红花EST-SSR分子标记开发与初步验证

Development and identification of SSR molecular markers based on transcriptome sequences of Safflower(Carthamus tinctorius L.)

旨在开发红花EST-SSR标记,为红花分子辅助育种和种质资源评价提供有力工具.先对红花转录组进行测序,选用MISA软件筛选鉴定SSR位点.在云南红花(YH)转录组中,共获得46 016个SSR位点,分布频率为32.09%,平均每3.11 kb有一个SSR.云南红花SSR位点以二,三核苷酸重复基序为主,其优势重复基序分别为AG/CT(44%)和AAG/CTT(24%).SSR位点重复次数相差较大,其中重复为5次比率最高,SSR位点数为9 369(26.63%),其次为6次(8 148, 23.16%).同时,利用Primer3.0进行引物设计,随机筛选27对引物,在60份不同来源红花种质中进行多态性验证,获得12对具有多态性稳定的引物,多态性引物比率为44.4%.结果表明,基于红花转录组序列开发SSR标记是可行的,开发的标记具有稳定扩增性,丰富多态性等优点,开发的SSR标记将有助于红花功能基因挖掘,遗传连锁图谱构建和遗传多样性分析.

The aim of this study was to develop EST-SSR molecular markers, providing a powerful tool for molecular marker assisted breeding and germplasm resource assessment for safflower.Based on the sequencing of safflower transcriptome, the SSR loci were searched by MISA software, and the polymorphism of some SSR primers were identified. In the YH safflower transcriptome, a total of 46 016 SSRs were identified through software analysis, at a frequency of 32.09%, with one SSR locus occurring in per 3.11 kb. Among the repeat types identified, dinucleotide and trinucleotide were the dominant repeat types, representing AG/CT and AAG/CTT, accounting 44% and 24%, respectively. SSR repeat frequencies were largely different, and the largest number of repeats was 5, accounting for 26.63% of the total repeat types, followed by 6 repeats(23.16%). Meanwhile, 27 primer pairs were randomly selected and synthesized, which were verified by PCR amplification of genomic DNA from 60 safflower samples. The results showed that 12 primer pairs produced expected bands. The ratio of polymorphic primers was 44.4%. In conclusion, the final results indicated that it was feasible to develop SSR markers based on transcriptome sequencing of safflower. The developed markers have the advantages of steady amplification and high polymorphism. The developed SSR markers will contribute to functional gene mining, genetic linkage mapping and genetic diversity analysis of safflower.