Development and polymorphism of Vigna unguiculata ssp. unguiculata microsatellite markers used for phylogenetic analysis in asparagus bean (Vigna unguiculata ssp. sesquipedialis (L.) Verdc.)

Asparagus bean (V. unguiculata ssp. sesquipedialis), a specific form of cowpea (V. unguiculata L. Walp.), is cultivated as a vegetable crop throughout eastern and southern Asia for its tender long pods. Little is known about the genetic relationship between asparagus bean and the broader species, particularly the dominant ssp. unguiculata. We report here the development and transferability of simple sequence repeat (SSR) markers, over 40% of which are EST-derived, from ssp. unguiculata to asparagus bean and the use of a subset of the polymorphic markers to assess the genetic diversity of asparagus bean cultivars from diverse geographic origins across China. A total of 410 EST derived SSR (eSSR) markers and 600 SSR markers derived from cowpea genespace sequences (GSS) were developed, with a cross-subspecies transferability of 100% and 98.5%, respectively. In a recombinant inbred line population of asparagus bean, a 1:1 segregation was observed for most loci. Principal coordinate analysis (PCA) and phylogenetic clustering based on 62 alleles detected by 14 polymorphic SSR markers distinguished ssp. unguiculata and sesquipedialis into separate groups. Improved asparagus bean cultivars in China generally have a narrow genetic basis compared with landraces varieties. This suggests that asparagus bean breeding programs need to consider utilizing landrace germplasm to enhance genetic variability and ensure long-term gains from selection and reduce genetic vulnerability to pathogen/pest epidemics. Because of their transferability across subspecies, the SSR markers described in this study could be effectively employed in cross-subspecies trait introgression breeding from ssp. unguiculata to sesquipedialis.     

60份豇豆品种资源的耐盐能力评判

运用《豇豆基因型耐盐能力技术鉴定方法》(专利号ZL200810048423.5),对60份豇豆品种的耐盐能力进行了鉴定评判。试验材料经苗期培养和盐胁迫处理后,分别测定处理组和对照组的植株叶片叶绿素含量、过氧化氢酶活性、可溶性蛋白质含量、细胞伤害率以及单株鲜重等指标并计算其SRI值,运用专利技术的预测模型,得出各品种耐盐综合评价值(D值)。依据评判标准,紫魁、玉农农家宝特优、抗病芦花等11个品种的综合评价值D≥0.68,归为盐耐受型品种;之豇14、白皮、矮紫尾青等8个品种的综合评价值D≤0.48,归为盐敏感型品种;其余41个品种的D值介于0.48~0.68之间,属于中度耐盐品种。本研究结果扩大了豇豆种质耐盐能力鉴定评判份数,丰富了耐盐种质鉴定信息。     

Genetic analysis of salt stress responses in asparagus bean (Vigna unguiculata (L.) ssp. sesquipedalis Verdc.)

Salt stress responses of 23 asparagus bean cultivars were evaluated using 14-day-old seedlings after 15-day exposure to 75 mM NaCl in a hydroponics culture system. Salt-induced changes in plant growth and morphology, photosynthetic capacity, cell membrane integrity, and cellular protection enzyme systems as well as other physiological and biochemical traits were investigated to identify genotypic variability in salt response. This study also analyzed heredity parameters and correlation of the salt response index (SRI). Salt stress suppressed seedling growth and simultaneously reduced leaf area, content of chlorophyll and soluble proteins, net assimilation rate, as well as dry matter accumulation. In contrast, leaf blade cell membrane relative permeability, content of malondialdehyde, and antioxidant enzyme activity were elevated after salt treatment. Analysis of the heredity parameters has identified that the 18 investigated traits have different genotypic variance of the SRI values. Based on Ward's distances estimated for the sum of square variance in the SRI values, all the cultivars were classified into 2 discrete salt-tolerant and salt-sensitive clusters. Findings from this study will provide theoretical bases for identification and breeding of salt-tolerant cultivars in asparagus bean.     

Agrobacterium mediated transformation of Vigna sesquipedalis Koern (asparagus bean)

Agrobacterium mediated transformation of Vigna sesquipedalis was achieved using cotyledonary node explants prepared from 5 days old seedlings germinated on B5 basal medium, and transformed using Agrobacterium tumefaciens strain EHA101, carrying the phosphinothricin-N-acetyltransferase gene and neomycin-3-phosphotransferase-II gene as selectable markers and GUS gene as a screenable marker. Gene transfer was achieved by inoculation of cotyledonary node explants with a bacterial suspension and a further cocultivation with Agrobacterium suspension for 3 days on B5 basal medium. Only 10% of the explants were transformed with EHA101 and exhibited transient expression of GUS genes, while 2% of shoots exhibited stable integration of genes and developed into plants. Transgenic character of tissues was confirmed by GUS assay and Southern analysis. Histological analysis of GUS gene expression directly after cocultivation revealed a high competence of subepidermal cell layers of cotyledonary node and associated cotyledons for transformation with Agrobacterium.     

The genetics of domestication of yardlong bean, Vigna unguiculata (L.) Walp. ssp. unguiculata cv.-gr. sesquipedalis

Background and Aims

The genetics of domestication of yardlong bean [Vigna unguiculata (L.) Walp. ssp. unguiculata cv.-gr. sesquipedalis] is of particular interest because the genome of this legume has experienced divergent domestication. Initially, cowpea was domesticated from wild cowpea in Africa; in Asia a vegetable form of cowpea, yardlong bean, subsequently evolved from cowpea. Information on the genetics of domestication-related traits would be useful for yardlong bean and cowpea breeding programmes, as well as comparative genome study among members of the genus Vigna. The objectives of this study were to identify quantitative trait loci (QTLs) for domestication-related traits in yardlong bean and compare them with previously reported QTLs in closely related Vigna.

Methods

Two linkage maps were developed from BC₁F₁ and F₂ populations from the cross between yardlong bean (V. unguiculata ssp. unguiculata cv.-gr. sesquipedalis) accession JP81610 and wild cowpea (V. unguiculata ssp. unguiculata var. spontanea) accession TVnu457. Using these linkage maps, QTLs for 24 domestication-related traits were analysed and mapped. QTLs were detected for traits related to seed, pod, stem and leaf.

Key Results

Most traits were controlled by between one and 11 QTLs. QTLs for domestication-related traits show co-location on several narrow genomic regions on almost all linkage groups (LGs), but especially on LGs 3, 7, 8 and 11. Major QTLs for sizes of seed, pod, stem and leaf were principally located on LG7. Pleiotropy or close linkage of genes for the traits is suggested in these chromosome regions.

Conclusions

This is the first report of QTLs for domestication-related traits in yardlong bean. The results provide a foundation for marker-assisted selection of domestication-related QTLs in yardlong bean and enhance understanding of domestication in the genus Vigna.     

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.     

Genome wide linkage disequilibrium in Chinese asparagus bean (Vigna. unguiculata ssp. sesquipedialis) germplasm: implications for domestication history and genome wide association studies

Association mapping of important traits of crop plants relies on first understanding the extent and patterns of linkage disequilibrium (LD) in the particular germplasm being investigated. We characterize here the genetic diversity, population structure and genome wide LD patterns in a set of asparagus bean (Vigna. unguiculata ssp. sesquipedialis) germplasm from China. A diverse collection of 99 asparagus bean and normal cowpea accessions were genotyped with 1127 expressed sequence tag-derived single nucleotide polymorphism markers (SNPs). The proportion of polymorphic SNPs across the collection was relatively low (39%), with an average number of SNPs per locus of 1.33. Bayesian population structure analysis indicated two subdivisions within the collection sampled that generally represented the 'standard vegetable' type (subgroup SV) and the 'non-standard vegetable' type (subgroup NSV), respectively. Level of LD (r(2)) was higher and extent of LD persisted longer in subgroup SV than in subgroup NSV, whereas LD decayed rapidly (0-2 cM) in both subgroups. LD decay distance varied among chromosomes, with the longest (≈ 5 cM) five times longer than the shortest (≈ 1 cM). Partitioning of LD variance into within- and between-subgroup components coupled with comparative LD decay analysis suggested that linkage group 5, 7 and 10 may have undergone the most intensive epistatic selection toward traits favorable for vegetable use. This work provides a first population genetic insight into domestication history of asparagus bean and demonstrates the feasibility of mapping complex traits by genome wide association study in asparagus bean using a currently available cowpea SNPs marker platform.     

Development of PCR-based chloroplast DNA markers that characterize domesticated cowpea (Vigna unguiculata ssp. unguiculata var. unguiculata) and highlight its crop-weed complex

In 1992, Vaillancourt and Weeden discovered a very important mutation for studying cowpea evolution and domestication. A loss of a BamHI restriction site in chloroplast DNA characterized all domesticated accessions and a few wild (Vigna unguiculata ssp. unguiculata var. spontanea) accessions. In order to screen a larger number of accessions, primers were designed to check this mutation using PCR RFLP or direct PCR methods. Using these new primers, 54 domesticated cowpea accessions and 130 accessions from the wild progenitor were screened. The absence of haplotype 0 was confirmed within domesticated accessions, including primitive landraces from cultivar-groups Biflora and Textilis, suggesting that this mutation occurred prior to domestication. However, 40 var. spontanea accessions distributed from Senegal to Tanzania and South Africa showed haplotype 1. Whereas this marker could not be used to identify a precise center of origin, it did highlight the widely distributed cowpea crop-weed complex. Its very high frequency in West Africa could be interpreted as a result of either genetic swamping of the wild/weedy gene pool by the domesticated cowpea gene pool or as the result of domestication by ethnic groups focusing primarily on cowpea as fodder.     

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.     

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.     

Analysis of phylogenetic relationship among five mulberry (Morus) species using molecular markers.

Species identification in mulberry (Morus) continues to be a point of great debate among scientists despite the number of criteria such as floral characters, wood, and leaf anatomical and biochemical characters used to identify the species within this genus. However, no consensus system of classification has emerged. Hence, an investigation was undertaken with inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers to find out the possibility of using these DNA markers to confirm the identity of genotypes in a particular species. Fifteen ISSR and 15 RAPD primers generated 86% and 78% polymorphism, respectively, among 19 mulberry genotypes. The polymorphism among the species varied from 50% to 57% in ISSR markers and 31% to 53% in RAPD markers. Similarity coefficients were higher among the genotypes of M. latifolia, M. bombycis and M. alba. Cluster analyses separated genotypes of M. laevigata and M. indica from those of the other species. Population structure analysis of these species further showed high genetic differentiation coefficients (GST), high heterozygosity between two species (DST), and total heterozygosity among populations (Ht) coupled with considerably low gene flow (Nm) when M. laevigata was paired with other species. Based on these parameters and the result of cluster analysis it is concluded that M. laevigata can be considered as a separate species of mulberry, whereas the other four species may be grouped together and treated as subspecies.     

Active aggregation among sexes in bean flower thrips (Megalurothrips sjostedti) on cowpea (Vigna unguiculata)

Male sexual aggregations are a common territorial, mating‐related or resource‐based, behaviour observed in diverse organisms, including insects such as thrips. The influence of factors such as plant substrate, time of day, and geographic location on aggregation of thrips is uncertain, therefore we monitored the dispersion of male and female bean flower thrips (BFT), Megalurothrips sjostedti (Trybom) (Thysanoptera: Thripidae), on cowpea, Vigna unguiculata (L.) Walp. (Fabaceae), over three cowpea growth stages and across three cowpea‐growing areas of Kenya. Our results indicated that for all the crop growth stages, the density of BFTs varied over the time of day, with higher densities at 10:00, 13:00, and 16:00 hours than at 07:00 hours. Thrips densities did not differ among blocks at the budding stage, but they did at peak flowering and podding stages. Dispersion indices suggested that both male and female BFTs were aggregated. Active male aggregation occurred only on green plant parts and it varied across blocks, crop stages, and locations. Similarly, active female aggregation was observed in peak flowering and podding stages. Such active aggregation indicates a semiochemical or behaviour‐mediated aggregation. Identification of such a semiochemical may offer new opportunities for refining monitoring and management strategies for BFT on cowpea, the most important grain legume in sub‐Saharan Africa.     

Molecular identification and phylogenetic relationship of green algae,Spirogyra ellipsospora (Chlorophyta) using ISSR and rbcL markers

Spirogyra is found in a wide range of habitats, including small stagnant water bodies, rivers, and streams. Spirogyra ellipsospora is common in northern Thailand. Species identification of the Spirogyra species based only on morphological characteristics can be difficult. A reliable and accurate method is required to evaluate genetic variations. This study aims to apply molecular approaches for the identification of S. ellipsospora using microsatellites and rbcL markers. Based on DNA sequencing, the rbcL gene was sequenced and the data was analyzed using the BLAST (Basic Local Alignment Search Tool) program in the NCBI (National Center for Biotechnology Information) database. The sequence of S. ellipsospora from this study revealed definitive identity matches in the range of 99% for the consensus sequences of S. ellipsospora. The 10 primers of ISSR could be amplified by 92 amplification fragments. The DNA fragments and the rbcL sequence data grouped the Spirogyra specimens into two distinct clusters.     

Genetic relatedness of Portuguese almond cultivars assessed by RAPD and ISSR markers

Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyse the genetic diversity of Portuguese Prunus dulcis cultivars and their relationship to important foreign cultivars. Of the primers tested, 6 (out of 60) RAPD and 5 (out of 18) ISSR primers were selected for their reproducibility and high polymorphism. Out of 124 polymerase chain reaction fragments that were scored, 120 (96.8%) were polymorphic. All the plants could be discriminated and constitute a very heterogeneous group. Five unidentified almond plants found in the region of Foz Côa (north Portugal) and wild almond (P. webbii) from Italy and Spain were also included. Four main groups of plants could be distinguished: P. dulcis cultivars; one Foz Côa plant; P. webbii; and P. persica (outgroup). The segregating Foz Côa plant may represent a feral individual or a hybrid between P. dulcis and P. webbii.Abbreviations dNTP Deoxynucleotide triphosphate - CTAB Cetyltrimethylammonium bromide - ISSR Inter-simple sequence repeats - PCR Polymerase chain reaction - RAPD Randomly amplified polymorphic DNA - RASTM Regional Agricultural Services of Trás-os-Montes - TE Tris-EDTA buffer - UPGMA Unweighted pair group method with arithmetical averagesCommunicated by P. Puigdoménech     

Histological studies of damage by pod-sucking bugs (Heteroptera: Coreoidea) associated with cowpea Vigna unguiculata ssp. unguiculata in Nigeria

Histological studies were conducted on cowpea pods fed upon by the coreoid pod-sucking bugs, Anoplocnemis curvipes (Fabricius), Clavigralla tomentosicollis St?l, C. shadabi Dolling, Riptortus dentipes (Fabricius) and Mirperus jaculus (Thunberg). Various degrees of tissue and cellular disruption were apparent, especially in the brachysclereids. They were manifested in terms of plasmolysis, cell enlargement and cell wall disintegration depending on the coreoid species involved. The feeding site and its periphery had all the cells of the brachysclereids plasmolysed, with the parenchyma having broken cell walls in the case of A. curvipes. Riptortus dentipes and M. jaculus showed similar patterns of feeding activity but less extensive levels of damage. The Clavigralla spp., however, caused cell enlargement in the brachysclereids, and broken cell walls in both the brachysclereids and the parenchyma. Damage symptoms were observed in cells far away from the feeding sites of the bugs, suggesting the possibility of sucrase activity which has been reported to cause osmotic pump feeding in the Coreidae.     

Genetic relationship among wild,landraces and cultivars of hazelnut (Corylus avellana) from Portugal revealed through ISSR and AFLP markers

The genus Corylus, a member of the birch family Betulaceae, includes several species that are widely distributed throughout temperate regions of the Northern Hemisphere. This study assesses the genetic diversity in 26 international cultivars and 32 accessions of Corylus avellana L. from Portugal: 13 wild genotypes and 19 landraces. The genetic relationships among the 58 hazelnuts (Corylus avellana L.) were analyzed using inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) markers. Eighteen ISSR primers and seven AFLP primer pairs generated a total of 570 unambiguous and repeatable bands, respectively, from which 541 (95.03 %) were polymorphic for both markers. Genetic similarity index values ranged from 0.239 for wild types and cultivars to 0.143 for landraces and wild types. The genetic relationships were presented as a Neighbor-Joining method dendrogram and a two-dimensional principal coordinate analysis (PCoA) plot. The Neighbor-Joining dendrogram showed three main clusters, and the PCoA analysis has shown to be congruent with the hierarchical analysis. Bayesian analysis clustered all individuals into three groups showing a good separation among wild genotypes, landraces and cultivars. The genetic diversity found on wild genotypes and Portuguese landraces may provide relevant information for the diversity conservation and it will be useful in breeding programs and to identify local selections for preservation.     

Comparative assessment of feeding damage by pod-sucking bugs (Heteroptera: Coreoidea) associated with cowpea, Vigna unguiculata ssp. unguiculata in Nigeria

Feeding trials were conducted on three (young, mid-fill and mature) developmental stages of cowpea Vigna unguiculata ssp. unguiculata pods in the screenhouse using fourth instar nymphs and adults of Anoplocnemis curvipes (Fabricius), Riptortus dentipes (Fabricius), Mirperus jaculus (Thunberg), Clavigralla tomentosicollis St?l and C. shadabi Dolling. Anoplocnemis curvipes was observed to be the most damaging coreoid species causing a yield reduction of 26.4-51.7% followed by R. dentipes (24.4-29.4%), M. jaculus (21.9-26.9%), C. tomentosicollis (17.9-22.4%) and C. shadabi (15.9-20.4%). The fourth instar nymphs of each pod-sucking bug species caused a significantly higher cowpea yield reduction than their respective adults. Similarly, infestation on young pods compared to mid-fill and mature stages resulted in significantly higher yield reduction. The results suggest that infestation levels of two fourth instar nymphs of A. curvipes or three fourth instar nymphs of the other four pod-sucking bug species per young pod should be adequate for screening of cowpea varieties for resistance to the coreoid bugs.     

Genetic diversity of mango cultivars estimated using SCoT and ISSR markers

Two molecular marker systems, SCoT and ISSR were used for identification and genetic comparison analysis of 23 mango germplasm accessions collected within Guangxi province of China. Using 18 selected SCoT primers 158 bands were generated, of which 104 (65.82%) were polymorphic. Eighteen selected ISSR primers amplified 156 bands with 87 (55.77%) being polymorphic. The cultivars of Xiang Ya Mango type and their progeny have high genetic similarity with each other. The 23 cultivars were clustered into two major groups based on the SCoT analysis and three major groups based on the ISSR analysis with UPGMA. These clusters are in accordance with their known origins and main phenotypic characteristics. Our results indicated that the SCoT analysis better represents the actual relationships than ISSR analysis, although both analyses give similar results. The results also demonstrate that the SCoT marker system is useful for identification and genetic diversity analysis of mango cultivars.     

Inheritance of seed protein content and other agronomic characters in long bean (Vigna sesquipedalis Fruw.)

Summary Seven varieties of long bean, which included three local and four exotic, were crossed in a complete diallel. This was an attempt to study the inheritance of crude protein content, protein yield, flowering date, pod yield and yield components.Both additive and non-additive gene effects were responsible for the genetic variation in the diallel population. However, dominance variance was more important than additive variance in crude protein content, number of pods per plant and number of seeds per pod. For seed weight and pod length, additive variance was more important.The crude protein content, protein yield and number of pods per plant appeared to be controlled by overdominance effects. Partial dominance seemed to be the case for flowering date, pod length and seed weight; complete to overdominance for pod yield. High protein appeared to be associated with recessive genes whereas there was a general trend of high yielding parents carrying more dominant genes.