基于果实品质与EST-SSR标记的刺梨居群遗传多样性分析及核心种质构建

通过分析评价野生刺梨(Rosa roxburghii Tratt.)资源居群遗传多样性和遗传结构,同时构建核心种质,为刺梨资源的保护和发掘利用提供科学依据。本研究利用10对EST-SSR引物和9个果实品质性状指标对收集的12个刺梨自然居群(共102份种质)的遗传多样性及遗传结构进行分析,同时结合原贵州省32份初级核心种质,采用位点优先取样策略进一步构建西南地区核心种质。结果表明,黔西(QX)居群拥有最高的Shannon信息指数I=0.6965,基因多样性指数h=0.3935,多态位点百分率p=84.62%;而古丈(GZ)居群则无论在分子数据还是表型数据都表现为遗传多样性最低;AMOVA分析表明刺梨居群内遗传变异在87%以上,居群间基因流Nem在3.5以上、平均Nei’s遗传距离(GD)0.223。构建的19份核心种质等位基因保留率和稀有等位基因保留率均为100%,能够代表原种质的遗传多样性。西南地区野生刺梨的遗传变异主要发生在居群内,居群间具有基因交流频繁、Nei’s遗传距离小等特点,构建的19份核心种质从等位基因保留率、稀有等位基因保留率及地理分布均能够较好地代表原种质的遗传多样性。自然居群以黔西(QX)居群遗传多样性最高。因此,野生刺梨的保护策略可采用就地保护黔西(QX)居群与迁地保护19份核心种质相结合的方法进行。     

基于转录组测序的槜李EST-SSR标记开发

为开发槜李EST-SSR标记,本研究利用MISA软件筛选了槜李花转录组测序获得的35584条Unigenes,对其SSR信息进行分析后,利用Primer Premier 3.0软件设计EST-SSR引物,并随机选取40对SSR引物对12个李品种进行EST-SSR引物筛选及多态性分析。结果发现,在槜李花转录组中共搜索到个10791个SSR位点,分布于8433条Unigenes,SSR发生频率为23.70%,平均每3.71 kb含有1个SSR;SSR重复基元中二核苷酸重复出现频率最高,占总SSR数量的52.98%,其次为三核苷酸重复(占24.00%)和单核苷酸重复(占20.95%);二核苷酸重复基元以AG/CT为主(85.95%),三核苷酸重复基元以AAG/CTT为主(31.24%)。利用Primer Premier 3.0软件共设计出9870对候选引物,随机选择40对引物对12个李品种进行SSR引物筛选及多态性分析。40对引物均能扩增出预期大小的条带,有效扩增效率为100%,40对引物中有5对引物在12个李品种中表现出多态性。本研究开发的EST-SSR标记可为李属植物遗传多样性分析提供丰富的候选标记,同时可为槜李发育相关功能基因定位、遗传图谱构建、及分子标记辅助育种等研究提供帮助。     

利用SSR﹑EST-SSR、SNP标记对鲤食物转化率、体厚、体质量3种性状的分析

利用Windows Map Manager2.0的标记回归法对鲤(Cyprinus carpio L.)F2群体进行单标记定位分析.用91个SSR标记、33个EST标记、364个SNP标记对鲤进行全基因组扫描,结果得到与食物转化率、体厚、体质量3个性状显著相关(P＜0.05)的标记,分别为27、35、27个,其中与食物转化率相关的标记SNP0041、SNP0044,其相关性达到极显著水平(P＜0.001),贡献率分别达到15%、16%.这些标记可作为分子标记辅助育种的参考标记.将得到的与食物转化率显著相关的EST座位HLJEl4、HLJE253和与体质量显著相关的HLJE253座位在NCBI上进行BLAST比对,结果显示HLJE253与斑马鱼上编码ORF2结构蛋白的基因相关,相关程度达61%.将得到的与3种性状相关的SNP标记在NCBI上进行序列查找,通过Blast比对找到相关的基因,并对可能的功能作了注释.     

胡椒EST-SSR标记的开发和应用

通过分析NCBI数据库中胡椒转录组数据,共得到unigene 23 524条,其中618条unigene中检测到643个EST-SSR位点,SSR发生频率为2.63%,平均分布距离为12.86 kb。在643个EST-SSR中,二、三核苷酸重复是主要重复类型,分别占27.84%和69.36%,其中(AG/CT)n、(AAG/CTT)n出现频率最高,(NNN/NNN)5类型占EST-SSR位点的42.90%。利用PRIMER3.0设计358对引物,随机合成45对引物,并选用其中11对扩增较好的多态性引物,对43份胡椒种质的遗传多样性进行初步检测,构建聚类分析树状图。结果说明,胡椒EST-SSR标记的开发是可行的,并能够有效的用于胡椒遗传分析研究,具有较高的应用价值。     

Identification of SSR loci in <Emphasis Type="Italic">Betula luminifera</Emphasis> using birch EST data

Expressed sequence tags(ESTs) are generated from single-pass sequencing of randomly picked cDNA clones and can be used for development of simple sequence repeat(SSR) markers or microsatellites.However,EST databases have been developed for only a small number of species.This paper provides a case study of the utility of freely available birch EST resources for the development of markers necessary for the genetic analysis of Betula luminifera.Based on birch EST data,primers for 80 EST-SSR candidate loci were ...     

日本落叶松EST-SSR标记开发及二代优树遗传多样性分析

利用SSR标记研究选自第1代育种试验林、拟纳入二代育种群体的264株日本落叶松二代优树的遗传变异情况。利用1620条落叶松EST序列进行EST-SSR标记的开发,共发现58条序列中含有67个SSR位点,占全部EST序列的3.58%。获得7个多态性EST-SSR标记,其中5个在朝鲜落叶松、长白落叶松、兴安落叶松和华北落叶松中均可扩增出预期目标片段,且具有多态性,表明这些标记在落叶松属内的通用性良好。利用6个EST-SSR标记和4个gSSR标记对日本落叶松二代优树群体开展遗传多样性分析,每个位点的平均等位基因数为4.6个,有效等位基因平均数为3.1个。群体观察和期望杂合度均值分别为0.5902和0.5702;Nei's基因多样度和Shannon多样性指数分别为0.5691和1.0966;全部单株间遗传相似系数0.1725～0.9667。说明该二代优树群体的遗传多样性较高,具有构建二代育种群体的潜力。     

Characterization of dinucleotide and trinucleotide EST-derived microsatellites in the wheat genome

Over the past decade microsatellites or simple sequence repeats (SSRs) have attracted a considerable amount of attention from researchers. The aim of the present paper was to analyse expressed sequence tag-derived SSR (EST-SSR) marker variability in wheat and to investigate the relationships between the number and type of repeat units and the level of microsatellite polymorphism. Two hundred and forty-one new EST-SSR markers available in a public database () were characterized in eight durum wheat cultivars (Svevo, Ciccio, Primadur, Duilio, Meridiano, Claudio, Latino, Messapia), two accessions of Triticum turgidum var. dicoccoides (MG4343, MG29896), one accession of T. turgidum var. dicoccum (MG5323) and in the common wheat cv. Chinese Spring. Of these, 201 primer pairs (83.4%) amplified PCR products successfully, while the remaining 40 (16.6%) failed to amplify any product. Of the EST-SSRs analysed, 45.2% of the primer pairs amplified one or two PCR products. Multiple discrete PCR products were observed among both di- and trinucleotide EST-SSR markers (31.2 and 40.5%, respectively). Markers based on dinucleotide microsatellites were more polymorphic than those based on trinucleotide SSRs in the 12 wheat genotypes tested (68.9 and 52.7%, respectively). An average of 2.5 alleles for dinucleotide and 2.0 alleles for trinucleotide SSRs was observed. The data reported in the present work indicate the presence of a significant relationship between motif sequence types and polymorphism. The primer set based on the AG repeat motif showed the lowest percentage of polymorphism (55.0%), while the primer set based on the AC repeat motif showed t he highest percentage (85.0%). Among trinucleotide SSRs, the AGG microsatellite markers showed the highest percentage of polymorphism (70.0%), and the ACG motif the lowest value (25.0%). The characterization of these new EST-SSR markers and the results of our studyon the effect of repeat number and type of motifs could have important applications in the genetic analysis of agronomically important traits, quantitative trait locus discovery and marker-assisted selection.     

Assessment of EST- and genomic microsatellite markers for variety discrimination and genetic diversity studies in wheat

It is likely that in the near future sequence information from sequencing programmes and EST libraries will generate an abundance of genic microsatellite markers. This study is focused on the assessment of their likely impact and performance vis-à-vis their genomic counterparts. Microsatellites from two sources were used to assess the genetic diversity in 56 old and new varieties of bread wheat on the UK Recommended List. A set of 12 microsatellite markers generated from genomic libraries and 20 expressed sequence tag (EST)-derived microsatellites were used in the study, and the performance of both marker sets assessed. The EST-derived or genic microsatellites delivered fingerprints of superior quality, amplifying clear products with few stutter bands. Diversity levels as revealed bygenic microsatellites are similar to the few published results. The PIC values for the genic markers were generally lower than those calculated for the genomic microsatellites, though advantages of both marker classes for variety identification applications are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

大豆基因组SSR和EST-SSR在黄芪中的通用性分析

分子标记的种间通用性可降低其开发成本,提高利用效率,促进遗传研究较薄弱物种的分子遗传学研究。为开发黄芪SSR分子标记,本研究利用大豆的66对基因组SSR(G-SSR)和43对EST-SSR引物在黄芪基因组中进行通用性分析,并选出其中条带清晰易辨的23对引物对6种不同来源的黄芪进行遗传分析。结果表明:大豆G-SSR、EST-SSR引物在黄芪中的通用性比例分别为31.82%、76.74%,多态性引物分别占18.18%、53.49%,大豆EST-SSR在黄芪的通用性高于基因组SSR,且通用的大豆SSR标记可以用于不同来源黄芪的遗传多样性分析。本研究发掘的多态性SSR引物可以有效用于黄芪的分子遗传多样性研究。     

Development of EST-SSR in foxtail millet (<Emphasis Type="Italic">Setaria italica</Emphasis>)

The development of EST-SSR in foxtail millet (Setaria italica) for polymorphism and transferability study was reported here. From 1213 EST sequences, 30 SSRs were obtained and primers were designed for 26 SSRs. Among them, four pairs of SSR primers amplified polymorphic products in 12 foxtail millet cultivars and one accession of Setaria viridis, a wild relative of foxtail millet, with 10 alleles detected for the four loci and 2.5 alleles per locus. In addition, ten SSR markers could be transferred to other nine Gramineae species. The putative functions of 11 ESTs containing polymorphic and transferable SSRs were also identified.