Development and characterization of SSR markers in Himalayan species Betula utilis

Abstract

Betula utilis D. Don. is an important species of alpine Himalaya and forms the major treeline component of western Himalaya. The different populations of B. utilis are declining and are under high risk. In the present study, novel expressed sequence tag–simple sequence repeat (EST–SSR) primers were developed from expressed sequence tag (EST) data of different Betula species. Of the 10,796 designed primers, the percentages of di-, tri-, tetra-, penta-, and hexa-repeats were 36%, 35%, 15%, 5.5% and 7.7%, respectively. For validation, 50 primers were synthesized randomly and were characterized in 20 different B. utilis accessions from north-western Himalaya. Of these, 45 primers amplified fragments in a range of 1–6. The 24 polymorphic primers produced 111 fragments in aggregate with 4.6 fragments on average. Polymorphism information content (PIC) ranged from 0.288 in marker BUMS-24 to 0.497 in BUMS-3 and BUMS-7, with an average of 0.447 among polymorphic markers. Dendrogram based on Jaccard’s similarity coefficient and UPGMA method showed that newly developed SSR markers distinguished twenty accessions of B. utilis into two groups. As no SSR markers were available in this species, the newly developed markers will foster molecular genetics research and conservation efforts for this species.

Keywords

Betula utilis / Simple sequence repeat (SSR) / Western Himalaya / Polymorphism information content (PIC) / Genetic diversity

References

[1] Allendorf F, Ryman N. Beissinger SR, McCullough DR. The role of genetics in population viability analysis. Population viability analysis, 2000, Chicago: The University of Chicago Press 50 85

[2] Carlton GC, Bazzaz FA. Regeneration of three sympatric birch species on experimental hurricane blowdown microsites. Ecol Monogr, 1998, 68: 99-120.

[3] Chauhan NS (1999) Medicinal and aromatic plants of Himachal Pradesh, New Delhi, India 1999, p 125

[4] DeJong PC (1993) An introduction to Betula: its morphology, evolution, classification and distribution, with a survey of recent work. In: Betula. Proceedings of the IDS Betula symposium 2–4 October 1992. International Dendrological Society, Sussex, England, pp. 7–18

[5] Doyle JJ, Doyle JL. A rapid total DNA preparation procedure for fresh plant tissue. Focus, 1990, 12: 13-15.

[6] Gaudeul M, Taberlet P, Till-Bottraud I. Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers. Mol Ecol, 2000, 9: 1625-1637.

[7] Gupta M, Sharma V, Singh SK, Chahota RK, Sharma TR. Analysis of genetic diversity and structure in a genebank collection of red clover (Trifolium pretense L.) using SSR markers. Plant Genet Resour, 2016

[8] Hagman M. On self- and cross-incompatibility shown by Betula verrucosa Ehrh. and Betula pubescens Ehrh. Commun Inst For Fenn, 1971, 73: 1-125.

[9] Hjelmroos M. Evidence of long-distance transport of Betula pollen. Grana, 1991, 30: 215-228.

[10] Holsinger KE, Gottlieb LD. Falk DA, Holsinger KE. Conservation of rare and endangered plants: Principles and prospects'. Genetics and Conservation of Rare Plants, 1991, New York: Oxford University Press 195 208

[11] Johnsson H. Genetic characteristics of Betula verrucosa Ehrh. and Betula pubescens Ehrh. Annales Forestales. Analiza Sumarstv, 1974, 6: 91-133.

[12] Jonsell B (2000) Flora nordica. Lycopodiaceae to polygonaceae. The Bergius Foundation, the Royal Swedish Academy of Sciences, Stockholm, Sweden, vol 1, pp 1–344

[13] Kaur K, Sharma V, Singh V, Wani MS, Gupta RC. Development of novel SSR markers for evaluation of genetic diversity and population structure in Tribulus terrestris L. (Zygophyllaceae). 3 Biotech, 2016, 6: 156.

[14] Kostova A, Todorovska E, Christov N, Hristov K, Atanassov A. Assessment of genetic variability induced by chemical mutagenesis in elite maize germplasm via SSR markers. J Crop Improv, 2006, 16: 37-48.

[15] Koul PM, Sharma V, Rana M, Chahota RK, Kumar S, Sharma TR. Genetic structure and interrelationships in lentil species using morphological and SSR markers. 3 Biotech, 2017, 7: 83.

[16] Masoudi-Nejad A, Tomura K, Kawashima S, Moriya Y, Suzuki M, Itoh M, Kanehisa M, Endo T, Goto S. EGassembler: online bioinformatics service for large-scale processing clustering and assembling ESTs and genomic DNA fragment. Nucleic Acids Res, 2006, 34: W459-W462.

[17] Perrier X, Jacquemoud-Collet JP (2006) DARwin software http://darwin.cirad.fr/darwin

[18] Powell W, Machray GC, Provan J. Polymorphism revealed by simple sequence repeats. Trends Plant Sci, 1996, 1: 215-222.

[19] Rana JC, Chahota RK, Sharma V, Rana M, Verma N, Verma B, Sharma TR. Genetic diversity and structure of Pyrus accessions of Indian Himalayan region based on morphological and SSR markers. Tree Genet Genom, 2015, 11: 821.

[20] Rana JC, Rana M, Sharma V, Nag A, Chahota RK, Sharma TR. Genetic diversity and structure of pea (Pisum sativum L.) germplasm based on morphological and SSR markers. Plant Mol Biol Rep, 2016

[21] Roldan-Ruiz I, Dendauw J, Bockstaele V, Depicker A, Loose M. AFLP markers reveal high polymorphic rates in rye grasses (Lolium spp.). Mol Breed, 2000, 6: 125-134.

[22] Rozen S, Skaletsky HJ. Krawetz S, Misener S. Primer3 on the WWW for general users and for biologist programmers. Bioinformatics methods and protocols: methods in molecular biology, 2000, Totowa: Humana Press 365 386

[23] Sakai A, Larcher W. Frost survival of plants: response and adaptation to freezing stress. Ecological studies, 1987, Berlin: Springer 62

[24] Samant SS, Pant S, Singh M, Lal M, Singh A, Aman Sharma A, Bhandari S. Medicinal plants in Himachal Pradesh, North western Himalaya, India. Int J Biodivers Sci Manag, 2007, 3: 234-251.

[25] Schemske DW, Husband BC, Ruckelshaus MH, Goodwillie C, Parker IM, Bishop JG. Evaluating approaches to the conservation of rare and endangered plants. Ecology, 1994, 75: 584-606.

[26] Selvam ABD. Inventory of Vegetable Crude Drug samples housed in Botanical Survey of India. Howrah. Phcog Rev, 2008, 2(3): 61-94.

[27] Sharma RK, Gupta P, Sharma V, Sood A, Mohapatra T, Ahuja PS. Evaluation of rice and sugarcane SSR markers for phylogenetic and genetic diversity analyses in bamboo. Genome, 2008, 51: 91-103.

[28] Sharma V, Bhardwaj P, Kumar R, Sharma RK, Ahuja PS. Identification and cross-species amplification of EST derived SSR markers in different bamboo species. Conserv Genet, 2009, 10: 721-724.

[29] Sharma S, Sharma V, Chhabra M, Rathour R, Sharma KD, Kapila RK. Characterization of novel polymorphic microsatellite markers in Dactylorhiza hatagirea: a critically endangered orchid species from western Himalayas. Conserv Genet Resour, 2014

[30] Sharma V, Rana M, Katoch M, Sharma PK, Ghani M, Rana JC, Sharma TR, Chahota RK. Development of SSR and ILP markers in horsegram (Macrotyloma uniflorum), their characterization, cross-transferability and relevance for mapping. Mol Breed, 2015, 35: 102.

[31] Sharma V, Sharma TR, Rana JC, Chahota RK. Analysis of genetic diversity and population structure in horsegram (Macrotyloma uniflorum) using RAPD and ISSR markers. Agric Res, 2015, 4: 221-230.

[32] Sharma V, Wani MS, Singh V, Kaur K, Gupta RC. Development and characterization of novel microsatellite markers in Trillium govanianum: a threatened plant species from North-Western Himalaya. 3 Biotech, 2017, 7: 190.

[33] Soule M, Simberloff D. What do genetics and ecology tell us about the design of nature reserves?. Biol Conserv, 1986, 35: 19-40.

[34] Temnykh S, DeClerck G, Lukashova A, Lipovich L, Cartinhour S, McCouch S. Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential. Genome Res, 2001, 11: 1441-1452.

[35] Tu M, Lu BY, Zhu YY, Wang YY. Abundant within-varietal genetic diversity in rice germplasm from Yunnan province of China revealed by SSR fingerprints. Biochem Genet, 2007, 45: 789-801.

[36] Zaki M, Sofi MS, Kaloo ZA. A reproducible protocol for raisingclonal plants from leaf segments excised from mature trees of Betula utilis a threatened tree species of Kashmir Himalayas. Int Multidiscip Res J, 2011, 1(5): 7-13.

[37] Zobel DB, Singh SP. Himalayan forests and ecological generalizations. Bioscience, 1997, 47: 735-745.