Conservation Genet Resour DOI 10.1007/s12686-014-0361-y MICROSATELLITE LETTERS Characterization of novel polymorphic microsatellite markers in Dactylorhiza hatagirea: a critically endangered orchid species from western Himalayas Shilpa Sharma • Vikas Sharma • Meenu Chhabra Rajeev Rathour • Kamal Dev Sharma • Rakesh Kumar Kapila • Received: 27 September 2014 / Accepted: 14 October 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Dactylorhiza hatagirea (D. Don) Soo, family Orchidaceae is an endangered medicinal herb inhabiting higher altitudes of western Himalayas. Due to reckless exploitation for its roots, it is of high conservation concern. In the present study fifteen microsatellites were developed and characterized across twenty collections of D. hatagirea. The total numbers of alleles amplified by these microsatellites were 64 with an average of 4.2 alleles per marker. Average observed and expected heterozygosity values for polymorphic loci were 0.623 and 0.631, respectively. Mean polymorphism information content value of the polymorphic markers was 0.532. Of the fourteen polymorphic microsatellites, 7 deviated from Hardy–Weinberg equilibrium. Microsatellites reported here can be utilized to address questions related to genetic characteristics in this species. Keywords Dactylorhiza hatagirea
Microsatellite markers
Orchids Introduction Dactylorhiza hatagirea (D. Don) Soo, an orchid of high economic importance is endemic to western Himalayan region (2,500–5,000 m asl). It is an erect herb with Electronic supplementary material The online version of this article (doi:10.1007/s12686-014-0361-y) contains supplementary material, which is available to authorized users. S. Sharma
V. Sharma
M. Chhabra
R. Rathour
K. D. Sharma
R. K. Kapila (&) Department of Agricultural Biotechnology, CSKHP Agricultural University, Palampur 176 062, H.P, India e-mail: rkkapila@gmail.com tuberous roots, lanceolate leaves and robust stem. The species is an important traditional medicinal plant with uses as aphrodisiac, demulcent and nervine tonic. Due to its high demand for therapeutic uses, unscientific exploitation of the species is in practice. The specialized mycorrhizal associations for growth and specific pollinator requirement make it a slow growing and/propagating plant. Due to sharp decline in its natural populations, D. hatagirea has been listed as critically endangered species by Conservation Assessment and Management Plan (CAMP status), critically rare by International Union for Conservation of Nature and Natural Resources (IUCN) and is listed by Convention on International Trade in Endangered Species (CITES) under appendix II (Bhatt et al. 2005). Little effort has been made to study its genetic diversity and population structure at molecular level due to paucity of DNA-based markers. Only 14 microsatellite markers have been developed so far (Lin et al. 2014) and there is need to develop more markers. In the present study 784 nucleotide sequences of Dactylorhiza species available at National Centre for Biotechnology Information database were utilized for developing microsatellite markers. The sequences were down loaded, processed for redundancy removal, microsatellite search and primers designing following Sharma et al. (2009). The markers were validated across a panel of twenty ecotypes of D. hatagirea collected from various locations in western Himalayan region of India. The genomic DNA was extracted and PCR performed in 10 lL reaction volumes containing 25 ng of template DNA, 15 ng of each primer, 200 lM of each dNTP, 0.5 U of Taq DNA polymerase and PCR buffer (HiMedia Pvt. Ltd., Bombay, India). The PCR conditions were: 1 cycle of 5 min at 94 °C, 35 cycles of 1 min at 94 °C, 1 min at respective annealing temperature (see Table 1), 1 min at 123 123 Table 1 Characteristics of 15 microsatellite markers developed in the present study Marker name Primer sequence (50 –30 ) Repeat motif Ta (°C) Size range (bp) No. of alleles Ho He P PIC Gene bank accession no.£ KSSR-02 F-GGTCCAGGGGGATAAGTTCT (TTC)4 53 200–290 8 0.800 0.821 0.001* 0.751 AY283535 KSSR-04 F-CGCGAAGTCAAGATTGAAAA (TA)6 50 150–250 2 0.500 0.394 0.354 0.305 EF594447 (TA)6 51 205–220 7 0.900 0.813 0.164 0.504 DQ022918 F-TCCTCTGCAGTCTTGTTCCA R-GAGAAAGAACGCCAAAGACG (TTC)4..(TTCCTC)3 53 360–480 6 0.900 0.763 0.029* 0.680 AY283532 F-CAGGGGGATAAGTTCTCGAC (AGA)3 53 205–400 8 0.625 0.892 0.007* 0.816 AY283531 (TTC)4 53 320–380 8 0.800 0.858 0.013* 0.790 AY283526 (TA)6 50 150–320 4 0.800 0.774 0.046* 0.685 EU884288 (GAA)6 52 230 1 – – – – DQ986361 (AGA)6 51 190–200 3 0.400 0.353 0.929 0.303 DQ986360 (TCT)8 56 130–150 2 0.400 0.337 0.502 0.269 DQ986357 R-AGAAAGAACGCCAAAGACGA R-CCCGGCCAGTACTTAACCAG KSSR-07 F-AAACAAACATGCCCCAGTTA R-GAGCCGGACATGAGAGTTTC KSSR-11 KSSR-12 R-AGAAAGAACGCCAAAGACGA KSSR-15 F-GGTGTTCCTAACTGCCCACT R-GAGAAAGAACGCCAAAGACG KSSR-18 F-CGCGAAGTCAAGATTGAAAA R-GGGAAATGAACCTTTTGCAC KSSR-20 F-CGCCGACAAACTCTACATCG R-CGATCCTCATCCTGTTTTGC KSSR-21 F-CTGGAAGTAGGGGGAGCAAT R-CTCAATCATCCAAAGGGACAA KSSR-22 F-AAGGTACCACGCTTCGTCAG R-GACTGCAGGTAAGGGCTCAG KSSR-30 F-GCCCGCGAACACTTTATTTA (TA)8 54 550–570 2 0.600 0.442 0.223 0.332 EU176076 KSSR-32 R-CTCCTCGCGAATGAAATGAT F-CGATGGAAGCTGTTCTAACGA (CAA)3 53 390–405 2 1.00 0.611 0.029* 0.492 GQ244815 (GAA)6 56 230–360 2 0.400 0.337 0.502 0.269 JQ229975 (TA)8 53 210–230 4 0.100 0.658 0.000* 0.551 DQ022926 (TA)15 50 190–205 5 0.500 0.789 0.051 0.709 DQ022920 4.2 0.623 0.631 R-TGGGACTCTCTCTTTATTCTCGTC KSSR-35 F-TCAGCGGAGGAGAGGTAGAA R-TGGCCACTTGTAGTGAGCTG KSSR-37 F-CATGCCCCAGTTATCCACTT KSSR-39 F-TAAACAAACATGCCCCAGTT R-CTCCTCGCGAATGAAATGAT Mean 0.532 Ta annealing temperature, Ho observed heterozygosity, He expected heterozygosity, P probability that genotype proportions conform to Hardy–Weinberg equilibrium, PIC polymorphism information content * The deviation from Hardy–Weinberg equilibrium £ Gene bank accession no. of the sequence from which primers were designed Conservation Genet Resour R-GAGCCGGACATGAGAGTTTC Conservation Genet Resour 72 °C and final extension for 7 min at 72 °C. The amplicons were separated on 6 % denaturing polyacrylamide gels in 19 TBE buffer and visualized by silver staining. Polymorphic alleles for each marker were scored manually and the data were utilized for computing various diversity indices. Expected heterozygosity (He), Observed heterozygosity (Ho) and Hardy–Weinberg equilibrium were obtained using POPGENE version 1.32 (Yeh and Boyle 1997) and polymorphism information content (PIC) was calculated as per Botstein et al. (1980). From the 784 nucleotide sequences, 35 primer pairs flanking microsatellite motifs were designed. Out of the primer pairs tested across a panel of 20 D. hatagirea ecotypes, only 15 amplified unambiguous amplicons. Of the 15 markers giving reliable amplification, 14 were polymorphic (Table 1). These 15 primers amplified 64 alleles with a mean value of 4.2 alleles in a size range of 130–570 bp. Ho and He values for polymorphic loci varied from 0.100 (KSSR-37) to 1.000 (KSSR-32) and 0.337 (KSSR-35) to 0.892 (KSSR-12) with average of 0.623 and 0.631, respectively. Minimum PIC value of 0.269 was recorded for the markers KSSR-22 and KSSR-35, whereas a maximum PIC value of 0.816 was recorded for KSSR-12 with an overall mean PIC of 0.532. Seven markers significantly deviated from Hardy–Weinberg equilibrium at P value of 0.005. These values of diversity indices clearly indicated their potential for uncovering the genetic diversity of D. hatagirea. The new set of microsatellite markers developed in this study can be used to study population genetics and the extent of genetic diversity in D. hatagirea for addressing the conservation and related issues of this critically endangered rare orchid species. Acknowledgments We thank University Grant Commission (UGC), GOI for financial support under the scheme ‘‘Major Research Projects’’. References Bhatt A, Joshi SK, Gairola S (2005) Dactylorhiza hatagirea (D. Don) Soo—a west Himalayan orchid in peril. Curr Sci 89(4):610–612 Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Human Genet 32:314–331 Lin P, Zeng L, Yang Z, Liu R, Zhong Y (2014) Development and characterization of polymorphic microsatellitemarker for Dactylorhiza hatagirea (D. Don) Soo. Conserv Genet Res. doi:10. 1007/s12686-013-0053-z Sharma V, Bhardwaj P, Kumar R, Sharma RK, Sood A, Ahuja PS (2009) Identification and cross-species amplification of EST derived SSR markers in different bamboo species. Conserv Genet 10:721–724 Yeh FC, Boyle TJB (1997) Population genetic analysis of codominant and dominant markers and quantitative traits. Belg J Bot 129:157 123