用于绿豆种质资源遗传多样性分析的SSR及STS引物的筛选

目前能够用于绿豆(Vigna radiate)种质资源遗传多样性分析的PCR引物极其有限。通过12份农艺性状差异较大的绿豆种质对绿豆以及小豆(Vigna angularis)、豇豆(Vigna unguiculata)、菜豆(Phaseolus vulgaris)等近缘食用豆中的PCR引物进行筛选,结果表明41对绿豆SSR引物中能够有效扩增的有35对,6对有多态性;28对绿豆STS引物中有23对能够有效扩增,2对有多态性;8对小豆SSR引物能够有效扩增的有6对,但均无多态性;27对豇豆SSR引物能够有效扩增的有17对,1对有多态性;24对菜豆SSR引物能够有效扩增的有9对,1对有多态性。这些多态性引物的获得将有助于中国绿豆种质资源的遗传多样性分析。     

Variation in the nod gene RFLPs, nucleotide sequences of 16S rRNA genes, Nod factors, and nodulation abilities of Bradyrhizobium strains isolated from Thai Vigna plants

The analysis of nod genes and 16S rRNA gene regions, Nod factors, and nodulation abilities of Brady rhizobium strains isolated from tropical Thai Vigna species is reported. A total of 55 Bradyrhizobium strains isolated from two cultivated and six wild Vigna species growing in central and northern Thailand were evaluated. Thai Vigna spp. Bradyrhizobium strains showed higher levels of nod gene RFLP diversity compared with Thai soybean Brady rhizobium strains or temperate strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Analysis of the 16S rRNA gene region using selected strains also suggests a high genetic diversity of the Thai Vigna-Bradyrhizobium association. Based on thin-layer chromatography analysis, Nod factors produced by tropical Thai Vigna spp. Brady rhizobium strains are more diverse than temperate Japanese and US strains of B. japonicum and B. elkanii. Thai Vigna spp. Bradyrhizobium strains showed variation in nodulation ability and affinity, estimated by the number of normal nodules versus green nodules in an inoculation study. There are some Bradyrhizobium-host combinations that could not form any nodules, suggesting that some genetic differentiation has evolved in their host range. However, most of the Thai Vigna spp. Bradyrhizobium strains formed nodules on the cultigens soybean (Glycine max), mungbean (Vigna radiata), azuki bean (Vigna angularis), and cowpea (Vigna unguiculata). This is the first study on Bradyrhizobium strains associated with a range of cultivated and wild Vigna and reveals that these Bradyrhizobium strains are diverse and may provide novel sources of useful variation for the improvement of symbiotic systems.     

浙江省地方豇豆种质资源的鉴定与评价

“第三次全国农作物种质资源普查与收集行动”浙江项目组从全省32个县(市/区)共收集到豇豆地方资源76份.本研究通过对76份种质43个基本农艺性状的田间调查,发现76份种质包括30份长豇豆(Vigna unguiculata(L.)Walp.ssp.sesquipedialis)、46份普通豇豆(Vigna unguic...     

Efficiency of three PCR-based markers in assessing genetic variation among cowpea (Vigna unguiculata subsp. unguiculata) landraces.

The main objective of this study was to investigate the efficiency of RAPD, AFLP, and SAMPL marker systems in detecting genetic polymorphism in cowpea landraces (Vigna unguiculata subsp. unguiculata (L.) Walp.) that probably share a similar genetic pool. A second objective was to determine the level of diversity among landraces from a restricted area, to define the most appropriate strategy of on-farm conservation. Each marker system was able to discriminate among the materials analysed, but a clear distinction between all the local varieties was only obtained with AFLP and SAMPL markers. The average diversity index was quite similar for each marker system, but owing to the differences in the effective multiplex ratio values the marker index was higher for the AFLP and SAMPL systems than for the RAPD system. The AFLP and SAMPL techniques appear to be more useful than the RAPD technique in the analysis of limited genetic diversity among the cowpea landraces tested. The significant correlations of SAMPL similarity and cophenetic matrices with those of the other markers, and the lower number of primer combinations required, indicate that this technique is the most valuable. The low genetic similarity detected among landraces suggests that all the cowpea landraces should be maintained on the respective farms from which they came.     

Phylogeny of bradyrhizobia from Chinese cowpea miscellany inferred from 16S rRNA, atpD, glnII, and 16S-23S intergenic spacer sequences

The cowpea (Vigna unguiculata L.), peanut (Arachis hypogaea L.), and mung bean (Vigna radiata L.) belong to a group of plants known as the "cowpea miscellany" plants, which are widely cultivated throughout the tropic and subtropical zones of Africa and Asia. However, the phylogeny of the rhizobial strains that nodulate these plants is poorly understood. Previous studies have isolated a diversity of rhizobial strains from cowpea miscellany hosts and have suggested that, phylogenetically, they are from different species. In this work, the phylogeny of 42 slow-growing rhizobial strains, isolated from root nodules of cowpea, peanut, and mung bean from different geographical regions of China, was investigated using sequences from the 16S rRNA, atpD and glnII genes, and the 16S-23S rRNA intergenic spacer. The indigenous rhizobial strains from the cowpea miscellany could all be placed in the genus Bradyrhizobium , and Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense were the main species. Phylogenies derived from housekeeping genes were consistent with phylogenies generated from the ribosomal gene. Mung bean rhizobia clustered only into B. liaoningense and B. yuanmingense and were phylogenetically less diverse than cowpea and peanut rhizobia. Geographical origin was significantly reflected in the phylogeny of mung bean rhizobia. Most cowpea rhizobia were more closely related to the 3 major groups B. liaoningense, B. yuanmingense, and Bradyrhizobium elkanii than to the minor groups Bradyrhizobium japonicum or Bradyrhizobium canariense . However, most peanut rhizobia were more closely related to the 2 major groups B. liaoningense and B. yuanmingense than to the minor group B. elkanii.     

Use of intrinsic antibiotic resistance for characterisation and identification of rhizobia from nodules of Vigna unguiculata (L) Walp. and Phaseolus vulgaris (L)

Intrinsic resistance to low concentrations of antibiotics was used to characterise 83 isolates from nodules of cowpea (Vigna unguiculata) and field bean (Phaseolus vulgaris). Characterisation and differentiation of isolates from cowpea was made difficult by associated fast-growing bacteria inside the nodule tissue. Thus, reliable pure culture was difficult to secure without repeated isolation and even via nodulation of the appropriate homologous host. Although the technique may be satisfactory for differentiation and identification of fast-growing rhizobia, it is rated inferior to serology on aspects of facility, time and accuracy where rhizobia from cowpea nodules are concerned. Fingerprint patterns of isolates revealed considerable heterogeneity amongst the populations even where there was commonality of location and/or host plant. Pure cultures of slow-growing rhizobia from V. unguiculata nodules were generally more resistant to the concentrations of antibiotics used than fast-growing nodule bacteria from P. vulgaris.     

Molecular evolution and phylogenetic implications of internal transcribed spacer sequences of nuclear ribosomal DNA in the Phaseolus-Vigna complex.

Molecular phylogeny based on internal transcribed spacer (ITS) sequences was studied to resolve the taxonomic contradiction in Vigna and its relation to Phaseolus. The ITS region of the 18S-26S nuclear ribosomal DNA repeat was sequenced for 29 Vigna species, selected from five of the nine subgenera, and 9 species of Phaseolus. The length of ITS-1 ranged from 187 to 243 bp and 217 to 290 bp, and that of ITS-2 from 187 to 219 bp and 225 to 243 bp, within Vigna and Phaseolus species, respectively. Phylogenies derived from ITS sequences based on maximum-parsimony and neighbor-joining methods gave trees essentially of similar topology. The ITS phylogeny was generally congruent with recent classifications based largely on morphological, biochemical, cytogenetical, and palynological features, except that subgenus Plectotropis of Neotropical origin was revealed to be closely related to subgenus Vigna instead of forming a link between African (subgenus Vigna) and Asiatic (subgenus Ceratotropis) vignas, and subgenus Sigmoidotropis, featuring morphological characters of both Vigna and Phaseolus, was placed as the sister group to the Phaseolus taxa. The ITS sequences were shown to be useful for identifying wild progenitors of V. mungo, V. radiata, V. umbellata, and V. unguiculata and for clarifying taxonomy-related problems in many previously controversial cases. This study also affirms that V. umbellata and V. angularis are the diploid progenitors of the only tetraploid species (V. glabrescens) known in the genus.     

Genome conservation among three legume genera detected with DNA markers.

A set of 219 DNA clones derived from mungbean (Vigna radiata), cowpea (V. unguiculata), common bean (Phaseolus vulgaris), and soybean (Glycine max) were used to generate comparative linkage maps among mungbean, common bean, and soybean. The maps allowed an assessment of linkage conservation and collinearity among the three genomes. Mungbean and common bean, both of the subtribe Phaseolinae, exhibited a high degree of linkage conservation and preservation of marker order. Most linkage groups of mungbean consisted of only one or two linkage blocks from common bean (and vice versa). The situation was significantly different with soybean, a member of the subtribe Glycininae. Mungbean and common bean linkage groups were generally mosaics of short soybean linkage blocks, each only a few centimorgans in length. These results suggest that it would be fruitful to join maps of mungbean and common bean, while knowledge of conserved genomic blocks would be useful in increasing marker density in specific genomic regions for all three genera. These comparative maps may also contribute to enhanced understanding of legume evolution.     

CHLOROPLAST DNA POLYMORPHISM SUGGESTS NIGERIAN CENTER OF DOMESTICATION FOR THE COWPEA,VIGNA UNGUICULATA (LEGUMINOSAE)

Twenty-one independent chloroplast DNA polymorphisms were identified in Vigna unguiculata defining 19 different chloroplast DNA molecules (plastome types). Two plastome types, differing by a single character, were found among 32 accessions of cultivated cowpea (Vigna unguiculata ssp. unguiculata). Eighteen different plastome types were found among 26 accessions of wild cowpea (V. unguiculata ssp. dekindtiana). The very low level of chloroplast DNA diversity found in cultivated accessions relative to wild cowpea suggests that 1) the domesticated form was derived from a narrow selection of the wild germplasm and 2) chloroplast gene flow between wild and cultivated types has been very limited. Cladistic analysis of the cpDNA data generated a robust tree completely lacking homoplasy. Three wild accessions from Nigeria possessed a plastome type indistinguishable from one present in cultivated accessions, suggesting that Nigeria represents one center of domestication of the cowpea. The other plastome type within the cultivated germplasm was not found among wild accessions.     

A SNP and SSR based genetic map of asparagus bean (Vigna. unguiculata ssp. sesquipedialis) and comparison with the broader species

Asparagus bean (Vigna. unguiculata ssp. sesquipedialis) is a distinctive subspecies of cowpea [Vigna. unguiculata (L.) Walp.] that apparently originated in East Asia and is characterized by extremely long and thin pods and an aggressive climbing growth habit. The crop is widely cultivated throughout Asia for the production of immature pods known as 'long beans' or 'asparagus beans'. While the genome of cowpea ssp. unguiculata has been characterized recently by high-density genetic mapping and partial sequencing, little is known about the genome of asparagus bean. We report here the first genetic map of asparagus bean based on SNP and SSR markers. The current map consists of 375 loci mapped onto 11 linkage groups (LGs), with 191 loci detected by SNP markers and 184 loci by SSR markers. The overall map length is 745 cM, with an average marker distance of 1.98 cM. There are four high marker-density blocks distributed on three LGs and three regions of segregation distortion (SDRs) identified on two other LGs, two of which co-locate in chromosomal regions syntenic to SDRs in soybean. Synteny between asparagus bean and the model legume Lotus. japonica was also established. This work provides the basis for mapping and functional analysis of genes/QTLs of particular interest in asparagus bean, as well as for comparative genomics study of cowpea at the subspecies level.     

Examination of the Possibility of the Involvement of the Alternative Respiration in Seed Germination

The possibility that the alternative respiration (AR) is involvedin seed germination was followed during and after imbibitionby noting the changes in the sensitivity to the respiratoryinhibitors, KCN and salicylhydroxamic acid (SHAM). This wascarried out by measuring oxygen uptake of mitochondrial preparationsfrom storage cotyledons and of the whole axis in a selectionof legumes (Vigna mungo, V. radiata, V. angularis, V. sinensisPhaseolus vulgaris, Pisum sativum and Dolichos lablab). Theextent of participation of AR in the respiration of cotyledonarymitochondria was variable according to species and time afterimbibition, and varied even within the same genus ( Vigna).In some species, AR was not observed. As to the axis, SHAM treatmentby itself had little effect on the rate of respiration, but,when applied with KCN, reduced oxygen uptake to an extent greaterthan KCN treatment alone. It was suggested that AR was not necessarilyimportant for seed germination. Key words: Alternative respiration, mitochondria, seed germination     

Identification and profiling of novel microRNAs in the Brassica rapagenome based on small RNA deep sequencing

Background

Cowpea is a highly inbred crop. It is part of a crop-weed complex, whose origin and dynamics is unknown, which is distributed across the African continent. This study examined outcrossing rates and genetic structures in 35 wild cowpea (Vigna unguiculata ssp. unguiculata var. spontanea) populations from West Africa, using 21 isozyme loci, 9 of them showing polymorphism.

Results

Outcrossing rates ranged from 1% to 9.5% (mean 3.4%), which classifies the wild cowpea breeding system as primarily selfing, though rare outcrossing events were detected in each population studied. Furthermore, the analyses of both the genetic structure of populations and the relationships between the wild and domesticated groups suggest possibilities of gene flow that are corroborated by field observations.

Conclusions

As expected in a predominantly inbred breeding system, wild cowpea shows high levels of genetic differentiation and low levels of genetic diversity within populations. Gene flow from domesticated to wild cowpea does occur, although the lack of strong genetic swamping and modified seed morphology in the wild populations suggest that these introgressions should be rare.     

Biodiversity studies in Phaseolus species by DNA barcoding

The potential of DNA barcoding was tested as a system for studying genetic diversity and genetic traceability in bean germplasm. This technique was applied to several pure lines of Phaseolus vulgaris L. belonging to wild, domesticated, and cultivated common beans, along with some accessions of Phaseolus coccineus L., Phaseolus lunatus L., and Vigna unguiculata (L.) Walp. A multilocus approach was exploited using three chloroplast genic regions (rbcL, trnL, and matK), four intergenic spacers (rpoB-trnC, atpBrbcL, trnT-trnL, and psbA-trnH), and nuclear ITS1 and ITS2 rDNA sequences. Our main goals were to identify the markers and SNPs that show the best discriminant power at the variety level in common bean germplasm, to examine two methods (tree based versus character based) for biodiversity analysis and traceability assays, and to evaluate the overall utility of chloroplast DNA barcodes for reconstructing the origins of modern Italian varieties. Our results indicate that the neighbor-joining method is a powerful approach for comparing genetic diversity within plant species, but it is relatively uninformative for the genetic traceability of plant varieties. In contrast, the character-based method was able to identify several distinct haplotypes over all target regions corresponding to Mesoamerican or Andean accessions; Italian accessions originated from both gene pools. On the whole, our findings raise some concerns about the use of DNA barcoding for intraspecific genetic diversity studies in common beans and highlights its limitations for resolving genetic relationships between landraces and varieties.     

Genetic relationship of cowpea (Vigna unguiculata) varieties from Senegal based on SSR markers

Genetic diversity and phylogenetic relationships among 22 local cowpea (Vigna unguiculata) varieties and inbred lines collected throughout Senegal were evaluated using simple sequence repeat molecular markers. A set of 49 primer combinations were developed from cowpea genomic/expressed sequence tags and evaluated for their ability to detect polymorphisms among the various cowpea genotypes. Forty-four primer combinations detected polymorphisms, with the remaining five primer sets failing to yield PCR amplification products. From one to 16 alleles were found among the informative primer combinations; their frequencies ranged from 0.60 to 0.95 (mean = 0.79). The genetic diversity of the sample varied from 0.08 to 0.42 (mean = 0.28). The polymorphic information content ranged from 0.08 to 0.33 (mean = 0.23). The local varieties clustered in the same group, except 53-3, 58-53, and 58-57; while Ndoute yellow pods, Ndoute violet pods and Baye Ngagne were in the second group. The photosensitive varieties (Ndoute yellow pods and Ndoute violet pods) were closely clustered in the second group and so were inbred line Mouride and local cultivar 58-57, which is also one of the parents for inbred line Mouride. These molecular markers could be used for selection and identification of elite varieties for cowpea improvement and germplasm management in Senegal.     

Genetic diversity of the Bambara groundnut (Vigna subterranea (L.) Verdc.) as assessed by SSR markers

Bambara groundnut ( Vigna subterranea (L.) Verdc.) is an important African legume crop. In this study, a collection consisting of 240 accessions was analyzed using 22 simple sequence repeat (SSR) markers. In total, 166 alleles were detected, with a mean of 7.59 alleles per locus. Allelic and gene diversities were higher in the west African and Cameroon/Nigeria regions with 6.68 and 6.18 alleles per locus, and 0.601 and 0.571, respectively. The genetic distance showed high similarity between west African and Cameroon/Nigeria accessions. Principal coordinate analyses and neighbor-joining analysis consistently revealed that the majority of west African accessions were grouped with Cameroon/Nigeria accessions, but they were differentiated from east African, central African, and southeast Asian accessions. Population structure analysis showed that two subpopulations existed, and most of the east African accessions were restricted to one subpopulation with some Cameroon/Nigeria accessions, whereas most of the west African accessions were associated with most of the Cameroon/Nigeria accessions in the other subpopulation. Comparison with SSR analysis of other Vigna cultigens, i.e., cultivated azuki bean ( Vigna angularis ) and mungbean ( Vigna radiata ), reveals that the mean gene diversity of Bambara groundnut was lower than azuki bean but higher than mungbean.     

我国豇豆和绿豆根瘤菌的数值分类及16S rDNA PCR-RFLP研究

对分离自中国14个不同省(自治区)的79株豇豆和绿豆根瘤菌及12株参比菌株进行了唯一碳、氮源利用,抗生素抗性,抗逆性和酶活性等128个表型性状的测定,并用MINTS软件进行聚类分析。表型性状测定结果发现,所有菌株都有极其广泛的碳、氮源利用谱,大多数菌株可在较宽的pH(pH5·0~11·0)值范围内生长,大部分菌株能在37℃高温条件下生长,个别菌株能耐受60℃高温较长时间(20~45min)的热激。聚类分析结果表明,全部供试菌株在63·5%的相似性水平上分为两大群:一个群为慢生菌群,另一群为快生和中慢生菌群;在79%的相似性水平上分为7个亚群。在数值分类的基础上,又将参比菌株增加到22株,对79株待测菌株进行了16SrDNAPCR-RFLP分析,16SrDNAPCR产物经HaeⅢ、HinfⅠ、MspⅠ和AluⅠ4种内切酶酶切共产生34种遗传图谱类型,经GelComparⅡ软件聚类后,在79%的相似性水平上也可划分为7个亚群,与数值分类的结果有很好的一致性。     

Serological and Biological Comparisons of Blackeye Cowpea Mosaic and Cowpea Aphid-borne Mosaic Potyvirus Isolates Seed-borne in Vigna unguiculata (L.) Walp. Germplasm

Serological and biological comparisons were made among 45 seed-borne isolates of blackeye cowpea mosaic (BICMV) and 54 seed-borne isolates of cowpea aphidborne mosaic (CAbMV) potyviruses derived from cowpea seedlots or young nursery-grown seedlings comprising 2112 germplasm accessions or pre-introduction seedlots of Vigna unguiculata. Isolates were identified by DAS-ELISA using polyclonal immunoglobulin G specific for these viruses. Twenty isolates of BICMV and 32 isolates of CAbMV were also compared by ACP-ELISA with selected monoclonal antibodies and by SDS-immunodiffusion. By all approaches, isolates of BICMV were clearly distinguished from CAbMV isolates. Isolate comparisons on selected cowpea genotypes (TVu-401, TVu-1582, TVu-2657, and TVu-3433) partitioned most isolates into two distinct groups. A few isolates seed-borne in Indian cv. Pusa Phalguni, however, were clearly BICMV by all serological tests, but behaved as CAbMV in definitive cowpea genotypes. Although BICMV is generally considered to be a 'new world virus', both BICMV and CAbMV occurred in V. unguiculata seedlots originating in 'old-world regions', including Afghanistan, Botswana, Nigeria, Senegal, and South Africa. Seedborne CAbMV was isolated from 6 of 155 tested US V. unguiculata Germplasm accessions originating, respectively, in Afghanistan (2), Botswana (2), India (1), and Pakistan (1).     

Production of intraspecific F1 hybrids between wild and cultivated accessions of cowpea (Vigna unguiculata (L.) walp.) using conventional methods

Cowpea (Vigna unguiculata (L.) Walp.) is an important food legume in the tropics. It belongs to the Phaseoleae (L.) tribe (Fabaceae family), it is diploid and its chromosome number is 22. Its gene pool includes the cultivated cowpea and its wild relatives, which are connected with Vigna subgenus, Catiang section. Cowpea has a great potential in increasing food legume production. The cowpea varieties, however, are susceptible to a number of insect pests, especially the pod borer Maruca testulalis and a pod sucking-bug complex (e.g.: Clavigralla tomentosicollis, Anoplocnemis curvipes and Riptortus dentipes), which cause severe damage. The crossing programme presented here exploits the variability existing in the wild African germplasm of V. unguiculata and cultivated cowpea. To incorporate the insect pest resistance into the cultivated cowpea economically, reciprocal crosses between wild forms and cowpea varieties were performed, using the stigmatic pollination methods at anthesis. Some barriers were found in these intraspecific crosses. In the majority of reciprocal crosses, the growth of the pollen tubes was arrested in the stigmatic tissue. Only 16.01% of the ovules were fertilised. In these ovules, embryo development was normal at about 20-25 days after pollination. The failure of the intraspecific crosses in about 80.7% of the cases is thus the result of the lack of fertilisation and the unfertilised ovules. There seems to exist considerable incompatibility within the primary cowpea gene pool. The breeding programme carried out under controlled conditions has proved to be less successful in developed cowpea intraspecific F1 hybrids. Further studies should concentrate on germplasm from Africa with documented resistance to major insect pests. In addition, the application of techniques for bypassing barriers to hybridisation of parent genotypes should enable these embryos to grow to plants.     

Genetic structure and differentiation in cultivated grape,Vitis vinifera L

222 cultivated (Vitis vinifera) and 22 wild (V. vinifera ssp. sylvestris) grape accessions were analysed for genetic diversity and differentiation at eight microsatellite loci. A total of 94 alleles were detected, with extensive polymorphism among the accessions. Multivariate relationships among accessions revealed 16 genetic groups structured into three clusters, supporting the classical eco-geographic grouping of grape cultivars: occidentalis, pontica and orientalis. French cultivars appeared to be distinct and showed close affinity to the wild progenitor, ssp. sylvestris from south-western France (Pyrenees) and Tunisia, probably reflecting the origin and domestication history of many of the old wine cultivars from France. There was appreciable level of differentiation between table and wine grape cultivars, and the Muscat types were somewhat distinct within the wine grapes. Contingency chi2 analysis indicated significant heterogeneity in allele frequencies among groups at all loci. The observed heterozygosities for different groups ranged from 0.625 to 0.9 with an overall average of 0.771. Genetic relationships among groups suggested hierarchical differentiation within cultivated grape. The gene diversity analysis indicated narrow divergence among groups and that most variation was found within groups (approximately 85%). Partitioning of diversity suggested that the remaining variation is somewhat structured hierarchically at different levels of differentiation. The overall organization of genetic diversity suggests that the germplasm of cultivated grape represents a single complex gene pool and that its structure is determined by strong artificial selection and a vegetative mode of reproduction.     

Vignin diversity in wild and cultivated taxa ofVigna unguiculata (L.) walp. (fabaceae)

Eighty-one cultivated and 55 wild accessions were characterized using one- and two-dimensional isoelectric focusing (1D and 2D IEF)/SDS-PAGE and immuno-detection techniques to examine genetic diversity present within cultivated and wild taxa inVigna unguiculata (L.) Walp. Twenty-seven unique banding patterns were identified in the vignin (or Gl) fraction of the major seed storage proteins. These patterns were controlled by at least four interacting genes, two of which were tightly linked and a third which also may be linked. Due to the tremendous amount of variation in these wild taxa it is not possible to make definitive statements about either the taxonomic or geographic distribution of vignin types. These results give no final answer to the identity of the progenitor of cowpea or the center of domestication, but do pose some interesting questions to be asked as the genetic relationships among the wild and cultivated taxa in this species are unraveled.