Genetic localization of a bruchid resistance gene and its relationship to insecticidal cyclopeptide alkaloids, the vignatic acids, in mungbean (Vigna radiata L. Wilczek)

Bruchid resistance, controlled by a single dominant gene (Br) in a wild mungbean accession (TC1966), has been incorporated into cultivated mungbean (Vigna radiata). The resistance gene simultaneously confers inhibitory activity against the bean bug, Riptortus clavatus Thunberg (Hemiptera: Alydidae). The resultant isogenic line (BC₂₀ generation) was characterized by the presence of a group of novel cyclopeptide alkaloids, called vignatic acids. A linkage map was constructed for Br and the vignatic acid gene (Va) using restriction fragment length polymorphism (RFLP) markers and a segregating BC₂₀F₂ population. By screening resistant and susceptible parental lines with 479 primers, eight randomly amplified polymorphic DNA (RAPD) markers linked to Br were identified and cloned for use as RFLP probes. All eight RAPD-based markers, one mungbean, and four common bean genomic clones were effectively integrated around Br within a 3.7-cM interval. Br was mapped to a 0.7-cM segment between a cluster consisting of six markers and a common bean RFLP marker, Bng110. The six markers are closest to the bruchid resistance gene, approximately 0.2?cM away. The vignatic acid gene, Va, cosegregated with bruchid resistance. However, one individual was identified in the BC₂₀F₂ population that retained vignatic acids in spite of its bruchid susceptibility. Consequently, Va was mapped to a single locus at the same position as the cluster of markers and 0.2?cM away from Br. These results suggest that the vignatic acids are not the principal factors responsible for bruchid resistance in V. radiata but will facilitate the use of map-based cloning strategies to isolate the Br gene.     

RFLP mapping of a major bruchid resistance gene in mungbean (Vigna radiata,L. Wilczek)

Summary Bruchids (genus Callosobruchus) are among the most destructive insect pests of mungbeans and other members of the genus, Vigna. Genetic resistance to bruchids was previously identified in a wild mungbean relative, TC1966. To analyze the underlying genetics, accelerate breeding, and provide a basis for map-based cloning of this gene, we have mapped the TC1966 bruchid resistance gene using restriction fragment length polymorphism (RFLP) markers. Fifty-eight F₂ progeny from a cross between TC1966 and a susceptible mungbean cultivar were analyzed with 153 RFLP markers. Resistance mapped to a single locus on linkage group VIII, approximately 3.6 centimorgans from the nearest RFLP marker. Because the genome of mungbean is relatively small (estimated to be between 470 and 560 million base pairs), this RFLP marker may be suitable as a starting point for chromosome walking. Based on RFLP analysis, an individual was also identified in the F₂ population that retained the bruchid resistance gene within a tightly linked double crossover. This individual will be valuable in developing resistant mungbean lines free of linkage drag.     

Differences in responses to iron deficiency among various cultivars of mungbean (Vigna radiata (L.) Wilczek)

Ten mungbean cultivars were evaluated for their resistance to iron deficiency in view of chlorosis symptoms, plant growth and seed yield under field conditions on a calcareous soil in Thailand. The KPS2 cultivar was highly susceptible; the KPS1, PSU1 and Pag-asa 1 cultivars were somewhat susceptible; the VC1163B cultivar was moderately tolerant; the CN36, CN60, UT1 and CNM-I cultivars were tolerant; and the CNM8509B cultivar was very tolerant to iron deficiency. Foliar application of a solution of 5 g L^-1 ferrous sulphate was effective in correcting chlorosis that was induced by iron deficiency, and it enhanced both the growth and the yield of susceptible cultivars. Compared with the susceptible cultivar KPS2, the tolerant cultivar UT1 had a greater ability to lower the pH of the nutrient solution in response to iron deficiency. The root-associated Fe³⁺-reduction activity of UT1 that had been grown in -Fe medium was similar to that of the plants grown in +Fe medium when the acidification of the medium occurred. Acidification of the medium in response to iron deficiency might contribute to the efficient solubilization of iron from calcareous soils, and it related more closely to the resistance to iron deficiency than Fe³⁺ reduction by roots in mungbean cultivars.     

Mapping of the QTL (quantitative trait locus) conferring partial resistance to leaf blast in rice cultivar Chubu 32

The rice cultivar Chubu 32 possesses a high level of partial resistance to leaf blast. The number and chromosomal location of genes conferring this resistance were detected by restriction fragment length polymorphism (RFLP) linkage mapping and quantitative trait locus (QTL) analysis. For the mapping, 149 F₃ lines derived from the cross between rice cultivar Norin 29, with a low level of partial resistance, and Chubu 32 were used, and their partial resistance to leaf blast was assessed in upland nurseries. A linkage map covering six chromosomes and consisting of 36 RFLP markers was constructed. In the map, only one significant QTL (LOD>2.0) for partial resistance was detected on chromosome 11. This QTL explained 45.6% of the phenotypic variation. The segregation ratio of the F3 lines was 3:1 for partial resistance to susceptibility. These results suggest that the partial resistance in Chubu 32 is controlled by a major gene. Received: 15 March 2001 / Accepted: 13 August 2001     

Mapping the genome of rapeseed (Brassica napus L.). I. Construction of an RFLP linkage map and localization of QTLs for seed glucosinolate content

A linkage map of the rapeseed genome comprising 204 RFLP markers, 2 RAPD markers, and 1 phenotypic marker was constructed using a F₁ derived doubled haploid population obtained from a cross between the winter rapeseed varieties Mansholt's Hamburger Raps and Samourai. The mapped markers were distributed on 19 linkage groups covering 1441 cM. About 43% of these markers proved to be of dominant nature; 36% of the mapped marker loci were duplicated, and conserved linkage arrangements indicated duplicated regions in the rapeseed genome. Deviation from Mendelian segregation ratios was observed for 27.8% of the markers. Most of these markers were clustered in 7 large blocks on 7 linkage groups, indicating an equal number of effective factors responsible for the skewed segregations. Using cDNA probes for the genes of acyl-carrier-protein (ACP) and -ketoacyl-ACP-synthase I (KASI) we were able to map three and two loci, respectively, for these genes. The linkage map was used to localize QTLs for seed glucosinolate content by interval mapping. Four QTLs could be mapped on four linkage groups, giving a minimum number of factors involved in the genetic control of this trait. The estimated effects of the mapped QTLs explain about 74% of the difference between both parental lines and about 61.7 % of the phenotypic variance observed in the doubled haploid mapping population.     

Three QTLs from Lycopersicon peruvianum confer a high level of resistance to Clavibactermichiganensis ssp. michiganensis

Lycopersicon peruvianum LA2157 originates from 1650 m above sea level and harbours several beneficial traits for cultivated tomatoes such as cold tolerance, nematode resistance and resistance to bacterial canker (Clavibacter michiganensis ssp. michiganensis). In order to identify quantitative trait loci (QTLs) for bacterial canker resistance, a QTL mapping approach was carried out in an F₂ population derived from the interspecific F₁ between Lycopersicon esculentum cv Solentos and L. peruvianum LA2157. Three QTLs for resistance mapped to chromosomes 5, 7 and 9 respectively. The resistance loci were additive and co-dominant with the QTL on chromosome 7 explaining the largest part of the variation for resistance in the F₂ population. The combination of this QTL with either of the other two QTLs conferred a resistance similar to the level in the resistant parent L. peruvianum. Some RFLP markers flanking this QTL on chromosome 7 were converted into SCAR markers allowing efficient marker-assisted selection of plants with high resistance to bacterial canker. Received: 26 February 1999 / Accepted: 12 March 1999     

Genetic mapping of a new semi-dwarf gene, <Emphasis Type="Italic">sd-t(t)</Emphasis>, in indica rice and estimating of the physical distance of the mapping region

Application and functional study of dwarf and semi-dwarf genes are of great importance to both crop breeding and molecular biology. A new semi-dwarf gene, sd-t(t), non-allelic to sd-1, had been identified in an indica rice variety, Aitaiyin 2. In this study the gene was genetically mapped by using an F₂ population, which consisted of 474 individuals developed from a cross between Aitaiyin 2 and B30. The sd-t(t) gene was located between the RFLP markers R514 and R1408B with a distance of 1.1 cM to R514, and 4.5 cM to R1408B on chromosome 4. A physical contig covering the sd-t(t) mapping region was further constructed by screening a BAC library with R514 and R1408B as probes, and the physical distance between R514 and R1408B was estimated at approximately 147 kb. This result will facilitate map-based cloning of the sd-t(t) gene.     

Vegetative multiplication and patch colonisation of Asarum europaeum subsp. europaeum L. (Aristolochiaceae) inferred by a combined morphological and molecular study

Asarum europaeum subsp. europaeum (Aristolochiaceae) is a rhizomatous herb forming distinct patches in calcareous broadleaved forests. Within natural stands, patches were mapped. In two regions, at least four patches were dug out, and connections between leaf modules through rhizomatous spacers were checked for signs of clonal reproduction (decay, breaking). Modules were sampled for amplified fragment length polymorphism (AFLP) fingerprinting to test whether they represent unique genets or are merigenets of a larger genet (split up by clonal reproduction), respectively.Morphologically, merigenet-relationships were only revealed in few cases with disrupted spacers between modules. With the obtained AFLP profiles for two primer combinations, the samples could be assigned to genets; clonal descendants of the same genet were readily identified. In one patch analysed in detail, 18 samples from 17 unconnected “plants” belonged to only two genets, which were morphologically divided into two and 15 merigenets, respectively. These two genets probably belonged to different maternal lineages and came into contact after lateral spread from the established clones. They showed divergent affinities to samples from adjacent patches (which all represented unique genets).The findings support the suitability of the combined morpho-ecological and molecular approach: compared to either method alone, it allows a more detailed analysis and interpretation of the fine-scale clonal structure, patch colonisation and especially of vegetative multiplication (with morpho-ecological studies to discern clonal growth and clonal reproduction and AFLP fingerprinting for genet and merigenet identification, respectively).     

Mapping of RFLP and qualitative trait loci in Brassica rapa and comparison to the linkage maps of B. napus,B. oleracea,and Arabidopsis thaliana

A linkage map of restriction fragment length polymorphisms (RFLPs) was constructed for oilseed, Brassica rapa, using anonymous genomic DNA and cDNA clones from Brassica and cloned genes from the crucifer Arabidopsis thaliana. We also mapped genes controlling the simply inherited traits, yellow seeds, low seed erucic acid, and pubescence. The map included 139 RFLP loci organized into ten linkage groups (LGs) and one small group covering 1785 cM. Each of the three traits mapped to a single locus on three different LGs. Many of the RFLP loci were detected with the same set of probes used to construct maps in the diploid B. oleracea and the amphidiploid B. napus. Comparisons of the linkage arrangements between the diploid species B. rapa and B. oleracea revealed six LGs with at least two loci in common. Nine of the B. rapa LGs had conserved linkage arrangements with B. napus LGs. The majority of loci in common were in the same order among the three species, although the distances between loci were largest on the B. rapa map. We also compared the genome organization between B. rapa and A. thaliana using RFLP loci detected with 12 cloned genes in the two species and found some evidence for a conservation of the linkage arrangements. This B. rapa map will be used to test for associations between segregation of RFLPs, detected by cloned genes of known function, and traits of interest.     

Salt stress alleviation in transgenic Vigna mungo L. Hepper (blackgram) by overexpression of the glyoxalase I gene using a novel Cestrum yellow leaf curling virus (CmYLCV) promoter

A reproducible and efficient transformation system utilizing the nodal regions of embryonal axis of blackgram (Vigna mungo L. Hepper) has been established via Agrobacterium tumefaciens. This is a report of genetic transformation of Vigna mungo for value addition of an agronomic trait, wherein the gene of interest, the glyoxalase I driven by a novel constitutive Cestrum yellow leaf curling viral promoter has been transferred for alleviating salt stress. The overexpression of this gene under the constitutive CaMV 35S promoter had earlier been shown to impart salt, heavy metal and drought stress tolerance in the model plant, tobacco. Molecular analyses of four independent transgenic lines performed by PCR, Southern and western blot revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 2.25% and the time required for the generation of transgenic plants was 10–11 weeks. Exposure of T1 transgenic plants as well as untransformed control plants to salt stress (100 mM NaCl) revealed that the transgenic plants survived under salt stress and set seed whereas the untransformed control plants failed to survive. The higher level of Glyoxalase I activity in transgenic lines was directly correlated with their ability to withstand salt stress. To the best of our knowledge this is the only report of engineering abiotic stress tolerance in blackgram. Prasanna Bhomkar, Chandrama P. Upadhyay are contributed equally. An erratum to this article can be found at     

Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the housefly (Musca domestica)

The voltage-sensitive sodium channel is generally regarded as the primary target site of dichlorodiphenyl-trichloro-ethane (DDT) and pyrethroid insecticides, and has been implicated in the widely reported mechanism of nerve insensitivity to these compounds. This phenomenon is expressed as knockdown resistance (kdr) and has been best characterised in the housefly where several putative alleles, including the more potent super-kdr factor, have been identified. We report the isolation of cDNA clones containing part of a housefly sodium channel gene, designated Msc, which show close homology to the para sodium channel of Drosophila (99% amino acid identity within the region of overlap). Using Southern blots of insect DNA, restriction fragment length polymorphisms (RFLPs) at the Msc locus were identified in susceptible, kdr and super-kdr housefly strains. These RFLPs showed tight linkage to resistance in controlled crosses involving these strains, thus providing clear genetic evidence that kdr, and hence pyrethroid mode of action, is closely associated with the voltage-sensitive sodium channel.     

Development of leucine-rich repeat polymorphism, amplified fragment length polymorphism, and sequence characterized amplified region markers to the Cronartium ribicola resistance gene Cr2 in western white pine ( Pinus monticola )

White pine blister rust (WPBR), caused by Cronartium ribicola, is a devastating disease in Pinus monticola and other five-needle pines. Pyramiding a major resistance gene (Cr2) with other resistance genes is an important component of integrated strategies to control WPBR in P. monticola. To facilitate this strategy, the objective of the present study was to identify leucine-rich repeat (LRR) polymorphisms, amplified fragment length polymorphisms (AFLPs), and sequence characterized amplified region (SCAR) markers linked to the western white pine Cr2 (BSA) gene for precise gene mapping. Bulked segregant analysis and haploid segregation analysis allowed the identification of 11 LRR polymorphisms and five AFLP markers in the Cr2 linkage. The closest LRR markers were 0.53 Kosambi cM from Cr2 at either end. After marker cloning and sequencing, AFLP marker EacccMccgat-365 and random polymorphic DNA marker U570–843 were converted successfully into SCAR markers. For a potential application in marker-assisted selection (MAS), these two SCAR markers were verified in two western white pine families. This study represents the first report of LRR-related DNA markers linked to C. ribicola resistance in five-needle pines. These findings may help further candidate gene identification for disease resistance in a conifer species.     

Development and utility of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLPs) linked to the Fom-2 fusarium wilt resistance gene in melon (Cucumis melo L.)

Fusarium wilt, caused by Fusarium oxysporum Schlecht f. sp. melonis Snyder & Hans, is a worldwide soil-borne disease of melon (Cucumis melo L.). Resistance to races 0 and 1 of Fusarium wilt is conditioned by the dominant gene Fom-2. To facilitate marker-assisted backcrossing with selection for Fusarium wilt resistance, we developed cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) markers by converting RAPD markers E07 (a 1.25-kb band) and G17 (a 1.05-kb band), respectively. The RAPD-PCR polymorphic fragments from the susceptible line ’Vedrantais’ were cloned and sequenced in order to construct primers that would amplify only the target fragment. The derived primers, E07SCAR-1/E07SCAR-2 from E07 and G17SCAR-1/G17SCAR-2 from G17, yielded a single 1.25-kb fragment (designated SCE07) and a 1.05-kb fragment (designated SCG17) (the same as RAPD markers E07 and G17), respectively, from both resistant and susceptible melon lines, thus demonstrating locus-specific associated primers. Potential CAPS markers were first revealed by comparing sequence data between fragments amplified from resistant (PI 161375) and susceptible (’Vedrantais’) lines and were then confirmed by electrophoresis of restriction endonuclease digestion products. Twelve restriction endonucleases were evaluated for their potential use as CAPS markers within the SCE07 fragment. Three (BclI, MspI, and BssSI) yielded ideal CAPS markers and were subsequently subjected to extensive testing using an additional 88 diverse melon cultigens, 93 and 119 F₂ individuals from crosses of ’Vedrantais’ x PI 161375 and ’Ananas Yokneam’×MR-1 respectively, and 17 families from a backcross BC₁S₁ population derived from the breeding line ’MD8654’ as a resistance source. BclI- and MspI-CAPS are susceptible-linked markers, whereas the BssSI-CAPS is a resistant-linked marker. The CAPS markers that resulted from double digestion by BclI and BssSI are co-dominant. Results from BclI- and MspI-CAPS showed over 90% accuracy in the melon cultigens, and nearly 100% accuracy in the F₂ individuals and BC₁S₁ families tested. This is the first report of PCR-based CAPS markers linked to resistance/susceptibility for Fusarium wilt in melon. The RFLP markers resulting from probing with a clone-derived 1.05-kb SCG17 PCR fragment showed 85% correct matches to the disease phenotype. Both the CAPS and RFLP markers were co-dominant, easier to score, and more accurate and consistent in predicting the melon phenotype than the RAPD markers from which they were derived. Received: 28 July 1998 / Accepted: 7 December 1998     

Genetic mapping and localization of a major QTL for seedling resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis)

Downy mildew caused by the fungus Peronospora parisitica is a serious threat to members of the Brassicaceae family. Annually, a substantial loss of yield is caused by the widespread presence of this disease in warm and humid climates. The aim of this study was to localize the genetic factors affecting downy mildew resistance in Chinese cabbage (Brassica rapa ssp. pekinensis). To achieve this goal, we improved a preexisting genetic map of a doubled-haploid population derived from a cross between two diverse Chinese cabbage lines, 91-112 and T12-19, via microspore culture. Microsatellite simple sequence repeat (SSR) markers, isozyme markers, sequence-related amplified polymorphism markers, sequence-characterized amplified region markers and sequence-tagged-site markers were integrated into the previously published map to construct a composite Chinese cabbage map. In this way, the identities of linkage groups corresponding to the Brassica A genome reference map were established. The new map contains 519 markers and covers a total length of 1,070 cM, with an average distance between markers of 2.06 cM. All markers were designated as A1–A10 through alignment and orientation using 55 markers anchored to previously published B. rapa or B. napus reference maps. Of the 89 SSR markers mapped, 15 were newly developed from express sequence tags in Genbank. The phenotypic assay indicated that a single major gene controls seedling resistance to downy mildew, and that a major QTL was detected on linkage group A8 by both interval and MQM mapping methods. The RAPD marker K14-1030 and isozyme marker PGM flanked this major QTL in a region spanning 2.9 cM, and the SSR marker Ol12G04 was linked to this QTL by a distance of 4.36 cM. This study identified a potential chromosomal segment and tightly linked markers for use in marker-assisted selection to improve downy mildew resistance in Chinese cabbage.     

Correlation between grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus and the genetic insert-deletion polymorphisms in promoter and intron of RIG-I gene

RIG-I (retinoic acid-inducible gene I) is an essential cytosolic pathogen recognition receptor that binds to a variety of viral RNA or DNA to induce type I interferons. In the present study, insert–deletion polymorphisms in promoter and introns of CiRIG-I (Ctenopharyngodon idella RIG-I) were explored, their associations with resistance/susceptibility to grass carp reovirus (GCRV) were analyzed. To this end, genomic sequence of CiRIG-I gene was obtained, and twenty pairs of primers were prepared for the detection of insert–deletion polymorphisms. Five insert–deletion mutations were found, a 2-bp mutation and an 8-bp mutation existed in the promoter and other three sizes in 74 bp, 146 bp and 53 bp were sited in the intron 8. After a challenge experiment, only the genotype and allele of − 740 insert–deletion mutation in the promoter and allele of 6804 insert–deletion mutation were significantly associated with resistance/susceptibility to GCRV among the five mutations (P < 0.05). To further identify this correlation, another independent challenge test was carried out. The result revealed that the cumulative mortality in ins/ins genotype individuals (43.75%) at − 740 insert–deletion mutation was significantly lower than that in ins/del (72.09%) and del/del (74.19%) genotypes (P < 0.05). Linkage disequilibrium and haplotype analysis showed 6610 insert–deletion mutation and 6804 insert–deletion mutation were linkage disequilibrium. The haplotype ins–ins (6610ins–6804ins) was significantly susceptible to GCRV, and ins–del (6610ins–6804del) was significantly resistant to GCRV (P < 0.05). Those could be potential gene markers for the future molecular selection of strains that are resistant to GCRV.