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SSR标记在海带群体遗传及胞质遗产研究中的应用
李秋莹
学位类型博士
导师段德林
2015-12
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词海带 Ssr标记 群体遗传 细胞质遗传
摘要海带是重要的经济褐藻,开展其群体遗传及重要经济性状遗传机制的研究对其品种改良具有重要价值,也对海带产业的发展具有重要意义。本研究在开发海带核基因组和细胞器SSR标记的基础上,对我国常见的海带养殖群体进行种群遗传结构的分析,另外,也对海带细胞器DNA的遗传模式进行研究,主要得出以下结果:
(1)   应用Illumina HiSeq 2000测序技术对海带基因组进行测序,从拼接的高质量基因组序列中开发SSR标记。使用MISA软件共检测到181,595个SSR位点。海带基因组中SSR重复类型十分丰富,其中单核苷酸(54%)和三核苷酸重复(26%)最为丰富。从设计的19,658对引物中合成600对引物,其中166个位点(28%)在三个检测的海带个体中呈现多态性。60个多态的引物被用于24个样品中进一步分析多态性,每个位点产生等位基因的范围为2~7个(平均3.7个等位基因),观测杂合度为0~0.625,期望杂合度为0.082~0.745。多态信息含量值的范围为0.08到0.69,其中16个位点为高信息含量位点(PIC > 0.5)。本研究开发的SSR标记将在海带遗传多样性分析,种群结构评估和QTL作图等方面起到重要作用。
(2)   应用23个SSR标记对中国17个养殖海带群体的遗传多样性和遗传结构进行评估。23个SSR在454个海带个体中共检测到184个等位基因,平均期望杂合度和多态信息含量值分别为0.5029和0.652,表明中国养殖海带整体遗传多样性呈中度水平。而单个种群水平的遗传多样性相对低些(平均期望杂合度为0.4026),南方养殖群体的遗传多样性普遍低于北方养殖群体,反映了受育种过程中不断的人工选择的影响。STRUCTURE聚类、PCA分析、UPGAM建树等结果均表明中国养殖海带主要分为5个类群:G1,包含10个北方养殖群体;G2包含4个南方养殖群体;G3、G4、G5分别对应NJ、DF2、DF3三个来自北方的养殖群体。G1和G2为主要的两大类群,而这两大类群也是基于南北地理区域划分。AMOVA与FST分析均表明南方海带养殖群体与北方养殖群体之间存在遗传分化,遗传变异主要来自种群内部。本研究结果可为后续海带育种和种质资源利用提供理论依据。
(3)   2个多态的叶绿体SSRs(cpSSRs)标记和2个多态的线粒体DNA序列标记(rpl6-rps2trnW-trnI)被用于海带叶绿体和线粒体DNA遗传模式的研究。对2套正反交的4对亲本配子体及76个杂交子代的单倍型进行分析。所有子代的单倍型均与其母本的单倍型一致,表明海带叶绿体和线粒体DNA均是母系遗传的,为海带细胞质母系遗传提供了分子证据。在开发的56个细胞器SSR标记中,用10个cpSSRs和4个mtSSR对24个海带样品进行多态检测。其中,2个cpSSRs和2个mtSSRs呈现多态性,可用于后续海带遗传研究。
其他摘要Saccharina japonica is an important commercial brown alga. Population genetic study and economic traits analysis for this kelp are crucial to its genetic improvement, and development of S. japonica industry. In this study, SSR markers from S. japonica nuclear and organellar genomes were developed, and were used for geneic analysis of common cultivated population in China. Moreover, inheritance pattern of organellar DNA in S. japonica was also investigated. The main results are as follows:
(1)   Illumina Hi-Seq 2000 next-generation sequencing (NGS) was used on S. japonica specimens to develop SSRs from high quality genomic sequences. 181,595 SSR loci were identified using the MISA program. SSR repeat types are plentiful in genomic sequences of S. japonica, with the most abundant types being mononucleotides (54%) and trinucleotides (26%). Six hundreds primer pairs were selected and synthesized from 19,658 primer pairs, showing 166 polymorphic SSR loci (28%) in three selected S. japonica individuals. 60 polymorphic SSRs were further analyzed in 24 S. japonica individuals, showing 2 to 7 alleles for each locus (average 3.7 alleles), with observed heterozygosities of 0~0.625 and expected heterozygosities of 0.082~0.745. Polymorphism information content (PIC) values ranged from 0.08 to 0.69. 16 loci were classified as informative markers (PIC > 0.5). Polymorphic SSR markers developed in this study should be useful for examining genetic diversity, assessment of population structure, and QTL mapping in S. japonica.
(2)   23 SSR markers were used for genetic diversity and genetic structure analysis of 17 S. japonica cultivated populations in China. Total 184 alleles were detected in the 454 individuals. The average expected heterozygosity and PIC values were 0.5029 and 0.652, respectively, indicated a moderate level of genetic diversity in Chinese cultivated S. japocnia. However, the genetic diversity at the single population level was relatively lower (average expected heterozygosity = 0.4026) and was lower in the South populations than in the North populations, reflected the influences of repeated artificial selection in the breeding. The results from Structure, PCA, and UPGAM showed that 17 cultivated populations were divided into five groups: G1, including ten North populations; G2, including four South populations; G3, G4, G5 correspond to NJ, DF2, DF3 three North populations. G1 and G2 are the two major groups, and were defined based on differences in geographical location. AMOVA and FST values showed genetic differentiation between North populations and South populations, and variation among individuals within populations account for majority of the total variation. This results could be helpful in further S. japonica breeding and utilization of germplasm resource.
2 simple sequence repeats from chloroplast DNA (cpSSRs) and 2 mitochondrial DNA markers (rpl6-rps2 and trnW-trnI) were polymorphic and used to investigate the inheritance patterns of the chloroplast and mitochondrial DNA in S. japonica. Haplotypes were tested in 4 pairs of parental gametophytes and 76 progeny sporophytes from two sets of reciprocal crosses. It is showed that both chloroplast and mitochondrial DNA were maternally inherited in all crosses, as the haplotypes of all progenies were consistent with that of the maternal parent, which showed molecular evidence for maternal inheritance of cytoplasm in S. japonica. In addition, 42 cpSSRs and 14 mtSSRs markers were developed, of which, 10 cpSSRs and 4 mtSSRs were tested for polymorphism. 2 cpSSRs and 2 mtSSRs exhibit polymorphism in 24 S. japonica individuals, which could be used for genetic studies in S. japonica.
学科领域海洋生物学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/86540
专题实验海洋生物学重点实验室
作者单位中国科学院海洋研究所
推荐引用方式
GB/T 7714
李秋莹. SSR标记在海带群体遗传及胞质遗产研究中的应用[D]. 北京. 中国科学院大学,2015.
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