ABSTRACT
Objective: EST-SSR loci were identified and analyzed based on the transcriptome sequencing data in Polygonatum cyrtonema, in order to develop SSR markers suitable for evaluation and application of germplasm resources on P. cyrtonema. Methods: SSR loci were identified and analyzed in all of 126 544 Unigenes by using MISA tool. SSR primers were designed by using Primer 3.0 software and 50 pairs of SSR primers were randomly selected for validation test. Results: A total of 12 317 SSR loci, including the types of 2-6 nucleotide repeats with occurring frequency of 1/5.91 kb, were identified from 9 982 Unigenes in P. cyrtonema transcriptome. The distribution frequency of SSRs was 9.73%. Dinucleotide repeat was the main type, accounting for as much as 53.14% of all SSRs, followed by trinucleotide repeat (33.31%). The validation test on 50 pairs of SSR primers showed that 29 of them (58%) generated fragments with expected molecular size from P. cyrtonema. The capillary electrophoresis using fluorescence-labeled SSR markers showed that nine genotypes were identified at seven SSR loci in P. cyrtonema, which further demonstrated the validity of these SSR primers. Conclusion: There are numerous SSRs in P. cyrtonema transcriptome with high frequency, rich repeat types and relatively high polymorphic, which will provide abundant valuable candidate markers for genetic diversity analysis and genetic mapping construction in P. cyrtonema, also can be used as a technical tool for molecular identification among Polygonatum species and for molecular marker assistant breeding in superior cultivars of P. cyrtonema.
ABSTRACT
In order to analyze the expression of genes involved in steroidal saponin biosynthesis pathway in Polygonatum cyrtonema tubers, it is very important to select internal reference genes that are stably expressed at different development stages and in response to abiotic stress. According to the previously established P. cyrtonema transcriptome database and reported internal reference genes in plant, this study systematically analyzed eight candidate internal reference genes including histone H2 A, glyceraldehyde-3-phosphate dehydrogenase, ACTIN, β-tubulin, ubiquitin-conjugating enzyme-E2-10, elongation factor 1-alpha isoform, 18 S rRNA and α-tubulin 4 for expression stability in P. cyrtonema tubers at different development stages and in response to methyl jasmonate(MeJA) stress by using Real time fluorescence quantitative PCR(qPCR). Based on the statistical analysis of qPCR results by using GeNorm, NormFinder and BestKeeper softwares, the expression of ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform are the most stable in P. cyrtonema tubes at different development stages and in response to MeJA stress. The two internal reference genes were further validated by analyzing the expression of 4 genes involved in steroidal saponin biosynthesis pathways. In conclusion, ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform can be used as the most appropriate internal reference genes for qPCR analysis in P. cyrtonema. This study also provide a foundation for future investigate the molecular mechanism of steroidal saponin biosynthesis pathways in P. cyrtonema.