Forty genotypes of Lablab purpureus were evaluated using random amplified polymorphic DNA as markers. Out of these forty genotypes, two of them, viz., 23 (HighWorth, from India) and 32 (Rongai from Kenya), were the only two commercial varieties used for forage in Australia. Five of them were wild genotypes characterized by these small seed sized and hard seededness. The rest aiming 39 accessions were randomly selected from accessions held by the Australian Tropical Forage Genetic Resource Centre, Division of Tropical Crops and Pastures, CSIRO. Fifteen of these were from Asia and 13 from Africa. The origin of the other five was not clear. The DNA from all the forty accessions was isolated from seeds soaked in water for 24 hours. RAPD analysis was performed using 58 decomer oligonucleotide primers. Out of the 58 primers used for amplification, 10 performed poorly, while each of the other 48 primers generated one or more scorable fragments. In all about 273 fragments were scored. Of these, 188 showed polymorphism between at least one pairwise comparison among the 40 accessions and the remaining 85 were monomorphic. There was a high level of genetic variation among the 40 Lablab genotypes mainly due to the inclusion of the five (1 – F value was 0.055) wild accessions. Moderate dissimilarities were observed among the cultivated genotypes. Most of the Asian collections showed higher genetic variation than the African collections. One accession CPI 31113 collected from Uganda was highly divergent. Four of the five wild accessions, two from Zimbabwe and two from Zambia were closely related. There was no agreement as to whether Asia or Africa is the centre of origin of Lablab purpureus. Inspite of the more intensive breeding work in India, L. purpureus collections from Asia seems to possess a higher level of variation. African and Asian collections did not form distinct sub–groups. Instead, they were mixed together in the cluster analysis.
A manose–binding lectin called FRIL was identified in extracts of kidney bean (Phaseolus vulgaris) and hyacinth bean (Dolichos lablab [Lablab purpureus]), and evidence is provided that this protein appears to satisfy properties of a stem cell preservation factor. FRIL was first identified based on its ability to stimulate NIH 3T3 cells transferred with FL+3, a tyrosine kinase receptor central to regulation of stem cells. Molecular characterization from polypeptide sequencing and identification of the cDNA of hyacinth bean FRIL shows 78% amino acid identity with a mannose–binding lectin of hyacinth beans. Treatment of primitive hematopoietic progenitors in suspension culture with purified hyacinth FRIL alone was able to preserve cells for 1 month without medium changes. In vitro progenitor assays for human hematopoietic cells cultured 3 weeks in FRIL displayed small blast–like colonies that were capable of serial replating and persisted even in the presence of cytokines known to induce differentiation. These results suggest that FRIL is capable of preserving primitive progenitors in suspension culture for prolonged periods. FRIL’s clinical utility involving procedures for stem cell transplantation, tumor cell purging before autologous transplantation and ex vivo cultures used for expansion and stem cell gene therapy currently are being explored. Nucleotide sequences reported here have been submitted to Gen Bank database under accession number AF067417.
A linkage map of Lablab purpureus consisting of 127 RELP and 91 RAPD loci was constructed in an F2 population of 119 individuals. This population was derived from a cross between “Rongai” (an annual cultivar) and CP 24973 (a perennial wild accession). The map comprises 17 linkage groups and covers 1610 centi Morgans (cM) with an average distance of 7 cM between markers. Severe segregation distortions were observed with the very extreme situation where no parental type was recovered from the mapping population. These results strongly suggest the presence of a gene conferring preferential transmission from the maternal parent “Rongai”. It was also clear that while the majority of RAPD markers are valuable when used together with RELP or other stringent marker systems, they could be problematic when used solely in mapping exercise. Konduri, et al., (2000): Genetic mapping of the Lablab purpureus genome suggests the presence of Cuckoo genes in this species.
In order to facilitate the breeding activities, a linkage map of Lablab purpureus consisting of 127 RFLP and 91 RAPD loci was constructed in an F2 population of 119 individuals obtained from a cross between annual cultivar Rongai and the wild accession CPI 24973.
The linkage map reported here consists of 17 linkage groups although the haploid chromosome number of Lablab is only 12. This suggested that a proportion of the Lablab genome had not been covered by the existing markers. Genetic distance between some of the markers in the current map could have been overestimated due to the inclusion of a large number of RAPD markers. It seems evident that some RAPD markers, although appearing to have a clear segregation, could not be scored properly. Evidence for this includes: (1) 11 of the 102 RAPD markers showed no linkage between themselves or to any other marker, while all the 127 RFLP markers were mapped, (2) inclusion of a RAPD markers dramatically increased genetic distances between some RFLP markers. The map comprising 17 linkage groups and covers 1610 centi Morgans (cM) with an average distance of 7 cM between markers.
Severe segregation distortions were observed with the very extreme situation where no parental type was recovered from the mapping population. These results suggest the presence of a gene conferring preferential transmission from the maternal parent ‘Rongai’. This gene is similar to “Cuckoo chromosome” found in several grass species.
The phylogenetic relationships among 102 Lablab bean accessions, including 8 wild accessions, were investigated using random amplified polymorphic DNA (RAPD) markers. One hundred and one RAPD fragments were generated using 11 single 12 mer primers of arbitrary nucleotide sequences. Two primers, CMN-A22 and CMN-A31, generated robust and easily interpretable markers between wild and cultivated accessions. A dendrogram constructed based on genetic similarity clearly separated wild type accessions from cultivated accessions. Small values of genetic dissimilarity between and among wild and cultivated accessions and diversity of cultivated accessions without relating to geographical distribution suggest the short history of breeding works in the crop.
Although several linkage maps of mungbean have been constructed upon which most marker research into this crop has been based, they do not provide the same level of gene saturation seen in many other species. Hence, in this study, a genetic linkage map of mungbean (Vigna radiata, 2n = 2x = 22) consisting of 255 RFLP loci were developed using recombinant inbred loci population of 80 individuals. The population is derived from an inter–sub–specific cross between the cultivated mungbean variety “Berken” and a wild mungbean genotype “ACC 41”. 214 probes identified as being polymorphic detected 260 loci. 255 of these loci were assigned to 13 linkage groups varying in length from 8.7cM to 100.7 cM. This spanned a total genetic distance of 737.9 cM with an average distance between genetic markers of 3.0 cM and a maximum distance of 15.4cM. The mungbean map was compared to a previously published map of Lablab (L. purpureus, 2n = 2x = 24) using a common set of 65 RFLP probes. It was observed that mungbean shares a high level of homology with Lablab. Twelve of the 13 linkage groups of mungbean were able to be compared to 17 of the Lablab groups. It was also seen that group 2 and 13 of Lablab and group I of mungbean appear to be involved in a complicated chromosomal rearrangement event. Markers LPCS328 and LPCS 314 both mapped to group I of mungbean, but showed no linkage to any of the markers on group 2 of Lablab as was expected by those surrounding markers common to both genomes. Instead, there too probes map to group 13 of Lablab indicating that linkage group 13 of Lablab is an extra chromosome. This study thus facilitates more powerful identification of genes conditioning traits of interest in both the species by the comparative mapping approach.
A set of 68 simple sequence repeat (SSR) markers were selected from existing databases (including Medicago, soyabean, cowpea and groundnut) for the purpose of exploiting the transferability of SSRs across species and/or genera within the legume family. Primers were tested for cross-species and cross-genus fragment amplification with an array of 24 different legume accessions. Nearly one-third (30.78%) of the SSR primers screened generated reproducible and cross-genus amplicons. One hundred and seventeen cross-species polymorphic amplicons were identified and could be used as DNA markers. These polymorphic markers are now being used for characterization and evaluation of our collected and donated legume germplasm. The transferability of SSRs, mis-/multiple-primings, homologous/heterologous amplifications, single/multiple-amplicons and application of these amplicons as DNA markers are discussed.
To improve understanding of diversity of Lablab purpureus and establish relationships among 103 germplasm accessions collected from diverse geographic origins, amplified fragment length polymorphism (AFLP) markers were used. Four primer sets selected out of 16 produced 289 clear, repeatable polymorphisms. UPGMA analysis of similarity data clustered the accessions according to their subspecific taxonomic organization, i.e. subsp. purpureus and subsp. uncinatus, as well as to cultivated and wild forms. The well-represented landraces from Africa and Asia, belonging predominantly to subsp. purpureus, displayed moderate genetic diversity. Wild forms from Africa showed far greater levels of diversity that would justify taxonomic reassessment of the wild subsp. uncinatus. The molecular analysis identified forms that were collected in the wild in India but were genetically placed intermediate between wild and cultivated forms. As these plant types did not exist among the African accessions, it is suggested that they might represent escapes from early attempts of domestication. These results support the suggested pathway of domestication and distribution of L. purpureus from Africa to Asia.
The work describes cloning of genomic components of whitefly-transmitted Gemini virus infecting Lablab purpureus syn. Dolichos lablab. The genome characterization using PCR with geminiviral degenerate primers and DNA sequencing were used to describe the bipartite virus associated with yellow mosaic disease of D. Lablab. Full-length DNA-A and DNA-B clones were obtained. The DNA-A sequence analysis showed that the isolate was similar to other Mungbean yellow mosaic India virus (MYMIV) isolates reported earlier. The nucleotide sequence analysis of the full length DNA-A of virus isolate revealed more than 97% homology with Mungbean yellow mosaic India virus- [cowpea] (AF481865), while the DNA-B also showed 95% homology with MYMIV-[Cp] (AF503580) and MYMIV-[sb] (AY049771). The phylogenetic analysis of the present isolate showed close relationship to legume Gemini viruses. The nucleotide sequence analysis showed presence of six open reading frames (ORFs) in DNA-A with 2 ORFs aligned in sense and 4 ORFs in antisense orientation. Similarly, DNA-B contained two open reading frames (ORFs), one in sense and another in antisense orientation.
Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection.
Two molecular marker types namely, AFLP and a number of EST–derived markers from a range of legumes were used to test about 78 Lablab purpureus accessions collected from various sources representing southern states of India and 15 accessions from CPI (Australia) and ILRI (Ethiopia) for their genetic diversity. AFLP was carried out using 3 and 6 primer combinations, while EST–derived PCR markers used as primers were designed from a range of legume species. AFLP diversity analysis clearly showed limited genetic variation within the accessions representing local collections from UAS, Bangalore compared to a more diverse set of 15 accessions from International collections. The study as a whole is centered around the transferability of markers from related species to L. purpureus and the results suggest that there is a good source of legume related primers in databases from a wide range of well characterized species readily available for Lablab diversity and genome analysis. Furthermore, molecular analysis with EST–derived markers from a range of legume sequences concerned with AFLP analysis. Both these markers serve as useful markers for mapping studies as well as for future breeding programmes.
The aim of the present study was to characterize genetic diversity within a subset of 22 selected Lablab purpureus accessions collected mainly from India using both morphological traits and amplified fragment length polymorphism (AFLP) markers. The high number of clusters formed from both morphological and AFLP analysis indicates considerable genetic diversity in the accessions used for the study. The clusters formed based on the AFLP data were not in agreement with the clusters formed based on morphological data found in the study, indicating the presence of discrepancy between the two methods. For selection of good parental material for heterosis breeding program, the diversity results obtained through AFLP can be used to correlate with the pedigree relationship and morphological traits for genetic improvement in dolichos crop.
Simple Sequence Repeat (SSR) markers were developed from soybean expressed sequence tag (EST) sequences obtained from The European Molecular Biology Laboratory (EMBL) database. Of the 1517 loci considered 764(50.4%) and 573(37.8%) constituted tri and dinucleotide and, 73(4.8%), 44(2.9%), 38(2.5%) and 25(1.6%) represented mono, hexa, tetra and pentanucleotide repeats, respectively. Fifty of these SSR makers exhibited 100% transferability in Lablab purpureus. Eight out of 50 loci (16%) displayed length polymorphisms. BLAST results showed that the function of only 15 EST sequences (30%) of the selected 50 SSR sequences were known. The novel EST-SSR will be useful for developing new molecular markers of L. purpureus.
RAPD marker was used to evaluate genetic relationships among 11 genotypes of country bean, including first three genotypes were photo-insensitive and the rests were sensitive. The genotypes were grouped into two major clusters where photo-insensitive genotypes remain in cluster I and sensitive genotypes remain in cluster II. A total of 26 bands were detected, of which 57.69% were polymorphic and the remaining were monomorphic across all genotypes. A highest level of genetic distance was observed between CB04 and CB06 while the lowest level of genetic distance showed between CB01 and CB03. The highest similarity index between the genotypes CB01 and CB03 indicated less divergence between them. Low similarity indices were observed between CB04 and CB06, which indicated more divergence. Crossing between the genotypes with low similarity coefficient will manifest high heterosis. The identified genetically distinct cultivars could be potentially important source of germplasm for further improvement of country bean.
Genetic improvement of the thirty Hyacinth bean cultivars from different races were evaluated using RAPD markers which is essential to enhance the crop for economically and agriculturally important traits. RAPD markers were efficient in separating cultivars according to different races. Twenty six decamer primers could generate a total number of 148 bands out which 70.27% (104) were polymorphic. The number of amplification products produced by each primer varied from 4 to 9 with an average of 5.69 bands per primer. The size of amplified fragments ranged from 250 to 3000 bp. RAPD based dendrogram showed similarity ranged from 0.38 to 0.96. Genetic distances were concluded using Sorenson Dices similarity coefficient. The genetic relationship obtained with these markers was dissimilar to that provided by morpho-agronomical trait. Genetically distinct cultivars were identified that could be potentially important source of germplasm for further crop improvement programme in the country.
Lablab (Lablab Purpureus) is a legume of high nutritional value and broad social acceptance ranging from human food to soil fertility improvement to foarge. Information on genetic worth and diversity of lablab genotypes in Kenya is scanty and this is a great impediment to the improvement of the crop hence its utilization. This research was conducted to determine genetic diversity in lablab germplasm in Kenya using SSR primers. Thirteen genotypes comprising of collections from farmers and gene bank were planted in individual lines in the green house at Chepkoilel University College. CTAB DNA extraction from two weeks old leaves was done followed by PCR amplification using twenty one primer sets from commonbeans (Phaseolus vulgaris) and products separated on agarose gel. Four primers showed diversity in lablab, mapping a total of 14 amplicons with an average of 3.5 bands per primer. Clustering by UPGMA and PCoA showed similarity between genotypes grown by farmers and gene bank accessions. Genetic distance computed using Popgene ranged between 0.000 and 0.620, suggesting narrow variability among the materials. The genetic base of cultivated lablab is relatively narrow and needs to be expanded.
The development of expressed sequence tags (ESTs) has provided a useful source for the mining of simple sequence repeat (SSR) markers. The hyacinth bean (Lablab purpureus L.) is one of the most ancient and important legume crops in the world, and is widely distributed in tropical and sub-tropical regions, but currently only limited molecular markers are available. The aimof the present study is to develop EST-SSRsfor hyacinth beans to investigate the genetic structure and diversity of different populations originating from China and Africa. A total of 459 hyacinth beanESTs from the National Center for Biotechnology Information (NCBI) database were downloaded and analyzed to search for SSRs with a minimum of 12 repeating nucleotides. Finally, 22 microsatellites were identified in 420 unigenes, indicating that merely 4.79% of the sequences contained SSRs. EST-SSR loci weresubsequently screened on 24 hyacinth bean accessionscollected from both China and Africa. Among 22 EST-SSRs, 11 loci showed polymorphism and revealed two to four alleles per locus. Tri-nucleotide motifs were the most common type of repeats, accounting for 54.55% of the total, followed by di-nucleotiderepeats.The polymorphicinformation content (PIC) values ranged from 0.0767 to 0.4864, with a mean of 0.286.Furthermore, both principal coordinate analysis (PCA) and phylogenetic tree analysisindicated that all accessions were clustered into two main groups, and that all 19 Chinese accessions were clustered into the singlegroup. These results suggest that there is a narrow genetic basis for Chinese hyacinth bean accessions. In summary, the results of this study suggest that the data mining of EST databases is a feasible method for generating EST-SSR markers for hyacinth bean, and these newly developed EST-SSR markers will be useful for further taxonomy, molecular breeding and comparative mapping studies.
Photoperiod is one of the important environmental factors that influence adaptation and productivity of dolichos bean through its effect on days to flowering. Thirty-six dolichos bean germplasm accessions were characterized for photoperiod-induced sensitivity to flowering time (PSFT) and were assayed using SSR markers during 2014 at Department of Genetics and Plant Breeding, University of Agricultural Sciences, Bengaluru. The germplasm accessions differed for their degree of PSFT, which varied from 0.63 to 1.00 depending on the extent of delay in flowering during long days compared to that in short days. The accessions were classified as photoperiod sensitive (>22 days delay in flowering), moderately photoperiod sensitive (9-22 days delay in flowering) and photoperiod sensitive (up to 8 days delay in flowering). The degree of PSFT was independent of growth habit, pod and seed traits. Twenty-three of 33 SSR markers among the 36 accessions were found polymorphic. The number of alleles per SSR locus varied from 1 to 3 with an average of 2.1 alleles. The estimates of PIC ranged from 0.00 to 0.62 with an average of 0.24. The three pairs of germplasm accessions such as GL 385 and GL 576, GL 385 and GL 506 and GL 382 and GL 576 were not only contrasting for PSFT but also polymorphic to most number (10) of SSR markers. It is suggested to use these pairs of germplasm accessions as putative parents for developing populations for SSR marker-assisted mapping genomic regions controlling PSFT in dolichos bean.