大豆EST—SSR标记开发及与Genomic—SSR的比较研究

对458220条大豆EST序列进行SSR搜索,共检测出EST—SSR序列39989条,经拼接得到无冗余EST—SSR序列8190条,包括357种重复基元。其中二、三核苷酸重复基元类型居多,分别占无冗余EST总数的11．13％和16％,统计得到二核苷酸重复类型12种,三核苷酸重复类型60种。以含有简单重复序列的元冗余EST序列设计200对引物,其中148对引物有清晰且单一条带扩增产物,以30份大豆品种资源进行引物筛选,获得多态性引物31对。以21份大豆不同基因型的基因组DNA为模板选取30对显示多态性的大豆EST—SSR引物和30对大豆基因组SSR引物进行扩增,带型统计结果显示：大豆EST—SSR与基因组SSR在供试基因型间多态性指数均值分别为0．55和0．44,二者揭示的多态性水平差异不大。从而说明利用生物信息学方法基于大豆EST开发SSR标记是切实可行的,大豆EST—SSR可以用于大豆遗传多样性分析,是大豆DNA分子标记体系的一个重要补充。     

2011-15-3等4个茶树新品系亲缘关系的 EST-SSR分析

目的对2011-15-3等4个茶树新品系进行亲缘关系分析,以期为新品系的识别、新品种登记及育种亲本选配提供可靠依据。方法利用文献开发的24对EST-SSR引物,对4个茶树新品系等7份材料进行PCR扩增,在所得相似系数的基础上进行UPGMA聚类,并绘制亲缘关系树状聚类图。结果 24对引物共扩增出69个等位位点,平均2.88;平均杂合度观测值(Ho)、平均杂合度期望值(He)、Shannon指数的平均值分别为0.42、0.54、0.75,相似系数变幅为0.47~0.89。槠叶齐、碧香早、高芽齐的后代与其母本相似系数值分别高达0.84、0.79、0.79、0.78。根据相似系数矩阵按UPGMA法进行聚类,在相似系数平均值0.66处可将参试的7份材料分为3类:第Ⅰ类为高芽齐和2011-34-1,第Ⅱ类为槠叶齐、2011-21-1和2011-21-3,第Ⅲ类为碧香早和2011-15-3。结论明确了4个茶树新品系与其他品种的亲缘关系及品种间的遗传距离。     

De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus densiflora)

Japanese red pine (Pinus densiflora) is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98%) unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%), Amborella trichopoda (9.83%), and Pinus taeda (4.15%). A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite) software, of which the tri-nucleotide repeats were most abundant (50.18%) and 629 EST-SSR (expressed sequence tag- simple sequence repeats) primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s) mapping for pine for P. densiflora.