保罗蓝睡莲花器官转录组SSR的分布及其序列特征分析

Distribution and Properties of SSR in Transcriptome of Nymphaea Paul Stetson Flowers

【目的】通过解析保罗蓝睡莲花转录组数据的SSR位点特征信息,开发新的与表型相关的SSR标记,为睡莲种质资源评价、新品种选育提供科学依据。【方法】以热带睡莲保罗蓝不同花器官部位(雌蕊、雄蕊、花瓣)的转录组数据为背景材料,用MISA软件检索序列的SSR位点,采用Excel对位点特征进行分析作图,Primer 3.0软件设计引物,TP-M13-SSR PCR筛选引物。【结果】在花器官转录组的39079条Unigenes中共检测到12365个SSR位点,位点发生频率为31.64%,平均每5.79 kb出现1个SSR位点。SSR位点的重复类型主要为二核苷酸重复碱基,占SSR位点总数的71.85%,优势重复类型为AG/CT,占碱基总数的61.34%;其次为三核苷酸重复碱基,为26.10%,优势重复类型是AAG/CTT,占比8.30%。SSR碱基重复次数在5~20次,序列长度为12~30 bp,平均长度18.38 bp。引物设计获得9212对引物,从中随机挑选100对进行PCR扩增验证,筛选出9对多态性好的引物,可将12份睡莲种质在遗传相似系数为0.735时聚为3支。【结论】热带睡莲保罗蓝花器官转录组中的SSR位点分布频率高、类型丰富,多态性较高,具较大的应用潜力。开发的9对SSR引物可将12份种质有效分开,进一步丰富了睡莲现有SSR标记,可为睡莲属植物的遗传多样性分析和分子辅助育种提供科学参考。

【Objective】SSRs in the transcriptome of Nymphaea Paul Stetson flowers were studied to generate new markers for evaluating germplasms and facilitating breeding of tropical waterlilies.【Method】SSR loci were retrieved from the transcriptomes of floral pistils,stamens,and petals of Nymphaea Paul Stetson using MISA.Characteristics of the loci were analyzed by Excel,and primers designed by Primer 3.0 and screened by TP-M13-SSR PCR.【Result】There were 12365 SSR loci found in the 39079 unigenes of the transcriptome at the frequency of 31.64%averaging one SSR locus per 5.79 kb.Most of the SSR loci had dinucleotide repeat motifs comprising 71.85%of total with AG/CT being the dominant unit that made up 61.34%of the motifs.Trinucleotide repeat motifs accounted for 26.10%of the sites with AAG/CTT being dominant at 8.30%.The repeating frequency was 5–20 times with a sequence of 12–30 bp averaging 18.38 bp long.Of the 9212 pairs of primers designed,100 were randomly selected for a validation by PCR amplification to arrive at 9 pairs with high polymorphism being used as the markers.Subsequently,the 12 germplasms were clustered into 3 branches under a genetic similarity coefficient of 0.7375.【Conclusion】The SSR loci in the Nymphaea Paul Stetson transcriptome were high on distribution frequency,rich in diversity,greatly polymorphic,and desirable for applications.The 9 pairs of SSR primers identified in this study extended the existing marker repertoire facilitating effective germplasm differentiation on waterlilies.