Development of genomic simple sequence repeats (g-SSR) markers in Tinospora cordifolia and their application in diversity analyses

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• 作者：Ritu Paliwal^a ; ^d ; Rajesh Kumar^a ; Debjani Roy Choudhury^a ; Amit Kumar Singh^a ; Sundeep Kumar^a ; Ashok Kumar^b ; K.C. Bhatt^c ; Rita Singh^d ; Ajay Kumar Mahato^e ; N.K. Singh^e ; Rakesh Singh^a ; ^{singhnbpgr@yahoo.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Tinospora cordifolia ; Genomic SSR ; Genetic diversity ; Cross species transferability ; Molecular markers
• 刊名：Plant Gene
• 出版年：2016
• 出版时间：March 2016
• 年：2016
• 卷：5
• 期：Complete
• 页码：118-125
• 全文大小：761 K

文摘

Tinospora cordifolia, a medicinal plant of economic importance, has very rich diversity and distributed across India growing in wild. However, for its judicious exploitation through targeted breeding program there is complete lack of genomic resources which is necessary for its desired utilization. In the present study, we have developed novel genomic-SSRs (g-SSR) markers, which due to their wider distribution across the genome find greater usefulness in assessing genetic diversity. For development of g-SSR markers, four microsatellite enriched libraries [(CT)₁₄, (AC)₁₀, (GT)₁₂ and (AAC)₈] were constructed from genomic DNA of T. cordifolia accession (IC-281965) by using a streptavidin-biotin based enrichment approach. The 536 random recombinant clones from the four SSR enriched libraries were further verified by colony PCR for the presence of insert. From such screening, 356 clones were found positive and were subjected to sequencing using Sanger sequencing method. The sequence analyses revealed that 114 clones (32%) were having SSR repeats. Further analysis, based on microsatellite library enrichment showed that 60% SSR repeats were found in clones from GT enriched libraries followed by 38%, 23% and 19% in CT, AAC and AC enriched libraries respectively. Altogether 90 SSR sequences were submitted to the National Center for Biotechnology Information (NCBI) with accession numbers from KT384079 to KT384168. For diversity study 42 primer pairs were designed and all primers reproducibly amplified across all accessions of T. cordifolia. However, when such markers were used in analysis for Tinospora rumphii and Tinospora sinensis, only 83.3% and 85.7% of the markers were transferred successfully. The alleles generated across 28 accessions of three Tinospora species were assessed for genetic relatedness and it grouped accessions into three major clusters suggesting sufficient diversity in Tinospora. The g-SSR markers generated in the present study are a valuable genomic resource for effective utilization in crop improvement of Tinospora germplasm.