玉米和高粱SSR标记在薏苡中的通用性研究

旨在分析玉米和高粱SSR标记在其近缘种薏苡中的通用性和多态性，以期筛选可用于薏苡种质资源和遗传学研究的分子标记。以11份薏苡资源为供试材料，利用SSR-PCR扩增和毛细管电泳检测方法，对364对SSR引物进行了测试和筛选。结果表明，364对玉米和高粱来源的标记中有163对能在薏苡中扩增并得到清晰的条带，其中44对标记在薏苡中表现出良好的多态性。高粱SSR的通用性和多态性比例分别为58.02%和30.85%，玉米SSR标记的通用性和多态性比例分别为23.45%和26.47%，高粱SSR标记在薏苡中通用性和多态性更好。44对引物在11份薏苡材料中扩增出110条多态性条带，每对引物扩增出的条带数在1～5条之间，平均为2.5条，PIC为0.15～0.79，其中15对引物的PIC值大于0.5，占全部多态性引物的34.09%。筛选出的通用SSR引物可为薏苡种质资源多样性和分子遗传学研究提供可用的遗传标记，也是高粱、玉米和薏苡3种作物之间比较基因组学研究的有力工具。     

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。     

新疆野樱桃李EST-SSR引物开发及通用性评价

野生樱桃李是中国濒临灭绝的树种之一。本研究从分子水平对野樱桃李进行研究,以期为制定保护策略提供理论依据。从NCBI 数据库中下载樱桃李EST 序列,用SSR Hunter l.3 查找SSR 位点,用Primer 6.0 设计了30 对引物并验证引物的通用性。结果表明：有20 对引物能扩增出有效条带,扩增率为66.67%,其中9 对引物能扩增出多态性条带,多态性比率为45%。用20 对有效引物在欧洲李和天山樱桃中进行扩增,均表现良好的通用性,且同属植物间的通用性明显高于同科植物间,说明引物通用性的高低与基因组的差异有关,亲缘关系越近引物通用性越高。     

棉花EST-SSRs在香蕉中的通用性

香蕉SSR分子标记开发和应用有限,通过对棉花SSR分子标记在香蕉中的通用性研究.用1343对棉花EST-SSR引物对贡蕉(AA)和野蕉(BB)两个材料进行转移扩增,得到226对有效扩增的引物.进一步用该226对引物对20个香蕉品种和4个野蕉材料扩增,得到了157对多态性引物.157对多态性引物共扩增到1188个条带,平均7.6个;引物的多态信息含量范围在0.58～0.91之间.依据获得的SSR数据,应用非加权类平均聚类方法,在相似系数0.60水平上,将24个品种分为两大类群,类群Ⅰ是以A基因组为主的品种群,类群Ⅱ是以B基因组和A基因组与B基因组杂交的品种群,证明了棉花EST-SSR分子标记在香蕉中具有通用性.     

基于苦瓜转录组序列的SSR分子标记开发

为了全面了解苦瓜转录组SSR位点特征和满足苦瓜分子育种对分子标记的需求,利用MISA软件对来自苦瓜材料‘大沥-11’6个组织的59740条转录组Unigene序列进行SSR位点搜索,共检测出31066个SSR位点,出现频率(发现的SSR个数与总Unigene数之比)为52.00%,平均每2.62 kb出现1个SSR位点。在苦瓜转录组SSR位点中,基元类型主要为三核苷酸,占总SSR位点的42.17%;其次是二核苷酸,占总SSR位点的31.66%。利用Primer 3软件对搜索到的苦瓜转录组SSR位点进行引物设计,然后把获得的引物序列比对回苦瓜的参考基因组,选择上下游引物序列都是唯一比对的序列并去除重复后共获得9135对特异SSR引物。选择MC00上的232对特异SSR引物对‘谭边大顶’和‘华艺320’两个苦瓜品种基因组DNA进行PCR扩增,其中有219对特异引物可扩增出清晰的条带,有效扩增率为94.40%;有31对特异引物扩增产物表现出多态性,多态率为13.36%。本研究开发的苦瓜转录组特异SSR引物为苦瓜的种质资源分析、基因定位、分子标记辅助选择等理论与应用研究提供了引物数据参考。     

近缘属SSR标记引物在光萼荷属植物中的通用性

为开发适合于光萼荷属植物的SSR标记引物,本文研究了凤梨属、丽穗凤梨属、铁兰属、艳红凤梨属和帝王凤梨属中共计84对SSR标记引物在光萼荷属植物中的通用性。结果表明,84对SSR标记引物中有68对在光萼荷属植物中的通用性较好,通用性比例高达81.0%。其中,凤梨属、丽穗凤梨属、铁兰属、艳红凤梨属和帝王凤梨属的SSR标记在光萼荷属植物中的有效扩增比例分别为80.7%、72.7%、80.0%、100.0%和75.0%。另外,利用两对SSR引物对部分光萼荷属植物F1杂交后代进行杂种鉴定,结果表明是可行的。本研究筛选出的通用性SSR标记为光萼荷属植物资源评价、杂种鉴定和今后的分子育种研究提供了新的研究手段。     

茄子ISSR和SSR分子标记的鉴定技术分析

以茄子为研究对象,初步建立起茄子的ISSR和SSR分子标记技术体系,包括DNA提取、退火温度、循环次数和引物筛选等。结果表明,ISSR分子标记体系中,从49条引物中筛选出3个条带清晰的引物对6个品种进行扩增,共检测出26条带,其中多态性带21条,多态性比例为80.77%;SSR分子标记体系中,从4对引物中筛选出3个条带清晰的引物对6个品种进行扩增,共检测出17条带,其中多态性带5条,多态性比例为29.41%,2种方法均能把6个茄子品种区分开来,本实验为茄子品种鉴定和新品种选育提供理论依据。     

绿豆基因组SSR引物在豇豆属作物中的通用性

分子标记的种间通用性可降低其开发成本,提高利用效率,也有助于促进遗传研究较薄弱物种的分子遗传学研究。本文选取绿豆、小豆、豇豆及饭豆材料各3份, 分析1 205对新开发的绿豆基因组SSR引物在这些材料中的扩增效果,结果显示绿豆基因组SSR引物在豇豆、小豆和饭豆中的通用性比率分别为50.0%、73.3%和81.6%;多态性比率分别为4.1%、1.7%和1.5%;在4个种间均通用的引物469对。这些通用性SSR引物将有助于这4种食用豆类在多样性评价、连锁图谱的构建、基因定位及比较基因组学等方面的研究。     

苦瓜SSR-PCR体系优化及其引物在瓜类作物中的穿梭性

为构建高密度苦瓜SSR分子标记遗传图谱,利用正交设计L16(45)对苦瓜SSR-PCR体系进行优化。采用该体系和本实验室开发的300对SSR引物对4种表型差异较大的苦瓜材料基因组DNA进行了多态性引物筛选,并用筛选出来的多态性引物对黄瓜、甜瓜、西瓜、丝瓜、冬瓜、南瓜进行了PCR扩增,研究了苦瓜SSR引物对6种瓜类作物扩增的通用性及多态性。优化后的SSR反应体系为:d NTPs(2.5 mmol/L)0.5μL、Taq DNA聚合酶(5 U/μL)0.1μL、10×PCR-Buffer 1.5μL、SSR引物(10μmol/L)0.6μL、模板DNA(100 ng/μL)70 ng,总反应体系为10μL。在苦瓜300对SSR引物中,筛选出49对条带清晰的多态性引物,多态性比率为16.3%。其中苦瓜SSR引物对黄瓜、甜瓜、西瓜、丝瓜、冬瓜、南瓜扩增的通用性分别为61.2%、51.0%、59.2%、57.1%、49.0%、59.2%;每个物种不同类型间的多态性比率排序为丝瓜(22.4%)西瓜(16.3%)甜瓜(14.3%)、冬瓜(14.3%)黄瓜(8.2%)南瓜(2.0%)。即有棱丝瓜和无棱丝瓜之间、圆形西瓜和椭圆形西瓜之间多态性较高,瓜用南瓜和叶用南瓜之间多态性比率最低。说明部分苦瓜的SSR标记在其他葫芦科作物中具有通用性,并且在不同物种间扩增多态性存在明显差异,本研究将为葫芦科其他作物分子标记研究及后续苦瓜分子标记辅助育种和比较基因组等方面的研究和应用提供依据。     

高粱SSR和EST-SSR标记在割手密中的通用性分析

目前, 割手密中开发的分子标记有限, 为增加其分子标记数量, 本研究对高粱分子标记在割手密中的通用性进行分析。选用高粱的 29对基因组 SSR标记和 20对 EST-SSR标记在割手密种质 GSM39中进行筛选, 以评价其通用性。进一步利用 14份割手密材料评价标记的多态性和遗传多样性。结果显示： 70%的 EST-SSR引物在割手密中得到成功扩增, 可用的 EST-SSR引物中多态性比率占 50%。SSR引物在割手密中的通用性比率只有 34.5%, 但多态性比率为 70%。14对多态性引物在 14份割手密中共检测到33个等位基因, 平均观测等位基因数为2.4286, 平均有效等位基因数为1.7, 平均观测表观杂合度为0.544, 平均期望杂合度为 0.3858, Nei’ s基因多样性指数平均值为 0.3716。结果表明, EST-SSR引物的通用性高于 SSR引物, 但 SSR引物的多态性更高。这对割手密遗传多样性研究和比较基因组学研究具有重要意义。     

Development and characterization of tomato SSR markers from genomic sequences of anchored BAC clones on chromosome 6

Simple sequence repeat motifs are abundant in plant genomes and are commonly used molecular markers in plant breeding. In tomato, currently available genetic maps possess a limited number of simple sequence repeat (SSR) markers that are not evenly distributed in the genome. This situation warrants the need for more SSRs in genomic regions lacking adequate markers. The objective of the study was to develop SSR markers pertaining to chromosome 6 from bacterial artificial chromosome (BAC) sequences available at Solanaceae Genomics Network. A total of 54 SSR primer pairs from 17 BAC clones on chromosome 6 were designed and validated. Polymorphism of these loci was evaluated in a panel of 16 genotypes comprising of Solanum lycopersicum and its wild relatives. Genetic diversity analysis based on these markers could distinguish genotypes at species level. Twenty-one SSR markers derived from 13 BAC clones were polymorphic between two closely related tomato accessions, West Virginia 700 and Hawaii 7996 and were mapped using a recombinant inbred line population derived from a cross between these two accessions. The markers were distributed throughout the chromosome spanning a total length of 117.6 cM following the order of the original BAC clones. A major QTL associated with resistance to bacterial wilt was mapped on chromosome 6 at similar location of the reported Bwr-6 locus. These chromosome 6-specific SSR markers developed in this study are useful tools for cultivar identification, genetic diversity analysis and genetic mapping in tomato.     

SSR标记与形态学方法鉴定杂交油菜纯度的比较研究

为了探讨SSR标记技术在油菜杂交种纯度鉴定中应用的可行性,本文采用SSR标记技术和形态学方法鉴定油菜杂交种纯度以比较两者的差异,用406对微卫星引物分别对杂交油菜品种"蜀杂六号"核不育系156AB的可育株及不育株、恢复系86Y17和杂种1代各自的形态学正常植株及杂株,进行了鉴定.首先从406对SSR引物中筛选出了多态性强且易识别的14对SSR引物.采用选出的14对引物能稳定扩增出蜀杂六号父母本的多态性,能够鉴定油菜杂种中的杂株,但结果表明SSR标记鉴定的杂株与形态学方法鉴定的杂株存在较大差异.     

利用水稻基因组序列数据开发SSR标记的方法

水稻基因组序列中存在着丰富的简单重复序列(SSR)，而已经开发利用的仅仅是一小部分。本文提出了通过搜索水稻基因组序列中的简单重复序列开发SSR标记的思路和方法，并介绍了应用本方法成功地在500kb范围内，获得22个SSR标记，其中10个在定位群体中扩增出多态带的SSR标记并将其定位，本实例验证了利用水稻基因组序列数据开发SSR标记是十分有效的。     

Assessment of the uniformity of wheat and tomato varieties at DNA microsatellite loci

By analysing a number (20–38) of individuals from selected varieties of wheat and tomato, we have been able to assess intra-varietal uniformity at certain micro satellite (simple sequence repeat, SSR) loci. In total, 45 varieties of wheat were analysed at between 7–9 different SSR loci, and 10 varieties of tomato were analysed at six loci. The results showed that there was variation both between varieties and between microsatellites in the degree of non-uniformity observed, and it was possible to identify a number of different probable sources of non-uniformity. Twenty-four of the wheat varieties and nine of the tomato varieties were sufficiently uniform to meet the standards currently applied for distinctness, uniformity and stability (DUS) testing using phenotypic characteristics. The implications for the potential future use of SSRs in DUS testing are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of genomic simple sequence repeat markers from an enriched genomic library of grass pea (Lathyrus sativus L.)

Simple sequence repeat (SSR) or microsatellite markers are a valuable tool for several purposes such as evaluation of genetic diversity, fingerprinting, marker‐assisted selection and breeding. In this study, a SSR genomic enriched library was developed in Lathyrus sativus (grass pea) by affinity capture of restriction fragments to biotinylated microsatellite oligonucleotides. About 400 randomly selected clones were sequenced, and SSRs were present in approximately 30% of them. Clones contained 75%, 9% and 16% of simple, interrupted and compound SSRs, respectively. Of the 10 SSRs tested, 7 primer pairs produced clearly distinguishable DNA banding patterns. Successively, SSR primer pairs were successfully tested to reveal polymorphism in a set of four different grass pea germplasm accessions. The transferability of SSR markers was high among three related species of Lathyrus, namely Lathyrus cicera, Lathyrus ochrus and Lathyrus tingitanus, and the legume crop, Pisum sativum. These results indicate that the novel SSR markers are informative and will be useful and convenient for genetic analysis in grass pea and related species.     

Identification and validation of a core set of microsatellite markers for genetic diversity analysis in watermelon, <Emphasis Type="Italic">Citrullus lanatus</Emphasis> Thunb. Matsum. &; Nakai

Watermelon, Citrullus lanatus Thunb. Matsum. & Nakai is an important vegetable crop worldwide. Due to its narrow genetic base, detection and utilization of the genetic variations, cultivar identification and increasing genetic diversity are some important tasks for watermelon breeders. Molecular markers, especially microsatellites or simple sequence repeats (SSRs) are playing increasingly important roles for these purposes. In the present study, a core set of 23 highly informative SSR markers was developed for watermelon genetic diversity analysis. Based on whole genome sequencing of 17 watermelon inbred lines, we identified 3.9 million single nucleotide polymorphisms (SNPs) which were used to construct a SNP-based dendrogram for the 17 lines. Meanwhile, from the sequenced genome, 13,744 SSRs were developed, of which 704 were placed on a high-resolution watermelon linkage map. To develop the core set SSR markers, 78 of the 704 mapped SSRs were selected as the candidate markers. Using the SNP-based dendrogram as calibration, 23 SSR markers evenly distributed across the genome were identified as the core marker set for watermelon genetic diversity analysis. Each marker was able to detect 2–7 alleles with polymorphism information content values ranging from 0.45 to 0.82. The dendrograms of 17 watermelon lines based on SNPs, the base set of 78 SSRs and the core set of 23 SSRs were highly consistent. The utility of this core set SSRs was demonstrated in 100 commercial watermelon cultivars and elite lines, which could be placed into six clusters that were largely consistent with previous classification based on morphology and parentage data. This core set of SSR markers should be very useful for genotyping and genetic variation analysis in watermelon.     

Simple sequence repeats as useful resources to study transcribed genes of cotton

Microsatellites or Simple Sequence Repeats(SSRs) are informative molecular genetic markers in many crop species. SSRs are PCR-based, highly polymorphic, abundant, widely distributed throughout the genome and inherited in a co-dominant manner in most cases. Here we describe the presence of SSRs in cDNAs of cotton. Thirty one SSR primer pairs of 220 (∼14%) tested led to PCR amplification of discrete fragments using cotton leaf cDNA as template. Sequence analysis showed 25% of 24randomly selected cDNA clones amplified with different SSR primer pairs contained repeat motifs. We further showed that sequences from the SSR-containing cDNAs were conserved across G. barbadense and G. hirsutum, revealing the importance of the SSR markers for comparative mapping of transcribed genes. Data mining for plant SSR-ESTs from the publicly available databases identified SSRs motifs in many plant species,including cotton, in a range of 1.1 to4.8% of the submitted ESTs for a given species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Validation of a set of informative simple sequence repeats markers for variety identification in Pak‐choi (Brassica rapa L. ssp. chinensis var. communis)

A core set of 21 simple sequence repeats (SSR) markers was developed for Pak‐choi (Brassica rapa ssp. chinensis var. communis) variety identification. We initially selected 74 SSR markers which exhibited high polymorphism and reproducibility in SSR detection from 2129 SSRs. Using the 74 SSR‐based dendrogram for 45 inbred lines as calibration, 21 core SSRs were selected out. The utility of this core set SSRs was firstly tested in 45 inbred lines and finally verified in 102 commercial varieties. We also constructed a molecular ladder for each core SSR as a reference standard. Diversity analysis of this core SSR panel in 102 varieties demonstrated that each marker generates 2–3 alleles (averaged 2.33), with polymorphism information content values ranging from 0.01 to 0.56 (averaged 0.31). The averaged values of Shannon information index, observed heterozygosity, expected heterozygosity and Wright's fixation index were 0.59, 0.43, 0.38 and −0.09, respectively. Furthermore, the 21 SSR‐based classifications for 102 varieties were consistent with traditional classification based on morphology. This core SSR panel represents an effective tool for genetic variation analysis in Pak‐choi.     

Characterization of microsatellite markers,their transferability to orphan legumes and use in determination of genetic diversity among chickpea (Cicer arietinum L.) cultivars

The microsatellite or simple sequence repeat (SSR) marker is the most preferred marker because of its many desirable properties. It is important to increase the genic and genomic resources particularly in legumes because the SSR markers currently available in chickpea, pigeonpea, horsegram, blackgram, and cowpea are very limited. In the present study, 201 pairs of SSR markers comprising of 172 genic and 29 genomic SSRs were screened against 11 chickpea genotypes, among which 153 produced monomorphic and 48 produced polymorphic bands. The polymorphic information content ranged from 0.152 to 0.373 for both genic and genomic SSRs. Among the polymorphic markers, two-three alleles were detected for genic and two-four alleles for genomic SSRs. A unique banding pattern could be found for all the genotypes within 48 polymorphic SSR markers and cultivar specific markers could be identified for seed purity test. We have also studied the ability of chickpea genic and genomic SSRs to amplify distantly related but important legumes viz., horsegram, blackgram, cowpea, pigeonpea, and soybean. Out of 201 chickpea SSR primer pairs, 66.7% in blackgram, 62.2% in horsegram, 61.7% in redgram, 54.7% in cowpea, and 62.7% in soybean produced amplification. The transferability of about 60.0% of the chickpea SSRs to distantly related legumes could be considered successful. In the present study, 134, 133, 126, 124, and 110 new SSR primers for blackgram, horsegram, soybean, redgram, and cowpea pulse crops, respectively, were identified. It is an important addition to the already available genomic resources in these crops. In addition, among genic primer pairs, 12 in horsegram, three in soybean, 13 in redgram, and eight in cowpea, and among genomic primer pairs, two in horsegram and four in redgram were polymorphic even in the two-three genotypes tested indicating their potentially for application in genetic studies and mapping.     

黄麻全基因组SSR鉴定与特征分析

黄麻是世界上重要的天然韧皮部纤维作物之一。然而, SSR标记的缺乏限制了黄麻的遗传改良。本研究从圆果种黄麻测序品种CVL-1的基因组、基因、CDS和cDNA中挖掘SSR信息,利用SSR Primer软件查找SSR位点,并分析其分布特征。结果表明,基于基因组序列共开发了153,242个基因组SSR,平均密度为467.20个SSRMb~(–1);基于cDNA序列开发了10,747个SSR,平均密度为260.85 SSR Mb~(–1)。大部分重复基元为二至四核苷酸,占76.91%,其中cDNA序列SSR中三核苷酸重复基元数量较多而基因组SSR中二核苷酸重复基元数量较多。对于不同类型的SSR重复基元,随着重复单元数量的增加,其基因组和cDNA的SSR分布频率呈现逐步降低特征。黄麻全基因组SSR标记鉴定,不仅可以丰富黄麻分子标记的数量,而且为剖析黄麻重要农艺性状的遗传机制奠定基础。