Assessment of genetic diversity in sesame (Sesamum indicum L.) genotypes, using EST-derived SSR markers

A total of 16,619 ESTs sequences (SSRs) of sesame (Sesamum indicum L.) were mined from Genbank. From sequences, 156 primer pairs were designed and characterized to determine the diversity among 49 sesame accessions. Twenty SSRs were found to be polymorphic and the number of alleles ranged from two to five per locus. The allele size varied from 101 to 399 bp. The average PIC value of the 20 SSR loci was 0.72 ranging from 0.49 (SEM-12-68) to 0.90 (SEM-12-27). Dendrogram analysis grouped the 49 genotypes into five separate clusters exhibiting a genetic similarity coefficient from 0.59 to 1.0. Hence, these EST-derived SSRs markers could be useful in assessing the diversity of sesame accessions and could also help in identifying diverse parents for sesame improvement programs.     

Genetic control of isozymes in Carica papaya L.

The genetic control of isozymes was investigated in papaya. Out of six buffer systems tested, Histidine-citrate pH 6.5 gave the most consistent and greatest number of well-resolved enzymes. Nine enzyme systems (ACO, IDH, MDH, PGM, PGI, SKD, TPI, UGP, 6-PGD) out of 28 were resolved. Based on the initial screening of 131 accessions of papaya from Central and South America for electromorph variants, parents were selected for various combinations of controlled crosses. These controlled crosses were then used to elucidate the inheritance of isozymes in papaya. Evidence of Mendelian inheritance was obtained for eight out of nine polymorphic loci: Aco-1, Aco-2, Pgi-2, Pgm-1, Skd, Tpi-1, Tpi-2, and Ugp. No controlled cross was available to study the genetic control of the polymorphic locus Pgm-2. Because they were not variable in all accessions included in this study, the loci Idh, Mdh-1, Mdh-2, and 6-Pgd, were also not examined by segregation analysis. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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。     

Comparison of isoenzymes in Prunus avium separated by two different electrophoretic techniques

Isoenzyme variation for seven systems revealed by two different electrophoretic procedures was compared in Prunus avium. Fourteen cultivars and 14 wild selections were analysed for acid phosphatase (ACP), isocitrate dehydrogenase (IDH), leucine aminopeptidase (LAP), malate dehydrogenase (MDH), phosphoglucomutase (PGM), shikimate dehydrogenase (SKD) and superoxide dismutase (SOD). Extracts were separated by isoelectric focusing (IEF) and by polyacrylamide gel electrophoresis (PAGE). For the eight loci that had been described previously in these enzyme systems on the basis of IEF analysis, we compared the variation revealed with IEF and PAGE. Similar variation was revealed for Acp‐1 and Pgm‐1, and the alleles revealed by PAGE could be identified directly with those reported for IEF. For Lap‐1, Mdh‐1 and Skd‐1, variation was seen with IEF but not with PAGE. For Mdh‐2, PAGE revealed additional variation not revealed by IEF. For Idh‐1, different patterns of variation were revealed by PAGE and IEF, and both procedures would be needed to genotype cherry accessions. We were unable to detect variation corresponding to that reported previously for Sod‐1 with either technique. The implications of these findings for allele labelling, for studies of genetic diversity and for linkage analysis are discussed.     

Phenotypic polymorphism of six enzymes in the grasspea (Lathyrus sativus L.)

Summary The phenotype variation in six enzymes, 6-phosphogluconate dehydrogenase (6-PGD), malate dehydrogenase (MDH), peroxidase (PRX), isocitrate dehydrogenase (IDH), galactose dehydrogenase (GD) and glutamate oxaloacetate transaminase (GOT), was investigated using horizontal starch gel electrophoresis in 52 accessions of the grasspea, Lathyrus sativus. Phenotypic polymorphism was observed for all six enzymes. High phenotypic polymorphism (Pj) was observed for PRX and 6-PGD, while there was little polymorphism for GOT, with only two accessions showing variation. There was no correlation between phenotypic polymorphism and region of origin, or groupings of accessions made on the basis of flower colour. Tentative genetic interpretations of banding patterns are given for five of the enzyme systems. The level of apparent heterozygosity was higher than expected in this predominantly autogamous species. The level of variation in the grasspea is discussed in terms of its potential for exploitation through plant breeding.     

新麦草Psathyrostachys juncea遗传变异的细胞学和分子的分析

本文用染色体C分带、同功酶和RAPD技术分析了来自不同地理分布的新麦草不同材料的单株,观察其c带的多态性,即同源染色体的配对,相同材料的不同个体之间,同一地区不同材料之间,以及不同来源的材料之间的多态性。10个同功酶揭示了14个假定标记位点,其中11个显示了多态性(平均66.2％,每个位点具2.6个等位基因)。大量的等位变化发生在多数的多态位点上。在所用试材中有近90％的等位变化。在优化条件下,200个测试引物中有55.5％的材料间产生了多态性的RAPDs带型。在同一材料中和不同材料间分别有55.7％和47.8％的扩增片断是多态的。C—带、同功酶和RAPD分析结果是互相确证而高度一致的,为新麦草的遗传变异提供了模式。 新麦草具有抗旱、耐盐碱和抗黄矮病的特性,可以在小麦育种工作上加以利用。     

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.     

Genetic variation and phylogenetic relationships in East and South Asian melons, Cucumis melo L., based on the analysis of five isozymes

Genetic structure and phylogenetic relationships in East and South Asian melons were analyzed, based on the geographical variation of five isozymes. The analysis of Indian melon accessions showed a continuous variation in seed length, ranging from 4 to 13 mm. Most of the East Asian melons, vars. makuwa and conomon, were classified as the small seed type with seed length shorter than 9 mm. The frequency of the small seed type increased from the west to the east in India. Allelic variation was detected at a total of nine loci of five isozymes among 114 melon accessions. Gene diversity calculated for the nine loci indicated that Indian melon was rich in genetic variation, which decreased from India towards the east. Clear geographical variation was detected in two enzymes, APS and6-PGDH. Pgd-1 ¹ and Ap-3 ¹ were frequent in India and Myanmar, while most of the melons in Laos, China, Korea and Japan carried Pgd-1 ³ and Ap-3 ³, except var. inodorusin China. Among the latter two alleles, the frequency of Ap-3 ³ was more than 50% in the small seed type in north and east India, indicating that vars. makuwa and conomon were related to the small seed type in these areas. It was also suggested that the small seed type with wet tolerance originated in central India and was selected under wet condition in the east. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Evaluation of cultivated barley (Hordeum vulgare) germplasm for the presence of thermostable alleles of β-amylase

A cleaved amplified polymorphic sequence marker was used to detect the alleles Bmy‐Sd2H and Bmy‐Sd3 identifying highly thermostable isoforms of the enzyme b‐amylase, which improves fermentability during brewing. Among the 889 accessions of barley (Hordeum vulgare) investigated, and two accessions of H. spontaneum a total of 166 accessions were identified carrying the superior b‐amylase alleles. These thermostable alleles of b‐amylase were most frequently observed in six‐rowed varieties originating from Asia, especially Japan, with 61.9% of the accessions from Asia carrying the alleles of interest. Additional six‐rowed barleys carrying the relevant alleles were identified among accessions from America, Africa and the Near East. In the European varieties, the percentage of accessions with the alleles of interest was 5.1% with a strong predominance in two‐rowed spring barleys. A pedigree analysis identified the cross ‘Binder’ x ‘Gull’ as the main source of the thermostable b‐amylase alleles in European varieties. The data suggest that an improvement of malting quality in barley could be achieved by introduction of the Bmy1‐Sd2H and Bmy1‐Sd3 alleles into the European breeding programmes.     

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 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.     

大豆引进种质抗胞囊线虫病、抗花叶病毒病和耐盐基因型鉴定及优异等位基因聚合种质筛选

我国从美国、俄罗斯、日本等26个国家或地区共引进大豆种质3218份, 仅对部分种质进行了大豆胞囊线虫病(Soybean cyst nematode, SCN)、大豆花叶病毒病(Soybean mosaic virus, SMV)和盐敏感性的抗性鉴定, 但基因型的系统分析尚未见报道。本研究针对大豆抗胞囊线虫病3个基因(rhg1、Rhg4、SCN3-11)和耐盐基因(GmSALT3)开发KASP标记5个, 结合与大豆花叶病毒抗性相关的1个SCAR标记(SCN11), 对1489份大豆引进种质进行基因型鉴定。结果表明, 具有优异等位基因的种质共1084份; 携带3个位点优异等位基因的种质19份, 包括抗胞囊线虫病3个位点(rhg1、Rhg4、SCN3-11)叠加(Peking型)种质3份, 聚合抗胞囊线虫病基因和抗花叶病毒病标记7份, 聚合抗胞囊线虫病和耐盐基因2份, 聚合抗胞囊线虫病、抗花叶病毒病和耐盐基因7份; 携带4个位点优异等位基因的种质9份, 包括聚合抗胞囊线虫病基因和抗花叶病毒病标记6份, 聚合抗胞囊线虫病和耐盐基因2份, 聚合抗胞囊线虫病、抗花叶病毒病和耐盐7份; 携带5个位点优异等位基因8份, 聚合了抗胞囊线虫病、抗花叶病毒病和耐盐优异等位变异。在这些携带优异等位变异的种质中, 44份已由前人证明具有相应的抗性。携带3个或3个以上优异等位基因的36份种质中, 有52.78%种质的一种或两种特性已被报道。在不携带抗性优异等位变异的种质中, 93份已证明有耐盐性或对SMV3号株系抗性, 这些种质可能存在新的抗性(等位)基因。本研究利用高通量分子标记筛选出的携带抗病、抗逆优异等位基因的种质为我国大豆资源表型鉴定、抗源的快速筛选及利用提供理论依据和新思路。     

Effectiveness of different classes of molecular marker for classifying and revealing variation in rice (Oryza sativa) germplasm

We have examined the effectiveness of similar numbers of markers from four molecular marker systems (AFLP, isozymes, ISSR and RAPD) for revealing genetic diversity and discriminating between infraspecific groups of Oryza sativa germplasm. Each marker system classifies the germplasm into three major groups (most effectively with isozymes and AFLPs), but with differences (primarily with ISSR) between the precise classifications generated. However, at the highest levels of genetic similarity there was only partial agreement as to relationships between individual accessions when different markers were used. When variance was partitioned among and within the three subspecific groups, although the differences were not significant, greater variation was found among than within groups using AFLP and isozymes, with the reverse for RAPD and ISSR. Measurement of polymorphism using average heterozygosity and effective number of alleles gave similar results for each marker system. These results are discussed in relation to various genetic resources conservation activities, and the advisability of extrapolating to other sets of germplasm particularly of other crop species. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

以湖南和湖北大豆[Glycine max（L.）Merr.]为例分析影响遗传多样性评价的因素

以不同来源大豆种质为材料进行基因组SSR分析,探讨大豆遗传多样性研究方法,旨在为科学评价大豆种质资源提供参考。当取样比例相同时,湖北大豆的遗传丰富度显著高于湖南大豆,但两省大豆遗传多样性指数之间没有显著差异。当取样量相同时,利用随机取样法比较,湖南大豆的遗传多样性显著高于湖北,而利用聚类取样比较,两省大豆的遗传多样性没有显著差异。在遗传多样性指数相同情况下,湖北大豆的遗传丰富度显著高于湖南大豆。取样数与三个遗传多样性评价参数（等位变异数、Shannon指数、He）之间均达到显著和极显著相关。以湖南大豆为例,估算出在大豆SSR遗传多样性分析中,至少需要50~60个样本,即占总体9.0%~10.8%的取样比例,才能代表总体的遗传变异。提出在评价大豆遗传多样性时,应用大豆样本数对其遗传多样性指数计算公式进行修正,建议将Shannon指数计算公式改为H＝－lnN ∑P_i lnP_i (N为样本数)。根据修正公式分析,结果表明,湖北大豆的遗传多样性高于湖南大豆。     

High level of natural variation in a groundnut (Arachis hypogaea L.) germplasm collection assayed by selected informative SSR markers

The ability to identify genetic variation is indispensable for effective management and use of genetic resources in crop breeding. Genetic variation among 189 groundnut ( Arachis hypogaea L.) accessions comprising landraces, cultivars, a mutant, advanced breeding lines and others (unknown genetic background) representing 29 countries and 10 geographical regions was assessed at 25 microsatellite or simple sequence repeat loci. A high number of alleles (265) were detected in the range of 3 (Ah1TC6G09) to 20 (Ah1TC11H06) with an average of 10.6 alleles per locus. The polymorphism information content value at these loci varied from 0.38 (Ah1TC6G09) to 0.88 (Ah1TC11H06) with an average of 0.70. A total of 59 unique alleles and 127 rare alleles were detected at almost all the loci assayed. Cluster analysis grouped 189 accessions into four clusters. In general, genotypes of South America and South Asia showed high level of diversity. Extraordinary level of natural genetic variation reported here provides opportunities to the groundnut community to make better decisions and define suitable strategies for harnessing the genetic variation in groundnut breeding.     

Allelic variation and genetic diversity of high molecular weight glutenin subunit in Chinese endemic wheats (Triticum aestivum L.)

The high molecular weight glutenin subunit (HMW-GS) compositions of 66 Chinese endemic wheats were determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Ten alleles at the Glu-1 loci were detected in 50 Tibetan weedrace wheat accessions which in combination resulted in seven different HMW-GS patterns. Four HMW-GS patterns were observed among 10 Yunnan hulled wheat accessions, and two patterns in six Xingjiang rice wheat accessions. Two novel alleles (Bx7** + By8, Bx7 + By8**) and two rare alleles (Dx2 + 1Dy12*, Dx2 + null) were found in Tibetan weedrace accessions, with one of the latter (Dx2 + Dy12*) also being found in Yunnan hulled wheat. The mean indices of genetic variation at the Glu-1 loci in Yunnan hulled wheat, Tibetan weedrace wheat and Xingjiang rice wheat were 0.2232, 0.1655 and 0.0926, respectively, showing that Yunnan hulled wheat and Tibetan weedrace wheat had higher genetic variation than Xingjiang rice wheat.