本研究基于大叶栎(Castanopsis fissa)转录组信息,开发大叶栎SSR分子标记,筛选及验证所开发SSR分子标记的有效性及多态性,对广西梧州大叶栎育种群体进行遗传多样性分析并构建指纹图谱.通过对大叶栎叶片的转录组测序结果进行分析,共挖掘到128 275个SSR位点,其中单核苷酸重复类型SSR位点占总位点的比例最高,为57.03％.通过引物设计和实验筛选,共计开发出225对SSR引物,有效扩增率为63.56％,最终筛选出扩增稳定、条带清晰的引物20对.用该组引物对广西大叶栎育种群体的79个样本进行遗传多样性分析.结果表明,该群体观测等位基因数(observed number of alleles,Na)、有效等位基因数(effective number of alleles,Ne)、Shannon 多样性指数(Shannon's diversity index,I)、观测杂合度(ob-served heterozygosity,Ho)变化范围分别为 2.000～5.000、1.163～2.838、0.266～1.092 和 0.150～0.763,平均值分别为 2.850、1.810、0.690和0.425.优选其中8对SSR引物构建育种群体的指纹图谱,可以实现对广西梧州大叶栎育种群体每个无性系的准确鉴定.综上认为,所开发的20对SSR引物可以满足开展大叶栎群体遗传学研究的需要,所构建的大叶栎育种群体指纹图谱为优良无性系分子鉴定和子代亲本分析提供了可靠的技术手段.
Development of SSR Primers for Castanopsis fissa and Construction of Breeding Population Fingerprint
In this study,based on transcriptome information of Castanopsis fissa,we developed SSR molecular markers,screened and verified the effectiveness and polymorphism of the developed SSR molecular markers,analyzed the genetic diversity of the breeding population in Guangxi,and constructed fingerprint.A total of 128 275 SSR loci were discovered by analyzing the transcriptome sequen-cing results of the Castanopsis fissa.The proportion of single nucleotide repeat type SSR loci was the highest,accounting for 63.56％.Through primer design and experimental screening,a total of 225 pairs of SSR primers were developed,the effective amplification rate was 57.10％,and finally 20 pairs of primers with stable amplification and clear bands were selected.The genetic diversity of 79 sam-ples from the breeding population in Guangxi was analyzed with these primers.The results showed that the observed number of alleles(Na),effective number of alleles(Ne),Shannon's diversity index(I)and observed heterozygosity(Ho)in this population ranged from 2.000 to 5.000,1.163 to 2.838,0.266 to 1.092 and 0.150 to 0.763,respectively.The mean values were 2.850,1.810,0.690 and 0.425,respectively.Selecting 8 pairs of SSR primers to construct the fingerprint of breeding population can realize the accurate identification of each clonal line of breeding population.In conclusion,the 20 pairs of SSR primers developed can meet the needs of population genetics research of Castanopsis fissa,and the constructed population fingerprint provides a reliable technical means for excellent clonal molecular identification and progeny parental analysis.
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