芝麻EST-SSR标记的开发和初步研究

为了加速分子标记在芝麻研究中的应用,利用网上现有的芝麻EST(expressed sequence tags)数据信息,开展了芝麻EST-SSR功能性标记的开发和利用研究。 在所有的3 328条芝麻EST序列中共确认得到1 785条非冗余EST序列。其中,在含有微卫星重复的148条序列中共检测有155个EST-SSR。非冗余EST序列总长为774.266 kb,平均每4.99 kb含有一个EST-SSR。EST-SSR的分布频率和特征分析表明,以AG/TC为重复基元(motif)的SSR出现最多,占总SSR的37.42%。利用这些序列,设计开发了50对EST-SSR引物,并分别选用36个芝麻、2个棉花、2个大豆和2个油葵进行多态性和通用性研究。其中44对引物在供试芝麻材料中扩增出条带,共产生108个位点,平均每对引物产生2.45个位点,多态信息含量(polymorphism information content, PIC)平均值为0.390。根据遗传相似性系数进行聚类,有26个芝麻材料聚类在两个大的亚类(III和IV)中,聚类结果表明芝麻的基因型与地理来源之间没有必然的联系。此外,分别有2对、3对和4对引物可以在棉花、大豆和油葵中进行通用性扩增。本研究证实这种全新开发的芝麻EST-SSR标记在芝麻遗传多样性分析、遗传图谱构建以及比较基因组等研究方面有广阔的利用前景。     

苦荞全基因组SSR位点特征分析与分子标记开发

目前苦荞SSR多态性标记数量较少,根据已发表的苦荞基因组测序数据,利用MISA软件对1~6核苷酸重复的SSR位点进行了查找和序列特征分析,批量设计引物并对引物进行了有效性和多态性检测。结果表明,苦荞基因组中共检测到1 640个SSR位点,其中三核苷酸重复型SSR最多,占比63.29%,五核苷酸重复型最少,仅占0.12%。AT/TA、AAG/CTT、ACC/GGT和ATC/GAT为出现频率较高的重复基序。苦荞基因组SSR序列长度变化范围为12~476bp,平均长度23.14bp,长度12~19bp的占比71.71%,长度≥20bp的占比28.29%。根据不同类型SSR位点设计并合成引物479对,选择200对引物对5份苦荞资源和3份甜荞资源进行多态性检测,有56对扩增出多态性条带,17对在苦荞种质中产生多态性条带,48对在甜荞种质中产生多态性条带,9对同时在两种种质中产生多态性条带。利用苦荞全基因组序列可实现SSR标记的批量开发,可鉴定出适用于苦荞和甜荞遗传多样性分析、遗传图谱构建和品种鉴定等研究的SSR引物。     

Study of genetic diversity in Indian and exotic sesame (Sesamum indicum L.) germplasm using random amplified polymorphic DNA (RAPD) markers

Fifty-eight accessions of sesame (Sesamum indicum L.), an important oil seed crop of the tropics and subtropics were analysed using random amplified polymorphic DNA (RAPD) technique. The material analysed comprised 36 collections from 18 different states of India and four adjoining countries of the Indian subcontinent, and 22 exotic accessions from 21 sesame growing countries around the world. The results from PCR amplifications with the selected 24 random 10-mer primers were statistically analysed. The value of Jaccard’s similarity coefficients ranged from 0.19 to 0.89. The results indicated the presence of high level of genetic diversity. However, the extent of genetic diversity was greater in the collections from Indian subcontinent as compared to the exotics. Among the Indian accessions, the collections from Rajasthan and North-eastern states were highly diverse. The phenetic analysis grouped 48 out of 58 accessions in six clusters and the remaining highly diverse accessions were placed outside these close-knit clusters. The Bootstrap estimates obtained by Wagner parsimony analysis were significant for seven out of 49 nodes in the majority-rule consensus tree (<95% occurrence). The results of both the analyses were, however, broadly comparable when the constitution of the individual clusters were considered. The principal components analysis indicated that the first two components accounted for only 21% of the total variations and in order to explain <75% of variations 18 components were required. The high level of genetic diversity prevalent among the Indian collections is probably indicative of the nativity of this crop species. Similarly, the relatively lower level of polymorphism in exotic germplasm could be ascribed to the comparatively recent introductions of limited germplasm of this crop into some of the non-traditional sesame growing countries. This revised version was published online in August 2006 with corrections to the Cover Date.     

应用SRAP标记分析黑芝麻核心种质遗传多样性

利用SRAP分子标记技术对中国芝麻资源核心收集品中的黑芝麻种质进行遗传多样性分析。结果表明,13对引物组合对100份黑芝麻核心种质共扩增出稳定清晰的条带182条,其中多态性条带126条,占69.2%,每对引物组合的条带数和多态性带数分别为14.0个和9.7个;供试材料间成对遗传相似系数介于0.469~0.986,平均为0.726,通过UPGMA法,在遗传相似系数为0.68处可将供试材料聚为5个类群,表明黑芝麻核心种质具有较丰富的遗传多样性,聚类结果与地理分布没有明显的关系;遗传多样性指数是南方黑芝麻核心种质(0.3557)＞中部种质(0.3415)＞北方种质(0.2986)。本研究结果较全面反映了中国保存的黑芝麻种质资源遗传多样性特点,为我国黑芝麻资源进一步考察收集和引进,以及优异黑芝麻基因资源挖掘和育种利用提供了理论依据。     

Genetic diversity in cultivated sesame (Sesamum indicum L.) and related wild species in East Africa

Genetic diversity of traditional sesame landraces and related wild species in East Africa remains largely unexplored. Knowing what fraction of the available genetic diversity is actually used by the farmers is of central importance for understanding how cultivation shapes the genetic structure of a crop and for the management of biodiversity preservation. Genetic diversity in cultivated sesame and related wild species in East Africa was determined using inter-simple sequence repeats (ISSR). Six reliable ISSR primers generated 51 amplification fragments of which 36 (70.6%) were polymorphic. The number of amplified fragments ranged from 7 to 12 with a mean of 8.5 fragments per primer. The overall gene diversity and Shannon’s index were 0.28 and 0.34, Jaccard’s similarity coefficient ranged from 0.26 to 0.96, with an average of 0.67. Forty-six accessions of sesame were divided into six clusters, although the clustering did not indicate any clear division among sesame accessions based on their geographical locations. Each wild species was more distant from cultivated sesame than from other wild species, indicating that no cross-pollination with these wild species occurred during sesame domestication. These results showed a relatively high genetic diversity in sesame and related wild species. Indian-1 and Indian-2 accessions showed a good amount of genetic divergence. The genetic diversity data uncovered in this study can be exploited to improve traditional landraces of sesame in East Africa.     

Development of EST‐SSR markers for diversity and breeding studies in opium poppy

All publicly available opium poppy expressed sequence tag (EST) sequences, totalling 20 885, were assembled into unigenes and examined for simple sequence repeats (SSRs). Nearly 19% of the 14 957 unigenes contained SSRs with 4% harbouring more than one SSR. Average density of the SSRs was 1 SSR per 3.6 kb of non‐redundant EST sequence. Trinucleotide SSRs were most frequently identified (39%), and many of the most prevalent motifs were AT‐rich. Flanking primers were designed for 86% of the SSRs and 67 primer pairs were tested on 37 opium poppy accessions and seven related species. All markers were transferable to the related species. Polymorphism information content (PIC) values for the markers were intermediate for comparisons within opium poppy (average of 0.27) and slightly higher for comparisons across species (average of 0.29). The markers were found to be useful for diversity analysis as they successfully distinguished among Turkish opium poppy accessions and land races.     

草棉EST-SSRs的遗传评价

根据GenBank中公布的247条草棉EST序列,搜索SSR并进行引物设计。其中的25条序列含有27个SSR,1~6碱基重复类型都存在,二碱基和三碱基重复的频率较高。为了明确在A、D和AD基因组中的可转移性,依据25条序列共设计25对EST-SSR引物,其中22对引物扩增出清晰可辨的DNA条带,产生92个多态性片段,平均每对引物产生3.64个多态性片段。引物的多态性信息含量(PIC)在0.49~0.91之间,平均为0.81。6对引物在BC₁种间作图群体[(鄂棉22 × Pima3-79) ×鄂棉22]中表现多态性,产生7个多态性位点,其中5个为共显性,2个为显性。除HAU230b标记在BC₁分离群体中不符合孟德尔式分离比例,其余引物表现正常分离。6个位点被整合到陆地棉和海岛棉种间BC₁遗传连锁图谱上的6条染色体：有4个位于A亚基因组的4条染色体上(Chr.6、10、11和12),2个位于D亚基因组的2条染色体(Chr.19和20)。     

Classification of a collection of sesame germplasm using multivariate analysis

Sesame (Sesamum indicum L.) is an important edible oil crop. Meteorological factors such as temperature, rainfall, and the amount of solar radiation determine the yield potential of sesame. To identify phenotypic diversity and to infer genotypic backgrounds in a collection of 250 sesame germplasm accessions, we classified the germplasm based on variation in morphological traits using principal component (PC) and cluster analysis. The sesame germplasm was grouped based on five PCs, which accounted for 82.3% of total variation. The first PC (PC1) was positively correlated with days to flowering, days to maturity, and number of capsules per plant, whereas the second PC (PC2) was negatively correlated with all characteristics except capsule-bearing stem length. The third component (PC3) was highly positively correlated with capsule length and plant height. We constructed a scatter diagram of the first two PCs (PC1 vs. PC2), revealing four distinct groups of eigenvectors. Most sesame germplasm was widely distributed among Groups I, II, III, and IV. Group III showed a wide range of distribution in the diagram. Otherwise, the distribution of the 250 germplasm accessions was more compact in a scatter diagram of PC1 vs. PC3 compared with PC1 vs. PC2. Groups I, II, III, and IV contained 142, 102, 2, and 3 sesame germplasm accessions, respectively. The two germplasm in Group III were collected from different regions, as were the three germplasm in Group IV. The results show that the distribution of sesame origin is wider than the regions examined in view of phenotypic diversity.     

Genetic relationships of sesame germplasm collection as revealed by inter-simple sequence repeats

Inter‐simple sequence repeats (ISSR) polymorphism was used to determine genetic relationships among 75 Sesamum indicum L. accessions of Korean and exotic sesame. Fourteen reliable ISSR primers were selected for the assessment of genetic diversity, yielding 79 amplification products. Of these polymerase chain reaction products, 33% revealed polymorphism among the 75 accessions. Genetic distances ranged from 0 to 0.255, with a mean genetic distance of 0.0687. The 75 accessions were divided into seven groups on the basis of unweighted pair‐group method with arithmetic averages (UPGMA) cluster analysis. The largest group consisted of 25 Korean cultivars, eight Korean breeding lines and 17 world‐wide accessions. The other groups included 25 accessions, several of which contained useful traits. The dendrogram did not indicate any clear division among sesame accessions based on their geographical origin. However, all Korean sesame cultivars except ‘Namsankkae’ were clustered in the same group, indicating a narrow gene pool. Some of the Korean breeding lines were spread along the dendrogram, showing enlargement of genetic diversity. The genetic diversity data uncovered in this study can be used in future breeding programmes.     

Assessment of genetic diversity amongst Ugandan sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) landraces based on agromorphological traits and genetic markers

Sesame (Sesamum indicum L.) is one of the most important ancient oilseed crops grown throughout the tropical and subtropical regions of the world. In Uganda, most of the cultivated sesame varieties are local landraces which are frequently traded between farmers. Although these traditional landraces are an important source of genetic diversity, knowledge of their genetic diversity is still limited.Agromorphological traits and a set of published and newly developed microsatellite markers were analyzed on a collection of 121 accessions of Ugandan sesame landraces. CpSSR analysis revealed four haplotypes, whereby haplotype B was present in 96% of the individuals. The analysis of nSSR markers from 6 non-coding regions revealed a mean PIC value of 0.56 whereas the PIC value of eight selected EST-derived SSRs was 0.26. Accession-wise, the expected heterozygosity (He) varied from 0 to 0.396. AMOVA revealed that the majority of the variance occurred among the individuals accounting for 75% of the total variation, only 6% was attributed to differences among the districts, pointing towards a high gene flow (Nm = 4.476). These results are supported by the PCoA analysis as well as the NJ tree both of which revealed no clustering of the accessions according to their geographic origin. Also the statistical analysis of 10 agromorphological traits indicated no clear pattern related to the geographic origin. Such a poor grouping, indicative of considerable gene flow across geographic domains, could be explained either by a high outcrossing rate, and/or through extensive seed trading.     

Genetic Diversity of Farmers’Preferred Sorghum Accessions and Improved Lines from ICRISAT Reveal a Disconnect Between Innovation and Technology Transfer

The genetic diversity of 65 accessions of sorghum [Sorghum bicolor (L.) Moench] collected from various farmers and germplasm lines from ICRISAT-Kenya were analyzed. Simple sequence repeats (SSR) markers were used in order to determine the extent and distribution of its genetic diversity. Twenty-nine (29) SSRs markers were polymorphic and a total of 192 alleles were detected which showed diversity. The number of alleles per primer ranged from 2 to 17, with an average of 6.62. The range of polymorphism information content (PIC) ranged from 0.03 to 0.86, with total average of 0.82. According to the results analyzed, estimates of the mean allelic pattern across the two populations was generated; expected heterozygosity (He; 0.45, 0.54), average observed alleles (Na; 3.40, 6.20), number of private allele (0.23, 3.03), and Shannon information index (I; 0.85, 1.13) for farmer and ICRISAT-Kenya germplasm, respectively. The expected heterozygosity (He) varied from 0 to 0.26 with an average of 0.05. The Neighbor-joining phenogram based on Nei’s genetic distance grouped the 65 accessions into three main groups. The analysis of molecular variance (AMOVA) revealed that 99% of the total genetic variation was within accessions in a population whereas the genetic variation among populations in accessions accounted for 1% of the total genetic variation. Genetic diversity in ICRISAT sorghum material compared to the farmer’s collection suggested little infiltration of improved germplasm to the farmers.     

红麻EST-SSR标记的开发及其多态性评价

红麻是最重要的自然纤维作物之一,然而SSR标记的匮乏限制了其遗传改良。本研究从红麻90 175个EST序列中挑出含有转录因子的EST,开发了94对SSR引物。以24份不同红麻种质资源的DNA为模板,利用9%非变性聚丙烯酰胺凝胶电泳检测多态性。结果表明,85对引物(占90.4%)至少在2个材料之间存在多态性,表明开发的EST-SSR具有很好的多态性。其中,三核苷酸重复所占比例最多,重复基元AAT和ATG的多态性较高。聚类分析表明,24份红麻种质资源的遗传相似系数变化在0.62~0.92之间,表现出丰富的遗传基础。这些结果不仅丰富了红麻的分子标记数量,而且为红麻的遗传分析提供资源。     

The Híbrido de Timor germplasm: identification of molecular diversity and resistance sources to coffee berry disease and leaf rust

The main phytosanitary problems affecting global coffee production are the fungal diseases known as rust, caused by Hemileia vastatrix Berkeley and Broome, and coffee berry disease (CBD), induced by Colletotrichum kahawae Waller and Bridge. The main disease control strategy is the use of resistant coffee cultivars. Híbrido de Timor is the most important source of resistant varieties used in breeding programs worldwide. The objective of this work was to characterize the diversity and disease resistance of 152 HdT genotypes from the germplasm collection at the Universidade Federal de Viçosa (UFV). Accessions were phenotyped with H. vastatrix races II and XXXIII. Molecular analysis was carried out with 29 random microsatellite markers or single sequence repeats (SSRs), and two SSRs associated with the CBD resistance gene Ck-1. All accessions in the germplasm collection were resistant to H. vastatrix race II, and 141 were resistant to H. vastatrix race XXXIII. Based on the presence of markers, there were 106 accessions containing the CBD resistance gene Ck-1. In the diversity study, the 152 accessions clustered into 21 different groups. A unique molecular profile (fingerprint) was determined for each individual, using 52 alleles from 22 SSR markers. The HdT germplasm of UFV was highly diverse, and included 99 accessions with multiple disease resistance genes, including the CBD resistance gene Ck-1, and others conferring resistance to H. vastatrix races II and XXXIII.     

Genetic variations of isozymes in cultivated sesame (Sesamum indicum L.)

Patterns of variation for seven enzyme systems were studied in 68 accessions of cultivated sesame (Sesamum indicum L.), 12 from Japan, 15 from Korea and 41 from Thailand. Only one enzyme system, isocitrate dehydrogenase (IDH), of these exhibited variation. The IDH isozymes were shown to be controlled by a single locus (Idh) with two alleles. The two alleles were widely distributed in the accessions from the three countries. As few gene markers which have simple genetic control are available in sesame, these IDH isozymes could contribute to a range of studies in the breeding and genetics of sesame. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spanish spelt: a separate gene pool within the spelt germplasm

The renewed interest in spelt (Triticum spelta L.) for wheat improvement programmes requires the study of the available genetic diversity. The purpose of this study was to assess genetic diversity within a Spanish spelt collection. Sixty‐six Spanish spelt accessions, 19 accessions of T. spelta and T. macha from different origins, three bread wheat cultivars (T. aestivum) and one accession of T. dicoccum were screened using simple sequence repeats (SSRs). The diversity observed within the Spanish group was comparable with that observed in the other wheat varieties, despite their broader geographical diversity. Indeed, the highest polymorphic information content value calculated with SSRs for Spanish material (0.90) is similar to that observed for the other wheat varieties (0.98). Principal component analysis explained 46.5% of the cumulative variation and confirmed the Spanish accessions as a separate group. This study showed the Spanish spelt collection to be a variable and unique genetic resource for wheat and spelt breeding programmes.     

Identification of SSR markers which could differentiate blast disease resistance accessions in finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

Microsatellites also known as SSR are the class of repetitive DNA sequences present throughout the genome of all eukaryotic organisms. The present study identified SSRs using biotinylated beads capture method. Ten sets of primers were designed based on sequences having at least (CT)10 repeats. A total of 27 accessions having a mix of both African (resistant) and Indian origin (resistant and susceptible) were assessed using 30 microsatellite markers. Amplification products were obtained for all 30 primers studied; 25 out of these primers were found to be polymorphic with 13 primers showing two alleles per locus. The current study identified markers which could differentiate between resistant and susceptible accessions and also segregate accessions based on geographical region. These informative SSR markers can be used in finger millet genetic improvement projects.     

Genetic diversity in groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) genotypes and detection of marker trait associations for plant habit and seed size using genomic and genic SSRs

Groundnut (Arachis hypogaea L.) an important oilseed crop in India is known to have narrow genetic base. Therefore, the assessment of genetic diversity and detection of marker-trait association are important objectives for the genetic improvement of groundnut. The present study involved the development of 192 SSR markers from Arachis genomic survey sequences. From these, seven polymorphic SSRs along with 15 other genomic SSRs, 19 genic SSRs, and three STS markers were used to detect genetic diversity among 44 groundnut genotypes. These polymorphic SSR markers amplified 155 bands (76 genomic and 79 genic), of these 128 bands (67 genomic and 61 genic) were polymorphic. The genomic SSR exhibited 88.1% and genic SSRs displayed 77.2% allelic polymorphism. The polymorphic information content (PIC) of the markers ranged from 0.04 to 0.95. The pair-wise genetic similarity ranged from 24.2 to 90.7% for genomic SSR and 32.9 to 97.9% for genic SSR markers. Cluster analysis based on the pooled data from both genomic and genic SSRs revealed a dendrogram which could distinguish all the genotypes. Further, the AMOVA analysis detected 16.7% genetic variation due to differences in seed size and 13.0% due to plant habit. Based on locus-by-locus AMOVA and Kruskal-Wallis ANOVA and further confirmation by discriminant analysis and general linear model, six markers were found to be associated with plant habit and four markers with seed size.     

Developing and validating microsatellite markers in elephant grass (<Emphasis Type="Italic">Pennisetum purpureum</Emphasis> S.)

Elephant grass [Pennisetum purpureum S.; syn. Cenchrus purpureus (Schumach.) Morrone] is an important global forage crop and is recognized for high yields of herbage with good nutritive value. It also has high biomass potential to be utilized as a biofuel feedstock. Whereas several previous genetic studies adapted simple sequence repeat (SSR) markers from pearl millet [Pennisetum glaucum (L.) R.Br.] for investigations in elephant grass, the present study developed SSR markers from 3536 DNA sequences derived from 16 elephant grass entries. A total of 3866 SSRs were identified including 1028 monomeric, 2019 dimeric, 735 trimeric, 49 tetrameric, 20 pentameric and 15 hexameric repeat motifs. Three hundred and seven sequences contained more than one repeated motif, and 154 SSRs were present in compound formation. Susequenctly,  four elephant grass and two pearl millet genotypes were chosen to validate 727 SSR markers. Of these, 628 markers produced visually detectable amplification products, including 73 (11.6%) polymorphic ones across all six genotypes. Polymorphism between the four elephant grass genotypes was revealed by 316 (50.6%) markers with diversity index values ranging from 0.75 to 0.38. Dimeric SSRs had the highest polymorphic rate (48.7%). These validated SSR markers had 58.6% (368 of 628) transferability rate to pearl millet. The availability of these polymorphic SSR markers will support advanced genetic studies in P. purpureum and its relatives.     

首批海岛棉基因组来源的微卫星标记的分离、评价和定位

海岛棉(Gossypium barbadense)是世界上最重要的栽培棉种之一。海岛棉纤维品质优良,是优质棉的重要产源。为了研究海岛棉的遗传多样性,为海岛棉育种提供参考依据,从海岛棉遗传标准系中分离基因组来源的微卫星标记用于海岛棉遗传评价。采用两种方法分离微卫星标记,一是用ISSR (inter simple sequence repeat) 引物扩增Pima3-79,克隆测序后从中开发微卫星标记;二是利用简并引物扩增Pima3-79,克隆测序后从中开发微卫星标记。共挑选1 447个克隆,筛选出239个独立克隆。测序后得到214个单一序列,其中包含微卫星并可用于引物设计的序列70个,获得86对引物。86对引物用于扩增56个海岛棉材料和4个陆地棉材料,16对引物没有扩增,43对引物在所有材料中没有多态性;27对引物在海岛棉和陆地棉之间有多态性,19对引物在海岛棉中表现多态性。利用Jaccard相似系数和UPGMA方法进行聚类分析可以明显区分陆地棉和海岛棉,并且将海岛棉分为4类。14对引物在BC₁群体中表现多态性,产生14个位点。9个位点整合到BC₁连锁图的7个染色体上,4个位于A亚基因组,5个位于D亚基因组。海岛棉微卫星标记扩展了棉花微卫星标记,有助于海岛棉遗传多样性的研究,有利于棉花遗传图谱的进一步丰富。     

Genetic relationship analysis and molecular fingerprint identification of the tea germplasms from Guangxi Province,China

The tea plant (Camellia sinensis) is an evergreen woody plant with a high economic value. Guangxi Province is adjacent to the origin center of the tea plant in southern China. It has abundant germplasm resources and is a historically important tea-producing province. However, there is little information about the genetic diversity, genetic introgression, and fingerprints of the tea germplasms from Guangxi Province. Here, we constructed a phylogenetic tree of 126 tea accessions from Guangxi Province using 20 SSR markers. This tree classified these tea accessions into three subgroups containing 19, 47, and 60 members, respectively. High genetic similarity was observed among the three subgroups, and the genetic diversity of the populations was ranked as follows: subgroup 3 > subgroup 2 > subgroup 1. Furthermore, we analyzed the genetic relationships among 168 tea accessions from Guangxi Province and neighboring provinces. The results of the population structure analysis were highly consistent with the clustering results, and genetic introgression was observed. We identified six SSRs as the core marker set, because they could sufficiently distinguish between all 126 tea accessions. The results provide a crucial theoretical basis for utilization and protection of tea germplasms from Guangxi Province, and will help improve the breeding and popularization of elite tea cultivars.