甜瓜(Cucumis melo L.)线粒体基因组标记的开发及应用

简单重复序列(SSR)是具有极大的信息含量且被广泛应用的一类分子标记。甜瓜线粒体基因组的公布为SSR标记的开发提供了契机。基于甜瓜线粒体基因组富含SSR位点及父系遗传特性,开发并研究线粒体基因组SSR标记在甜瓜育种及种质资源调查等方面的应用。在甜瓜线粒体基因组上共发现808个SSR位点,并对328个位点进行了标记的开发。使用11份甜瓜变种进行PCR筛选及分析后,获得了由89对高多态性引物组成的核心标记,成功应用于甜瓜品种‘夏蜜’的种子纯度鉴定。并基于线粒体SSR标记多样性构建了80份甜瓜材料的UPGMA聚类分析,结果显示粗网纹厚皮甜瓜类型形成一个独立的分支,其他分支具有明显的地域分布特色。本研究结果表明,同一地域不同亚类甜瓜早期可能存在广泛的杂交,人类的育种过程导致了甜瓜不同亚种资源的混杂,这将为后续甜瓜远缘杂交亲本的选择提供重要参考。     

利用紧密连锁抗白粉病基因的SSR标记鉴定甜瓜种质

在本研究中,利用12对紧密连锁甜瓜抗白粉病基因的SSR分子标记,对18份甜瓜品种进行遗传背景及亲缘关系分析,提出一种快捷简便的鉴别甜瓜抗病品种亲缘关系的方法。结果表明,遗传相似性系数较高且遗传背景相近的抗白粉病甜瓜品种聚为一类,故推测其携带相同抗白粉病基因。本研究使用的12对SSR标记,可用于甜瓜抗白粉病品种鉴定,能够完全区分18个甜瓜品种对白粉病的抗感性,为甜瓜种质资源评价、鉴定及新品种培育工作提供帮助。     

甜瓜栽培品种的遗传多样性研究

为了通过分子水平了解甜瓜品种的遗传多样性,利用36个SSR标记对浙江省种植的甜瓜品种进行遗传多样性研究。结果表明,33个具有多态性SSR标记共产生97个等位基因,每个标记平均产生2.9个等位基因。多态性信息含量(PIC)变化范围为0.029～0.746,平均值为0.412。聚类分析表明,33份材料可明显分成两大类,分别对应于厚皮甜瓜和薄皮甜瓜类型。调查了不同甜瓜品种的果实性状和品质性状,发现有些性状如果形、果重、香气、质地等在厚皮甜瓜和薄皮甜瓜间存在差异。研究结果表明,厚皮甜瓜品种遗传多样性较丰富,但需要利用更多的分子标记才能有效地鉴别不同薄皮甜瓜品种。本研究为高效合理利用品种资源提供了有用信息,为甜瓜育种提供了物质基础。     

甜瓜果肉颜色遗传分析及相关分子标记的开发

甜瓜果肉颜色是影响消费者选择的重要果实品质性状之一。为了研究甜瓜果肉颜色性状的遗传规律,以‘库克拜热’(绿肉)与‘西州蜜’(橙肉)构建BC1群体,‘库克拜热’与‘皇后’(橙肉)构建F2群体,利用集群分离分析(bulked segregation analysis, BSA)与简单重复序列(simple sequence repeats, SSR)分子标记相结合的方法,明确了在上述甜瓜品种中,果肉颜色受两个基因的调控,其中橙色果肉控制基因gf对白色果肉控制基因wf具有显性上位作用,绿色对白色为隐性。本研究同时开发出相应的多态性分子标记,其中分子标记SSR2021、SSR2103与gf紧密连锁,分子标记SSR2956、SSR2980与wf紧密连锁,可用于区分橙色、白色和绿色果肉甜瓜。本研究为不同果肉颜色甜瓜的选育提供一定的理论基础和技术支持。     

甜瓜SSR标记在葫芦科不同作物间的通用性分析

微卫星标记(SSR)在近缘物种间具有通用性,因其高效率、低成本可广泛用于跨物种间的遗传多样性分析、基因定位和图谱构建等。本试验基于甜瓜基因组测序结果开发了120对SSR标记,研究其在8种葫芦科作物间的通用性及作物内的多态性,并利用NTSYS软件分析37份不同瓜类作物材料间的遗传多样性。结果显示,114对标记均在甜瓜基因组中得到有效扩增,黄瓜中有108对标记有效扩增,通用性比率为94.7%,南瓜中能有效扩增标记最少,其通用性比率也达到了41.2%,在8种葫芦科作物间均通用的标记有29对;通过对通用性标记的多态性分析表明,甜瓜多态性比率最高为61.4%,西瓜、黄瓜、苦瓜、冬瓜、西葫芦、南瓜和丝瓜多态性比率分别为32.5%、14%、4.4%、8.8%、3.5%、13.2%和11.4%;不同作物间的平均遗传相似系数在0.70~0.83之间,其中甜瓜和黄瓜平均遗传相似系数较小,亲缘关系较近,西葫芦和南瓜间的平均遗传相似系数最小,亲缘关系最近。这些通用性标记将有利于提高葫芦科作物整体分子生物学研究水平。     

与甜瓜嫁接亲和性连锁分子标记鉴定

使用与南瓜类型砧木(“壮士”)嫁接亲和性差异明显的两个甜瓜自交系(85-1和B717)为亲本,配制F2∶3家系群体;利用BSA法构建亲和性优差池,结合PCR技术筛选与嫁接亲和性有关的分子标记.结果表明:使用葫芦科遗传连锁图谱上发表的750对SSR、SRAP和AFLP等分子标记引物扩增,最终筛选出在两个甜瓜材料之间具有多态性的382对引物,其中9对引物分别在嫁接亲和性优差DNA池中扩增出差异性片段.将这些标记用于F2群体的扩增,计算标记与嫁接亲和性遗传连锁距离,两对SSR引物CMN-0616和M103与嫁接亲和性的遗传距离分别为17.6 cm和9.3 cm.这2对引物有望成为鉴定甜瓜嫁接亲和性的辅助引物,从而将嫁接亲和性差异材料从分子角度区别开来,为甜瓜嫁接接穗育种等研究提供参考.     

甜瓜PI414723抗霜霉病基因SSR分子标记

为了研究野生甜瓜PI414723抗性遗传规律,找到与抗病基因连锁的分子标记,并对抗病基因进行定位。以野生甜瓜PI414723和新疆哈密瓜农家品种卡拉克赛为材料构建BCr、BCs和F2,苗期接种甜瓜霜霉病进行抗性鉴定、统计分析,基于ICu GI已构建遗传连锁图谱,应用集团分离法和1 090对甜瓜SSR引物进行连锁遗传分析。结果表明:PI414723对霜霉病的抗性由隐性单基因控制,引物DM0231扩增出的多态性条带与抗病基因表现连锁关系,该多态性片段大小为226 bp,遗传连锁距离为2.67 c M,并将抗病基因定位在LG9上。DM0231可作为甜瓜抗霜霉病分子育种的分子标记,也为进一步对抗霜霉病基因精细定位奠定了基础。     

甜瓜抗霜霉病基因SSR分子标记

为了研究甜瓜抗霜霉病资源T115抗性遗传规律,找到与抗病基因连锁的分子标记,并对抗病基因进行定位。以抗病资源T115和感病哈密瓜农家品种SP红心脆、F1、BCr、BCs、F2为材料,苗期接种甜瓜霜霉病进行抗性鉴定,基于ICu GI已构建遗传连锁图谱,应用集团分离法和1 090对甜瓜SSR引物进行连锁遗传分析。结果表明,T115对霜霉病的抗性属显性单基因控制,引物DM0073扩增出的特异性片段与抗病基因表现连锁关系,该片段大小为120 bp,与抗病基因遗传连锁距离为3.6 c M,并将抗病基因定位在LG1上。DM0073可作为甜瓜抗霜霉病分子育种的分子标记。     

SSR标记应用于烟草品种遗传多样性研究

SSR标记是进行植物遗传多样性研究的理想技术。本研究应用最近公布的烟草SSR标记分析了13个云南省烟草主栽品种的遗传多样性。对92对分布于烟草24个连锁群的SSR标记引物筛选发现20对在这些品种之间存在多态性。20个SSR位点上共检测到52个等位基因,平均每对引物等位基因数为2.6个。根据SSR标记多态性计算了不同品种之间的遗传距离（GD）,13个品种之间遗传距离介于0.0090-0.4286之间,平均距离为0.2237,说明品种间遗传差异较小。SSR标记技术将在烟草的基因标记定位及分子辅助育种中发挥重要的作用。     

SSR标记在中国花生研究中的应用

SSR标记是广泛分布于多种生物基因组中由2~6个核苷酸组成的多次重复的一段DNA序列。根据近几年来SSR标记在中国花生上的研究进行了综述,包括SSR标记在花生种质遗传多样性研究、分子标记辅助选择育种等领域的广泛应用情况。通过对花生SSR标记相关研究的分析得知,中国花生育成品种普遍存在遗传基础狭窄的问题,认为在未来研究过程中应设计和开发大量多态性好的SSR分子标记,将标记研究工作与实际育种相结合,从而有效指导育种,提高育种效率。     

Stability of seed oil quality traits in high and mid-oleic acid sunflower hybrids

Powdery mildew caused by Podosphaera xanthii is an important disease of melon, and race 2F is the predominant race in most areas of China. Resistance to P. xanthii race 2F in melon K7-1 was controlled by a dominant gene, designated Pm-2F, in a 106-member population of recombinant inbred lines derived from K7-1× susceptible K7-2. Using bulked segregant analysis with molecular markers, we have identified two polymorphic simple sequence repeats (SSR) to determine that Pm-2F is located on linkage group II. Comparative genomic analyses using mapped SSR markers and the cucumber genome sequence showed that the melon chromosomal region carrying Pm-2F is homologous to a 288,223 bp genomic region on cucumber chromosome (chr) 1. The SSR markers on chr 1 of cucumber, SSR02734, SSR02733 and CS27 were found linked with Pm-2F. Comparative mapping showed that two SSR markers (SSR02734 and CMBR8) flanked the Pm-2F locus and two nucleotide binding site-leucine-rich repeat resistance genes were identified in the collinear region of cucumber. A cleaved amplified polymorphic sequence (CAPS) marker was developed from the sequence of resistance genes and it delimits the genomic region carrying Pm-2F to 0.8 cM. The evaluation of 165 melon accessions and 13 race differential lines showed that the newly developed CAPS (CAPS-Dde I) marker can be used as a universal marker for effective marker assisted selection in melon powdery mildew resistance breeding. The putative resistance gene cluster provides a potential target site for further fine mapping and cloning of Pm-2F.     

Genetic diversity and population structure of Indian melon (Cucumis melo L.) landraces with special reference to disease and insect resistance loci

This study was aimed to examine the genetic diversity and population structure of Indian melon landraces with special reference to disease and insect resistance loci. Thirty‐six simple sequence repeat (SSR) markers along with seven markers at disease and insect resistance loci were used for this purpose on a panel of 91 accessions available at Indian Institute of Horticultural Research, Bengaluru, India. Model‐based structure analysis revealed the presence of four groups that were consistent with the results of principal coordinate analysis (PCoA). The delineation of populations was mostly based on geography with improved varieties as a separate group. Ten accessions have been identified to possess beneficial alleles at all the selected disease resistance loci and shall be useful for incorporating multiple disease resistance after phenotypic validation. The results obtained in the current study demonstrate the importance of the Indian melon group as a valuable genetic reservoir and the need to plan strategies for its conservation and utilization in breeding programmes.     

Genic microsatellite markers for discrimination of spinach cultivars

A set of simple sequence repeat (SSR or microsatellite) markers was used to discriminate a collection of 33 Spinacia oleracea hybrid cultivars from seven different breeding stations all over the world. All SSR markers were genic microsatellite markers located in coding or non-coding regions of genes of known function. Cluster analysis based on 13 of the SSR markers showed that the spinach hybrids grouped into three clusters. The first two clusters consisted of European spinach types, which were well discriminated according to their origin from different breeding stations. The third cluster was a mixture of Asian as well as European types of spinach. Subclusters in this group did not reflect differences in morphology, earliness or company origin. The data show that genic microsatellites are a powerful tool for discrimination of spinach cultivars.     

Genetic variability in melon based on microsatellite variation

A set of 18 simple‐sequence repeat (SSR or microsatellite) markers was used to study genetic diversity in a collection of 27 melon (Cucumis melo L.) accessions, representing a broad range of wild and cultivated melons. The materials studied were highly polymorphic for SSRs and a total of 114 alleles were detected (average of 6.3 alleles per locus). Cluster analysis suggests the division of these accessions into two major groups, largely corresponding to the division of C. melo in the two subspecies agrestis and melo. The assignment of the accession to the subspecies was generally in agreement with published reports, except for those corresponding to the ‘dudaim’ and ‘chito’ cultivar groups, which, according to the observed SSR variability, should be included in subspecies agrestis. Based on cluster analysis, five groups of accessions were defined. The two most divergent groups include mainly accessions from the Mediterranean which form one group, and accessions from China, Japan, Korea and India forming the other. Both groups shared a low level of intra‐accession variation compared with the other groups, which suggests an erosion of their genetic variability because of drift and/or inbreeding. The remaining accessions, mainly from Central Africa and India, were more variable and may be an important source of genetic variation for melon breeding.     

Association mapping of yield-related traits and SSR markers in wild soybean (Glycine soja Sieb. and Zucc.)

Wild soybean, the progenitor of cultivated soybean, is an important gene pool for ongoing soybean breeding efforts. To identify yield-enhancing quantitative trait locus (QTL) or gene from wild soybean, 113 wild soybeans accessions were phenotyped for five yield-related traits and genotyped with 85 simple sequence repeat (SSR) markers to conduct association mapping. A total of 892 alleles were detected for the 85 SSR markers, with an average 10.49 alleles; the corresponding PIC values ranged from 0.07 to 0.92, with an average 0.73. The genetic diversity of each SSR marker ranged from 0.07 to 0.93, with an average 0.75. A total of 18 SSR markers were identified for the five traits. Two SSR markers, sct_010 and satt316, which are associated with the yield per plant were stably expressed over two years at two experimental locations. Our results suggested that association mapping can be an effective approach for identifying QTL from wild soybean.     

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.     

Development of EST-SSR markers of Ipomoea nil

Although Japanese morning glory (Ipomoea nil (L.) Roth.) has been used intensively for genetic studies, DNA markers have not been developed in Ipomoea nil sufficient to cover all chromosomes. Therefore, we conducted microsatellite (simple sequence repeats, SSR) marker development in I. nil for future genetic studies. From 92,662 expressed sequence tag (EST) sequences, 514 unique microsatellite-containing ESTs were identified. Primer pairs were designed automatically in 326 SSRs. Of 150 SSRs examined, 75 showed polymorphisms among strains. A phenogram based on the SSR genotypes revealed the genetic relation among seven Japanese morning glories from five different regions of the world and an ivyleaf morning glory (I. hederacea Jacq.). The developed SSR markers might be applicable for genetic studies of morning glories and their relatives.     

Analysis of Genetic Polymorphic SSR Markers in Germplasm Resources of the Natural Colored Cotton

Short sequence repeats (microsatellite,SSR) and expressed sequence tags-SSR (EST-SSR) markers were employed to analyze the genetic diversity of natural colored cotton varieties.About 490 pairs of SSR markers spanning the 26 chromosomes were selected from the cotton microsatellite database,they were composed of the NAU,BNL,MUSS,and CIR markers,and there was one marker every 5 cM on average.     

Genetic diversity in soybean genotypes from north‐eastern China and identification of candidate markers associated with maturity rating

Random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to estimate the genetic relationships among 101 soybean cultivars developed in north‐eastern China. Fifty‐three fragments of the 100 RAPD markers and 35 SSR markers tested were polymorphic across the 101 soybean cultivars. Similarity values among these soybean cultivars ranged from 45.2% to 100% for RAPD data, and ranged from 36.1% to 100% for SSR data. The similarity matrices for SSR data and RAPD data were moderately correlated (r = 0.31, P < 0.05). Cluster analyses indicated that the cultivars released from the same seed company were mostly grouped together. A principal component analysis, based on the combined RAPD and SSR data, yielded a good separation of soybean varieties with different maturity ratings [represented by soybean Heat Unit (HU)]. The varieties with HU < 2200 were well separated from those with HU > 2200. Four RAPD markers and eight SSR markers were significantly associated with the maturity ratings of soybean.