem 17:. 203-207 (1993), incorporated herein by reference)) to be used to identify DNA sequences, the DNA sequences loci high level of identity with the sequence homology of the present invention . 可以比较高同一性的序列，以找到玉米品种的有用的多态性标记。 Can be relatively high sequence identity to find maize varieties useful polymorphic markers.

本发明进一步提供系统，特别是基于计算机的系统，其包含本文描述的序列信息。 The present invention further provides systems, particularly computer-based system, which contains sequence information described herein. 此类系统设计为鉴定本发明的核酸分子中商业上重要的序列片段。 Such system design for commercially important fragments of the sequence identified by the present invention is a nucleic acid molecule. 本文所用的"基于计算机的系统"指用于分析核苷酸序列信息的硬件，软件和存储器。 "Computer-based system" as used herein refers to the analysis of hardware, software and the nucleotide sequence information of the memory. 本领域技术人员可以容易地理解任一种当前可获得的基于计算机的系统适用于本发明。 Those skilled in the art can readily appreciate that any one of the currently available computer-based system suitable for the present invention.

如上所述，本发明的基于计算机的系统包含数据库（所述数据库中存储有本发明的多态性标记，遗传图镨和/或核酸分子的序列）以及支持和执行基因分型应用所必需的硬件和软件。 As described above, the present invention is a computer-based system includes a database (the database is stored polymorphic markers of the present invention, the genetic map praseodymium and / or a nucleic acid molecule sequence) and the support and the implementation of genotyping applications required hardware and software.

33实施例1 33 Example 1

本实施例举例说明通过使用对甲基化胞嘧啶残基敏感的酶以富集 Illustrated by the use of methylated cytosine residues Jimin sense to enrich the present embodiment, the enzyme

独特/编码序列基因组DNA而制备约化表示文库。 Unique / coding sequence of the genomic DNA library prepared about representation.

用于制备来自玉米（或其他植物）的基因组DNA的常规方法适用于构建约化表示的数据库。 Conventional methods for the preparation of genomic DNA from maize (or other plant) is applied to build the database about Representation. 存在商业可获得的试剂盒，例如来自Qiagen (Valencia, CA)的"DNeasy Plant Maxi试剂盒"。 The presence of commercially available kits, e.g. from Qiagen (Valencia, CA) the "DNeasy Plant Maxi Kit." 然而，最大化产量和便利的优选方法是用来自Life Technologies (Grand IslandNY)的"Plant DNAzol Reagent "提取DNA。 However, to maximize the yield and convenience preferred method is to use from Life Technologies (Grand IslandNY) The "Plant DNAzol Reagent" extracted DNA. 简言之，冷冻的叶组织用研钵和研杆在液氮中研磨。 Briefly, frozen leaf tissue with a mortar and pestle grinding in liquid nitrogen. 然后用DNAzol试剂提取研磨的组织。 Then extracted by grinding the tissue DNAzol reagents. 该步骤除去了细胞蛋白，细胞壁物质和其他碎片。 This step removes the cell proteins, cell wall material and other debris. 用该试剂提取后，沉淀DNA，沖洗，重悬，并用RNAse处理以除去RNA。 After the reagent extraction, precipitated DNA, wash, resuspended and treated with RNAse to remove RNA. 再次沉淀DNA，并在合适体积的TE(以便浓度为lMg/ul)中重悬。 The DNA was precipitated again, and resuspended (to a concentration of lMg / ul) in the appropriate volume of TE. 该基因组DNA预备用于文库构建。 The genomic DNA preparation for library construction.

分别用Pst I限制性核酸内切酶消化来自两种玉米品系的即将针对多态性检测进行比较的基因组DNA，使DNA片段的末端为粘性末端，该末端可以连接到具有相同限制性位点的质粒中。 Were digested with restriction enzymes forthcoming compare genomic DNA polymorphism detection for both maize lines from the restriction endonuclease Pst I, so that the end of the DNA fragment of cohesive ends, the ends can be connected to have the same restriction sites plasmid. 例如，将IOO单位的Pst I加入到20|ig DNA中并在37。 For example, the Pst IOO units I was added to 20 | ig DNA and incubated at 37. C温育8小时。 C incubated for 8 hours. 经消化的DNA产物通过在1°/。 The digested DNA products by 1 ° /. 低熔解温度琼脂糖凝胶上电泳分离，以通过大小分离DNA片段。 Low melting temperature agarose gel electrophoresis to separate DNA fragments by size. 来自两种玉米品系的经消化的DNA并排载于凝胶上（之间用一个泳道作为间隔）。 Digested DNA from two maize lines side by side on the gel contained in (with a lane as the interval between). 1KB DNA梯度标记和lOObp DM梯度标记载于两种玉米DM泳道每一侧。 1KB DNA markers and lOObp DM gradient gradient contained in two maize DM marked lanes on each side. 这些标记作为经消化的玉米DM的大小分离的指导。 These markers as corn DM digested size separate guidance. 500-3000bp范围内的片段递增地从凝胶上切离，以500-600bp、 600-700bp、 700-800bp、 800-900bp、 900-1100bp、1100-1500bp、 1500-2000bp、 2000-2500bp和2500-3000bp的大小级分。 Fragment within 500-3000bp range incrementally excised from the gel, to 500-600bp, 600-700bp, 700-800bp, 800-900bp, 900-1100bp, 1100-1500bp, 1500-2000bp, 2000-2500bp and 2500 -3000bp size fractions. 各级分中的DM用p-琼脂糖酶纯化并连接入pUC18的Pst I克隆位点。 Pst I cloning site of each fraction was purified by p- agarase DM and ligated into pUC18 in. 质粒连接产物通过电穿孔转化到DH10B大肠杆菌细菌宿主中，以产生约化表示文库。 Plasmid ligation product was transformed by electroporation into DH10B E. coli bacterial host to produce a library of approximately representation. 例如，约500纳克经大小选择的DNA连接到50ng去磷酸化pUC18栽体。 For example, about 500 ng of size-selected DNA was ligated to 50ng dephosphorylated pUC18 plant body.

转化通过电穿孔进行并且用于约化表示的Pst I文库的转化效率 Were transformed by electroporation and the conversion efficiency of about representation for the Pst I library

34为大约50, 000-300， OOO个转化体每l微升连接产物或1000-6000转化体/ng DNA。 34 is about 50, 000-300, OOO transformants per l microliter ligation product or 1000-6000 transformants / ng DNA.

用于评估质量的基本测试包括平均插入大小，叶绿体/线粒体DNA含量，和重复序列分数。 The basic test is used to assess the quality, including average insert size, chloroplast / mitochondrial DNA content, and repeat scores.

在文库构建过程中，评估文库的平均插入大小的检测结果。 In the library construction process, evaluation of the test results with an average insert size of the library. 测试 Test

每个连接以通过分析10-20克隆/个连接确定平均插入大小。 Each connection by analyzing 10-20 clones / connections determine the average insert size. 使用标准微制备方法使DM分离自重组克隆，用Pst I消化以从载体中释放插入物，然后用1%凝胶电泳确定大小（Maule， Molecular Biotechnology9:1 07-126 (1998)，其全文通过引用并入本文）。 The method makes use of standard micro preparation DM isolated from recombinant clones, was digested with Pst I to release the insert from the vector, then with 1% gel electrophoresis to determine the size (Maule, Molecular Biotechnology9: 1 07-126 (1998), in its entirety by incorporated herein by reference).

文库中的叶绿体/线粒体DNA含量，和重复序列的百分比通过对小量样品的克隆（400个）测序，并且对照多种序列数据库交叉核对所获得的序列而评估。 Chloroplast library / mitochondrial DNA content, and the percentage of repeat sequences cloned by small sample (400) sequencing, and sequence control multiple sequence database obtained by cross-checking and evaluation. 一些重复元件在数据库中不存在，但是通常可以通过大量拷贝的相同序列来鉴定。 Some repetitive elements does not exist in the database, but usually a large number of copies of the same sequence can be identified. 例如，在对400个克隆的组测序后，未被重复元件数据库过滤，但是在样品中的存在大于10倍的任何序列被认为是重复元件。 For example, after a group of 400 clones sequenced, not repeat element database filter, but any sequence is present in the sample is greater than 10 times the repetitive elements are considered.

本发明的玉米约化表示文库通过插入经富集的编码区DNA而构建，所述DNA来自如下玉米品系：B73， M017， LH82和5CM1。 Maize present invention showing the reduced library by inserting the coding region of DNA enriched constructed, the DNA is from a maize line: B73, M017, LH82 and 5CM1.

实施例2: Example 2:

本实施例举例说明来自实施例1中制备的约化表示文库中的克隆的玉米基因组DM序列的确定。 This example illustrates an embodiment of from about 1 represents determining prepared library clones DM maize genome sequence. 两种基本的方法可用于DM测序，链终止法（Sanger等人，Proc. Natl. Acad. Sci. USA 74:5463-5467(1977))和化学降解法（Maxam和Gilbert, Pro" Natl. Acad. Sci.USA 74: 560-564 (1977))。自动化和技术的进步，例如用基于荧光的测序法取代放射性同位素法，减少了DNA测序所需的精力（Craxton，Methods, 2:20-26 (1991)， Ju等人.Proc. Natl. Acad. Sci USA92:4347-4351 (1995)以及Tabor和Richardson, Proc. Natl. Acad.Sci USA 92: 6339-6343 (1995))。自动化测序仪可获自，例如，AppliedBiosystems， Foster City, California (ABI Prism® systems); Two basic methods can be used for DM sequencing, the chain termination method (Sanger et al., Proc Natl Acad Sci USA 74:.... 5463-5467 (1977)) and the chemical degradation method (Maxam and Gilbert, Pro "Natl Acad. . Sci.USA 74:. 560-564 (1977)) and advances in automation technology, for example, by fluorescence-based sequencing substituted radioisotope, reducing the effort required to sequence DNA (Craxton, Methods, 2: 20-26 (1991), Ju et al .Proc Natl Acad Sci USA92:... 4347-4351 (1995) and Tabor and Richardson, Proc Natl Acad.Sci USA 92:... 6339-6343 (1995)) can be automated sequencer obtained from, for example, AppliedBiosystems, Foster City, California (ABI Prism® systems);

35Pharmacia Biotech, Inc., Piscataway， New Jersey (Pharmacia ALF)，LI-COR， Inc., Lincoln, Nebraska (Ll-COR 4，000)和Millipore,Bedford, Massachusetts (Millipore BaseStation)。 35Pharmacia Biotech, Inc., Piscataway, New Jersey (Pharmacia ALF), LI-COR, Inc., Lincoln, Nebraska (Ll-COR 4,000) and Millipore, Bedford, Massachusetts (Millipore BaseStation).

另外，毛细管凝胶电泳的进步也减少了DNA测序所需的精力，并且此类进步提供了用于DNA样品测序的快速高效的技术方案(Swerdlow和Gesteland， Nucleic Acids Res. 75:1415-1419 ( 1990);Smith, Nature 349:812-813 (1991); Luckey等人，Methods Enzymol.218: 154-172 (1993); Lu等人，J. Chromatog. A. 680: 497-501(1994); Carson等人，Anal. Chem. 65:3219- 3226 (1993); H画g etal.. Anal. Chem. 64:2149-2154 (1992); Kheterpal et al.，Electrophoresis 17: 1852-1859 (1996); Quesada 和Zhang，Electrophoresis 17:1841-1851 ( 1996); Baba， Yakugaku Zasshi117:265-281 (1997)。 In addition, capillary gel electrophoresis DNA sequencing progress also reduces the required energy, and such progress provides fast and efficient technical solutions (Swerdlow and Gesteland, Nucleic Acids Res DNA sample for sequencing 75: 1415-1419 ( 1990); Smith, Nature 349: 812-813 (1991); Luckey et al., Methods Enzymol.218:.. 154-172 (1993); Lu et al., J Chromatog A. 680: 497-501 (1994); . Carson et al., Anal Chem 65: 3219- 3226 (1993); H Videos g etal .. Anal Chem 64:... 2149-2154 (1992); Kheterpal et al, Electrophoresis 17: 1852-1859 (1996). ; Quesada and Zhang, Electrophoresis 17: 1841-1851 (1996); Baba, Yakugaku Zasshi117: 265-281 (1997).

很多测序方法是本领域已知的，包括基于荧光的测序方法学。 Many sequencing methods are known in the art, including fluorescence-based sequencing methods of learning. 这些方法具有分析大量序列数据所需的检测、自动化和仪器化的能力。 These methods have analytical testing, automation and instrumentation capabilities required for a large number of sequence data. ABI Prism®377謹观'J序仪（Applied Biosyst面，Foster City, CA)允许快速电泳和收集数据。 ABI Prism®377 the honor concept 'J sequencer (Applied Biosyst face, Foster City, CA) allows rapid electrophoresis and data collection. 用这些类型的自动化系统，检测荧光染料标记的测序反应产物并且将数据直接输入计算机，产生色谱，其随后进行检查，存储并用相应的软件程序分析。 With these types of automated systems, detection of the fluorescent dye-labeled sequencing reaction product and the data entered into the computer, generating chromatography, which is subsequently inspected, is stored and analyzed with appropriate software program. 这些方法对本领域技术人员来说是已知的并且已被描述和评论(Birren等人.Genome Analysis:Analyzing DNA， 1， Cold Spring Harbor, New York (1999))。 These methods of the skilled artisan are known and have been described and Reviews (Birren et al .Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, New York (1999)).

通过获自CodonCode Corporation, Dedham， MA的PHRED对踪迹文件的序列碱基判断和质量得分赋值，PHRED由Brent Ewing，等人，"Base-cal 1 ing of automated sequencer traces using phred"， 1998，Genome Research, Vol. 8， pages 175- 185 and 186-194描述（其通过引用并入本文）。 By PHRED obtained from CodonCode Corporation, Dedham, MA bases of sequence trace files to determine the quality score and assignment, PHRED by Brent Ewing, et al, "Base-cal 1 ing of automated sequencer traces using phred", 1998, Genome Research , Vol. 8, pages 175- 185 and 186-194 description (which is incorporated herein by reference).

完成碱基判定后，通过切除低质量末端序列提高序列质量。 After the completion of base determination, by the end of the sequence to improve the removal of low-quality sequence quality. 如果得到的序列少于50bp,则将其删除。 If the resulting sequence is less than 50bp, it is deleted. 删除总的质量低于12. 5的序列。 Remove the total mass of less than 12.5 sequence. 并且，去除污染序列，例如，大肠杆菌BAC和载体序列和亚克隆载体。 Further, the decontamination sequence, e.g., E. coli and BAC vector sequence and subcloning vector.

36用获自DoubleTwist Inc. ， Oakland, CA的Pangea Clustering and Alignment Tools,通过针对重叠碱基比较序列对，组装重叠群。 36 obtained from using DoubleTwist Inc., Oakland, Pangea Clustering and Alignment Tools CA by comparing the nucleotide sequence for overlapping assembled contigs. 使用如下的高严紧性参数检测重叠：字大小（word size) = 8;窗口大小（window size) - 60; 且同一性为93%。 Use the following high stringency parameter detecting overlap: word size (word size) = 8; window size (window size) - 60; and 93% identity. 用获自CodonCode Corporation的PHRAP片段组装程序，使用0. 5或更低的"重复严紧性参数，，，重新组装聚簇。最终的组装产品包含序列集合，所述序列包括重叠群序列，其表示重叠聚类序列的共有序列（重叠群）和单碱基差异序列（其不存在于相关序列的任何聚簇中（单碱基差异））。 共同地，由DM集合产生的重叠群和单碱基差异序列称为岛。 Obtained from CodonCode Corporation with the PHRAP fragment assembly program, using 0.5 or less "stringency parameters ,,, repeated reassembly clustered. The final assembly product comprising the sequence set, the sequence comprising a sequence contigs, which represents Clustering consensus sequence overlapping sequences (contigs) and single nucleotide sequence difference (which does not exist in the related sequences clustered in any (single-base difference)). Collectively, the contig generated by the DM collection and monobasic base sequence difference is called Island.

实施例3 Example 3

本实施例举例说明SNP和Indel多态性的鉴定，通过比较来自至少两种单独的玉米品系的重叠群和单碱基差异序列（如实施例2中制备）的序列比对。 This example illustrates SNP and Indel polymorphisms identified by comparing from at least two separate maize lines contig sequences and single nucleotide difference (e.g., prepared in Example 2) sequence comparison. 将来自多种玉米品系的序列装配到具有一种或多种多态性（即SNP和/或Indel)的基因座中。 The sequence assembly from a variety of maize lines having one or more polymorphisms (i.e. SNP and / or Indel) of the locus. 候选的多态性由如下参数限制： Candidate polymorphisms limit by the following parameters:

U)就共有序列而言，重叠群和单碱基差异序列的最小长度为200个碱基。 U) on the consensus sequence, the minimum length of the contigs and single nucleotide sequence difference is 200 bp.

(b) 在候选SNP每一侧的15个碱基区域中，观察到的碱基的同一性百分比为75°/«。 Percent identity bases (b) in the candidate SNP 15 bases on each side of the region observed is 75 ° / «.

(c) 位于多态性位点的各个重叠群中的最低BLAST质量为35. (C) located at the polymorphic site of each contig lowest BLAST mass 35.

(d) 多态性位点每一侧的15个碱基区域中最低BLAST质量为20. 具有符合条件的多态性的大量基因座鉴定为具有共有序列，如 15 bp region (d) on each side of the polymorphic site in the lowest quality of 20. BLAST large number of polymorphic loci identified as having qualified having the consensus sequence, e.g.

SEQ ID NO: 1至SEQ ID NO: 10373所示。 SEQ ID NO: 1 to SEQ ID NO: 10373 in Fig. 各个基因座中的符合条件的SNP和Indel多态性鉴定于表1中。 Individual loci in qualifying SNP and Indel polymorphisms identified in Table 1. 更具体地，表l如下鉴定了多态性的类型和位置： More specifically, as identified in Table l type and location of the polymorphisms:

SEQ-NUM指多态性玉米DNA基因座的序列号，例如SEQ ID N0。 SEQ-NUM refers to polymorphism loci in maize DNA sequence number, for example SEQ ID N0. SEQ-ID指多态性玉米DM基因座的任意的（arbitrary)识别命 SEQ-ID refers to any polymorphism locus of maize DM (arbitrary) Identification life

37名。 37.

MUTATION-ID指各个多态性的任意的识别命名。 MUTATION-ID refers to an identification of any individual polymorphism named. START-POS指多态性玉米DNA基因座的核苷酸序列中多态性起始的位置。 START-POS refers to a nucleotide sequence polymorphisms in maize DNA polymorphism loci starting position.

END_P0S指多态性玉米DNA基因座中多态性终止的位置；对SNP 来说，START-POS和END-POS是相同的。 END_P0S refers to the location of maize DNA polymorphism loci polymorphism termination; for SNP speaking, START-POS and END-POS is the same.

TYPE指鉴定多态性为SNP或IND ( Indel )。 TYPE refers to the identification of polymorphism or SNP IND (Indel).

ALLELEn和STRAINn指特定等位基因的玉米品种中多态性的核苷酸序列。 ALLELEn and STRAINn refers to the nucleotide sequence of the maize varieties in particular allele polymorphism.

CHROMOSOME指经定位的多态性的染色体。 CHROMOSOME means positioned polymorphism chromosome.

POSITION指以cM测量的经定位的多态性与染色体5，末端之间的距离。 POSITION refers to the measurement of the distance in cM chromosomal localization polymorphism 5, between the ends.

实施例4 Example 4

本实施例举例说明了引物碱基延伸用于检测SNP多态性的用途， 即用SEQ ID NO: 5378的玉米基因座中的Mutation ID 3972，其在下面的表2中进行更具体地描述。 This example illustrates the base extension primer for detecting the SNP polymorphism use, i.e. with SEQ ID NO: maize locus 5378 in Mutation ID 3972, which is performed in the following Table 2 is more specifically described.

表2 Table 2

SEQ MUTATION START END TYPE ALLELE1/S ALLELE2/ SEQ MUTATION START END TYPE ALLELE1 / S ALLELE2 /

醒 ID POS POS TRAIN1 STRAIN2 Woke ID POS POS TRAIN1 STRAIN2

5738 3971 66 66 SNP A/b73 C/mol7 5738 3971 66 66 SNP A / b73 C / mol7

5738 3972 126 126 SNP A/mol7 G/b73 5738 3972 126 126 SNP A / mol7 G / b73

5738 3973 149 150 IND **/mol7 TG/b73 5738 3973 149 150 IND ** / mol7 TG / b73

5738 3974 338 338 SNP A/b73 G/mol7 5738 3974 338 338 SNP A / b73 G / mol7

扩增少量玉米基因组DNA(例如大约10ng),使用正向和反向PCR 引物，即分别为SEQ ID NO: 10379和SEQ ID NO: 10378，所述引物设计为对模板的退火温度为55°C,所述模板在Mutation ID 3972多态性附近的SEQ ID NO: 5738的基因座中，所述多态性为A/G SNP。 Amplification of small amounts of maize genomic DNA (e.g., approximately 10ng), using forward and reverse PCR primers, i.e. for SEQ ID NO: 10379 and SEQ ID NO: 10378, said primers are designed for the annealing temperature was 55 ° C template , the template in the vicinity of the polymorphism Mutation ID 3972 SEQ ID NO: 5738 in the locus, the polymorphism A / G SNP.

38将PCR产物加入到新的平板中，其中延伸引物SEQ IDN0: 10380共价连接到GBA平板的反应孔的表面。 38 The PCR product was added to a new plate, wherein the extension primer IDN0 SEQ: 10380 covalently attached to the reaction plate hole GBA surface. 延伸混合物包含DM多聚酶，两种不同的标记ddNTP，并且加入延伸緩冲液。 DM polymerase extends mixture comprises two different mark ddNTP, and adding the extension buffer. GBA平板在42。 GBA flat at 42. C温育15分钟以允许延伸。 C was incubated for 15 minutes to allow the extension. 通过用合适的緩沖液冲洗，从孔中移除反应混合物。 By washing with an appropriate buffer, the reaction mixture was removed from the wells. 通过用针对各个标记的第一和笫二检测试剂连续温育检测两种标记。 Incubated continuously detected by two markers for each marker and the undertaking of the first two detection reagents. 在本实施例中，通过与HRP-抗-FITC温育，然后冲洗孔，然后在含有HRP的发色底物的援沖液中温育测量ddATP-FITC的掺入。 In the present embodiment, by incubation with a HRP- Anti -FITC, then rinse holes, and then the red solution containing the aid HRP chromogenic substrate incubated measuring ddATP-FITC incorporation. 在适合HRP 反应产物的波长下，针对各个孔用分光光度法确定反应程度。 HRP reaction product at a suitable wavelength, the extent of the reaction was determined by spectrophotometry for each hole. 再次沖洗孔，并且用AP-链霉亲和素重复上述步骤，使用AP的发色底物，以及在适合AP反应产物的波长下的分光光度法。 Rinse again with holes, and repeat the above steps with AP- streptavidin-biotin, using the AP's chromogenic substrate and spectrophotometry AP reaction products under appropriate wavelength. 结果分析。 Results.

从对检测步骤特异标记的反应产物测量得到的吸光度，推断各个标记的ddNTP的掺入程度，并且从这些吸光度与已知的基因型和无模板对照反应的标准进行比较的比率，推断样品的基因型。 From the measurement of the reaction product of step to detect specific markers obtained absorbance, infer the degree of incorporation of each labeled ddNTP's, and compares the ratio of these absorbances known genotype and no template control reaction criteria, the sample to infer gene type. 在最普通的实践中，将各个数据点观察到的吸光度相互对应地在散点图中作图， 产生"对偶图"。 In the most common practice, the individual data points corresponding to each other is observed absorbance plotted in a scatter plot, a "dual graph." 使用本实施例的单碱基延伸测定的成功的基因分型测定提供了对偶图（如图2中所示），其中数据点分为四组：纯合子1 (例如，等位基因A),纯合子2 (例如，等位基因G)，杂合子（每个样品包含两种等位基因），和由无模板对照，或失败的扩增反应或检测产生的"无信号"组。 Use of this embodiment of the single base extension assay successful genotyping assay provides a dual graph (shown in FIG. 2), wherein the data points are divided into four groups: 1 homozygous (e.g., allele A), homozygous 2 (for example, allele G), the amplification reaction heterozygotes (each sample contains two alleles), and by no template control, or failure to produce or detect "no signal" group.

实施例5 Example 5

本实施例举例说明了标记探针降解测定用于检测在实例4中测定的SNP多态性（即SEQ ID NO: 5738基因座中的Mutation ID 3972 多态性）的用途。 This example illustrates the determination of a labeled probe for detecting degradation measured in Example 4 SNP polymorphism (i.e., SEQ ID NO: 5738 locus polymorphism Mutation ID 3972) purposes. 将一些玉米基因组模板DNA (例如约2-20ng)混合到5ul总体积中，与四种寡核苷酸一起，即正向引物SEQ ID N0: 10376, 反向引物SEQ ID NO: 10377和具有结合到5，末端的设计为VIC-TGTGTGAGCTGCTG的VIC报告子的杂交探针（其中探针的寡核苷酸片段具有SEQ ID NO: 10374 )，以及具有结合到5，末端的设计为 Some of the maize genomic template DNA (e.g., about 2-20ng) mixing to a total volume of 5ul, together with the four kinds of oligonucleotides, i.e., forward primer SEQ ID N0: 10376, reverse primer SEQ ID NO: 10377 and having a binding 5, the end of the design of VIC to VIC-TGTGTGAGCTGCTG reporter hybridization probe (probe wherein the oligonucleotide fragment having SEQ ID NO: 10374), and have binding to 5, the end of the design

39FAM-TTGTGTGGGCTGCT的FAM报告子的杂交探针（其中探针的寡核苷酸片段具有SEQIDNO: 10375 )，以及包含参比染料R0X的PCR反应緩沖液。 39FAM-TTGTGTGGGCTGCT the FAM reporter hybridization probe (probe oligonucleotide fragments which have SEQIDNO: 10375), and the reaction buffer containing ginseng than dye R0X of PCR. PCR反应使用6(TC的退火延伸温度进行35个循环。反应后，各个荧光团的荧光和参比的荧光在荧光计中进行检测。用参比的荧光值使各个荧光团的荧光值标准化。经标准化的值针对各个样品相互对应地进行作图以产生对偶图。使用本实施例的引物和杂交探针的成功的基因分型测定提供了对偶图，其数据点在清楚可分的组中，如图2中所示。 PCR reactions using 6 (annealing extension temperature TC of 35 cycles were performed. After the reaction, the fluorescence and reference fluorescence detecting each fluorophore in the fluorometer. Reference value with a fluorescence value of each of the fluorescent fluorophore standardization. The normalized values ​​are plotted against each other in correspondence of each sample to produce the dual graph. measured using the genotyping primers and the hybridization probes Examples of successful embodiment provides the dual graph, the data point in the group clearly separable , as shown in Fig.

为了证实测定产生了精确的结果，对代表三种可能的基因型（即两种纯合子等位基因和一种杂合子样本）中的每种的已知基因型身份的大量重复样品进行新的测定。 In order to confirm measurement produces accurate results, to represent the three possible genotypes (i.e., both homozygous and a heterozygous sample allele) in a large number of replicate samples of known genotype for each identity a new Determination. 为了有效的和有用的测定，其必须产生清楚可分的数据点的组，以便三种基因型之一可以赋值给至少90% 的数据点，并且可以观察赋值，以针对至少98%的数据点进行校正。 For effective and useful measurement, which must be generated clearly separable set of data points to be assigned to one of three genotypes of at least 90% of the data points, and the assignment can be observed, for at least 98% with data points correction. 在这个验证步骤之后，对两种高度近交的个体之间杂交的后代进行测定，以获得分离数据，其之后被用于计算多态性基因座的遗传图谱位置。 After this verification step, on the progeny of the cross between two highly inbred individuals were measured to obtain the separated data, which after being used to calculate polymorphic locus genetic map position.

实施例6 Example 6

本实施例举例说明本发明的基因座中的多态性的遗传图镨，所述图语基于超过1000个SNP的基因型，针对78个来源于玉米品系B73 和Mo17的杂交的重组近交系（RIL)。 Illustrate loci of the present invention of FIG praseodymium genetic polymorphism, the atlas-based over 1000 SNP genotypes, the recombinant inbred lines derived from corn for 78 B73 and Mo17 of the hybridization of the present embodiment (RIL). 所述基因型与针对约80个公开的在203个RIL上打分的核心SSR和RFLP标记的基因型结合。 The genotype and genotype combination for about 80 open scoring in the 203 RIL core SSR and RFLP markers. 定位前， 将任何表现出异常分离（对1: l分离比的卡方检验，P<0. Ol)的基因座去除。 Former position, showing any abnormal separation (for 1: l ratio chi-square test of the separation, P <0 Ol.) Loci removed. 稍后，可将这些基因座加入到图谦中，但是不允许它们改变标记次序。 Later, these loci can be added to the figure modest, but they are not allowed to change the tag order.

用JoinMap version 2. 0软件(由Stam， P. "Construction of integrated genetic linkage maps by means of a new computer package: JoinMap, The Plant Journal. 3: 739-744 (1993); Stam， P. 和van Ooijen， JW "JoinMap version 2.0: Software for the With JoinMap version 2. 0 software (by Stam, P. "Construction of integrated genetic linkage maps by means of a new computer package: JoinMap, The Plant Journal 3: 739-744 (1993); Stam, P. and van Ooijen. , JW "JoinMap version 2.0: Software for the

40calculation of genetic linkage maps (1995) CPR0-DL0， Wageningen 描述）构建图镨。 40calculation of genetic linkage maps (1995) CPR0-DL0, Wageningen description) construction diagram praseodymium. JoinMap执行加权最小二乘法以多点定位，其中并入了来自所有连锁基因座对（紧邻或不紧邻）的信息。 JoinMap weighted least squares method to perform a multi-point positioning, which incorporates a pair (adjacent or close to) the information from all the chain loci. 用5. 0的LOD 阈值形成连锁群。 Forming a linkage group with a LOD threshold of 5.0. SSR和RFLP公开标记用于将连锁群分配给染色体。 SSR and RFLP markers for assigning public linkage groups to chromosomes.

在图谱构建前合并染色体内的连锁群。 Construction of linkage groups in the pre-merger chromosome map.

Haldane的定位功能用于将重组分数转化为图谱距离。 Haldane targeting capabilities for the recombinant fraction into a map distance. 除了成对的连锁数据以外使用宽标准；只去除LOD不大于0. 001或重组分数不小于0.499的数据。 In addition to a pair of chain data using wide standard; only the removal of no more than 0.001 LOD score of not less than 0.499 or restructuring data. 为了对基因座排序，我们使用的跳变阈值（jump threshold )为5. 0,三联体阈值（triplet threshold )为7. 0和ripple 值为3。 To sort the locus transition threshold (jump threshold) that we use for the 5.0, triplets threshold (triplet threshold) is 7.0 and the ripple is 3. 在两轮图语构建中，约38%的基因座（424/1108 )被排序，使用5. 0的跳变阈值，其阻止往图镨添加基因座，如果此类添加导致吻合度标准跳变大于5.0。 Figure language construct in two rounds, about 38% of the loci (424/1108) are sorted using transition threshold of 5.0, which prevents Add to map loci praseodymium, if such standards add transitions leading to the degree of matching greater than 5.0. 将剩余的基因座加入到图谦中，而不使用该跳变阈值。 The remaining locus is added to FIG Qian, without the use of the transition threshold. 这些基因座的添加对初始的424个基因座的图谱次序和距离具有可以忽略不计的影响。 Adding these loci have negligible effect on the pattern and order from the initial 424 loci. 已定位的SNP多态性在表3中进行鉴定， 其中"Chromosome"和"Position"鉴定以cM测量的由"Mutation ID" 识别的SNP与玉米染色体5'末端的距离。 SNP polymorphisms have been identified location in Table 3, where "Chromosome" and "Position" identification with cM measured by the "Mutation ID" identified SNP and maize chromosome 5 'end of the distance. "Public Name"提供了涉及的公开标记的公布的名称，其不是本发明的部分。 "Public Name" provides the name of the publication relates to open-label, which is not part of the present invention. 对于在表3中列出的一些定位的多态性标记，Mutation ID不止一次被列出，其表示定位基于多次基因分型测定进行。 For some targeted polymorphic markers listed in Table 3, Mutation ID are listed more than once, which represents the position determination based genotyping performed repeatedly. 多次基因分型测定的图谱位置通常用于证实图谱位置，除图谱位置有分歧的情况外，例如由于测定的设计和操作中的误差。 Repeatedly genotyping assay map location is typically used to confirm the location map, in addition to map the location of divergent circumstances, e.g., due to the design and operation of the error in the measurement. 经定位的多态性的密度和分布在图1中示出。 After positioning and polymorphism density distribution shown in FIG. 1.

用于连锁谙图构建的基于找到基因座次序以最小化重组事件的总数的可选择的方法，由J"ansen， J. 等人"Constructing dense genetic linkage maps", Theor Appl Genet, (in press)描述。该方法在很多条件下产生最大可能图谱的接近值。由该方法建立的图i瞽与用JoinMap 2. O获得的图镨非常相符。 Alternative methods linkage map constructed based versed find locus order to minimize the total number of recombination events for the J "ansen, J., et al." Constructing dense genetic linkage maps ", Theor Appl Genet, (in press) is described. The method generates a value close to the maximum possible map in many conditions established by the method of FIG i blind FIG praseodymium JoinMap 2. O obtained with very consistent.

实施例7 Example 7

本实施例举例说明本发明的使用表1和SEQ ID NO: 1-10373的 This example illustrates the use of the present invention in Table 1 and SEQ ID NO: 1-10373 of

41DNA序列中公开的多态性的方法。 Method 41DNA sequence polymorphism disclosed.

分析具有不同遗传的玉米的育种群体，使用如实施例5中所示的基于SEQIDN0: l-10373的序列制备的引物对和探针对（其针对表l中鉴定的各个多态性）。 Analysis of Maize with different genetic breeding populations, use as shown in Example 5 based SEQIDN0: primer pair and probe sequences prepared l-10373 of (directed against the individual identified in Table l polymorphisms). 紧密连锁的多态性鉴定为在跨越玉米基因组的约8 厘摩的相邻基因组窗口中表征单倍型。 Identification of closely linked polymorphisms for the characterization of haplotypes across the corn genome, approximately 8% of the adjacent Mount genome window. 代表至少4%群体的单倍型与为玉米群体的各个成员鉴定的性状值关联，所述性状值包括针对产量， On behalf of at least 4% of the population haplotype associated with the trait value for each member identified populations of maize, the trait value, including for production,

成熟期，倒伏，植林高度，抗玉米锈病，耐旱性和低温萌发的性状值。 Maturity, lodging, planting height, anti-corn rust, drought tolerance trait values ​​and low germination. 各个单倍型的性状值排列在各个8厘摩的窗口中。 Trait value of each haplotype arranged in each 8 centiMorgans window. 就各个窗口中的单倍型的同一性分析来自群体中的随机交配的成员的后代种子。 Analysis of progeny seed from members of random mating populations on each window haplotype identity. 基于在所述种子中鉴定的单倍型的高性状值选择后代种子用于种植。 Based on the high trait values ​​identified in the seeds haplotype selection progeny seed for planting.

42 42