Identification of RFLP and NBS/PK profiling markers for disease resistance loci in genetic maps of oats

Two of the domains most widely shared among R genes are the nucleotide binding site (NBS) and protein kinase (PK) domains. The present study describes and maps a number of new oat resistance gene analogues (RGAs) with two purposes in mind: (1) to identify genetic regions that contain R genes and (2) to determine whether RGAs can be used as molecular markers for qualitative loci and for QTLs affording resistance to Puccinia coronata. Such genes have been mapped in the diploid A. strigosa × A. wiestii (Asw map) and the hexaploid MN841801-1 × Noble-2 (MN map). Genomic and cDNA NBS-RGA probes from oat, barley and wheat were used to produce RFLPs and to obtain markers by motif-directed profiling based on the NBS (NBS profiling) and PK (PK profiling) domains. The efficiency of primers used in NBS/PK profiling to amplify RGA fragments was assessed by sequencing individual marker bands derived from genomic and cDNA fragments. The positions of 184 markers were identified in the Asw map, while those for 99 were identified in the MN map. Large numbers of NBS and PK profiling markers were found in clusters across different linkage groups, with the PK profiling markers more evenly distributed. The location of markers throughout the genetic maps and the composition of marker clusters indicate that NBS- and PK-based markers cover partly complementary regions of oat genomes. Markers of the different classes obtained were found associated with the two resistance loci, PcA and R-284B-2, mapped on Asw, and with five out of eight QTLs for partial resistance in the MN map. 53 RGA-RFLPs and 187 NBS/PK profiling markers were also mapped on the hexaploid map A. byzantina cv. Kanota × A. sativa cv. Ogle. Significant co-localization was seen between the RGA markers in the KO map and other markers closely linked to resistance loci, such as those for P. coronata and barley yellow dwarf virus (Bydv) that were previously mapped in other segregating populations.     

A molecular linkage map of cultivated oat.

A molecular linkage map of cultivated oat composed of 561 loci has been developed using 71 recombinant inbred lines from a cross between Avena byzantina cv. Kanota and A. sativa cv. Ogle. The loci are mainly restriction fragment length polymorphisms detected by oat cDNA clones from leaf, endosperm, and root tissue, as well as by barley leaf cDNA clones. The loci form 38 linkage groups ranging in size from 0.0 to 122.1 cM (mean, 39 cM) and consist of 2-51 loci each (mean, 14). Twenty-nine loci remain unlinked. The current map size is 1482 cM and the total size, on the basis of the number of unlinked loci, is estimated to be 2932.0 cM. This indicates that this map covers at least 50% of the cultivated oat genome. Comparisons with an A-genome diploid oat map and between linkage groups exhibiting homoeology to each other indicate that several major chromosomal rearrangements exist in cultivated oat. This map provides a tool for marker-assisted selection, quantitative trait loci analyses, and studies of genome organization in oat.     

Inheritance and mapping of a powdery mildew resistance gene introgressed from Avena macrostachya in cultivated oat

The powdery mildew resistance from Avena macrostachya was successfully introgressed into hexaploid oat (A. sativa). Genetic analysis of F₁, F₂, F₃ and BC₁ populations from two powdery-mildew resistant introgression lines revealed that the resistance is controlled by a dominant gene, tentatively designated Eg-5. Molecular marker analysis was conducted using bulked-segregant analysis in two segregating F₃ populations. One codominant simple sequence repeats (SSR) marker AM102 and four AFLP-derived PCR-based markers were successfully developed. The SSR marker AM102 and the STS marker ASE41M56 were linked to the gene Eg-5, with genetic distances of 2 and 0.4 cM, respectively, in both mapping populations. Three STS markers (ASE45M56, ASE41M61, ASE36M55) co-segregated with Eg-5 in one population while two (ASE45M56, ASE36M55) of them linked to Eg-5 with a genetic distance of 1 cM in another population. The gene was further mapped to be in a region corresponding to linkage group 22_44+18 in the Kanota × Ogle (KO) hexaploid oat map by comparative mapping. To our knowledge, this is the first report of mapping powdery-mildew resistance in hexaploid oat. The new resistance source of A. macrostachya, together with the tightly linked markers identified here, could be beneficial in oat breeding programmes.     

A restriction fragment length polymorphism based linkage map of a diploid Avena recombinant inbred line population.

A population of 100 F6-derived recombinant inbred lines was developed from the cross of two diploid (2n = 14) Avena accessions, CI3815 (A. strigosa) and C11994 (A. wiestii). Restriction fragment length polymorphism (RFLP) probes previously mapped in other grass species were used to develop a framework linkage map suitable for comparative genetics. Nine linkage groups were identified among the 181 loci mapped, with an average interlocus distance of 5 cM, and a total genetic map length of 880 cM. A cluster of five tightly linked crown rust resistance genes (Pca) was localized on the map, as were five loci identified by disease resistance gene analogs from maize, sorghum, and wheat. None of the five loci identified by the gene analogs were linked to the Pca locus. The linkage map was compared with previously published diploid and hexaploid linkage maps in an attempt to identify homologous or homoeologous chromosomes between populations. Locus orders and linkage relationships were poorly conserved between the A. strigosa x A. wiestii map and other Avena maps. In spite of mapping complications due to duplications within a basic genome a well as the allopolyploid constitution of many Avena species, such map comparisons within Avena provide further evi dence of substantial chromosomal rearrangement between species within Avena.     

An integrated molecular linkage map of diploid wheat based on a <Emphasis Type="Italic">Triticum boeoticum</Emphasis> × <Emphasis Type="Italic">T. monococcum</Emphasis> RIL population

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum × Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A^m. With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A^m of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.     

A linkage map of diploid Avena based on RFLP loci and a locus conferring resistance to nine isolates of Puccinia coronata var. ‘avenae’

An F₂ oat population was produced by crossing the diploid (n=7) species Avena strigosa (CI 3815) with A. wiestii (CI 1994), resistant and susceptible, respectively, to 40 isolates of Puccinia coronata, the causal agent of crown rust. Eighty-eight F₂ individuals were used to construct an RFLP linkage map representing the A genome of cultivated hexaploid oat. Two hundred and eight RFLP loci have been placed into 10 linkage groups. This map covers 2416 cM, with an average of 12 cM between RFLP loci. Eighty-eight F₃ lines, derived from F₂ individuals used to construct the map, were screened for resistance to 9 isolates of P. coronata. One locus, Pca, was found to confer a dominant resistance phenotype to isolates 203, 258, 263, 264B, 290, 298, 325A, and 345. Pca also conferred resistance to isolate 276; however, an unlinked second gene may also be involved.Journal Paper No. 15143 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 3134 and 2447     

花椰菜遗传图谱的构建及NBS-LRR类抗性同源基因在图谱中的定位

利用AFLP和NBS profiling技术, 以花椰菜自交系“AD白花”与高代自交不亲和系“C-8”杂交得到的F1代自交产生的F2代分离群体为材料, 构建了第一个花椰菜遗传连锁图谱。该图谱由234个AFLP标记和21个NBS标记构成了9个连锁群, 总图距为668.4 cM, 标记间平均距离为2.9 cM。每个连锁群包含的位点数从12到47个, 相邻两标记之间的距离范围是0~14.9 cM。NBS标记分布在8个连锁群中, 这些标记大部分聚在一起。本研究为今后的基因定位及重要农艺性状的分析提供框架图。此外, 研究NBS profiling 方法在花椰菜中的稳定性和有效性以及NBS-LRR类RGA在花椰菜基因组中的分布和特点。     

Mapping of genome-wide resistance gene analogs (RGAs) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Isolation and mapping of genome-wide resistance (R) gene analogs (RGAs) is of importance in identifying candidate(s) for a particular resistance gene/QTL. Here we reported our result in mapping totally 228 genome-wide RGAs in maize. By developing RGA-tagged markers and subsequent genotyping a population consisting of 294 recombinant inbred lines (RILs), 67 RGAs were genetically mapped on maize genome. Meanwhile, in silico mapping was conducted to anchor 113 RGAs by comparing all 228 RGAs to those anchored EST and BAC/BAC-end sequences via tblastx search (E-value < 10⁻²⁰). All RGAs from different mapping efforts were integrated into the existing SSR linkage map. After accounting for redundancy, the resultant RGA linkage map was composed of 153 RGAs that were mapped onto 172 loci on maize genome, and the mapped RGAs accounted for approximate three quarters of the genome-wide RGAs in maize. The extensive co-localizations were observed between mapped RGAs and resistance gene/QTL loci, implying the usefulness of this RGA linkage map in R gene cloning via candidate gene approach. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Wenkai Xiao, Jing Zhao and Shengci Fan have contributed equally to this research.     

Microsatellite mapping of a Triticum urartu Tum. derived powdery mildew resistance gene transferred to common wheat (Triticum aestivum L.)

A powdery mildew resistance gene from Triticum urartu Tum. accession UR206 was successfully transferred into hexaploid wheat (Triticum aestivum L.) through crossing and backcrossing. The F₁ plants, which had 28 chromosomes and an average of 5.32 bivalents and 17.36 univalents in meiotic pollen mother cells (PMC), were obtained through embryos rescued owing to shriveling of endosperm in hybrid seed of cross Chinese Spring (CS) × UR206. Hybrid seeds were produced through backcrossing F₁ with common wheat parents. The derivative lines had normal chromosome numbers and powdery mildew resistance similar to the donor UR206, indicating that the powdery mildew resistance gene originating from T. urartu accession UR206 was successfully transferred and expressed in a hexaploid wheat background. Genetic analysis indicated that a single dominant gene controlled the powdery mildew resistance at the seedling stage. To map and tag the powdery mildew resistance gene, 143 F₂ individuals derived from a cross UR206 × UR203 were used to construct a linkage map. The resistant gene was mapped on the chromosome 7AL based on the mapped microsatellite makers. The map spanned 52.1 cM and the order of these microsatellite loci agreed well with the established microsatellite map of chromosome arm 7AL. The resistance gene was flanked by the microsatellite loci Xwmc273 and Xpsp3003, with the genetic distances of 2.2 cM and 3.8 cM, respectively. On the basis of the origin and chromosomal location of the gene, it was temporarily designated PmU.     

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.     

A composite map of expressed sequences and phenotypic traits of the sunflower (Helianthus annuus L.) genome

 A map of the sunflower genome, based on expressed sequences and consisting of 273 loci, was constructed. The map incorporates data from seven F₂ populations, for a total of 1115 individuals. Two hundred and fourty five loci corresponding to 170 anonymous cDNA markers and four loci for morphological markers were mapped. We also mapped 18 loci corresponding to previously described genes or to sequences obtained through homology cloning. The unit maps vary from 774 cM to 1060 cM, with an average value of 14 major linkage groups. The integrated map is arranged in 17 major linkage groups including 238 loci, plus four small segments with 2–5 marker loci; and covers 1573 cM with an overall average marker interval of 7 cM. Thirty five percent of the markers were dominant in nature and 30% showed inter-linkage group duplication without any indication of homoeologous linkage groups. Evidence is provided for the independence of two distinct fertility restoration genes, for the presence of two loosely linked branching loci, and for marker tightly linked to the Rf1 restoration locus. This map provides an efficient tool in breeding applications such as disease-resistance mapping, QTL analyses and marker-assisted selection. Received: 27 August 1998 / Accepted: 28 December 1998     

Restriction fragment length polymorphism analysis of loci associated with disease resistance genes and developmental traits in Pisum sativum L.

An F₂ population of pea (Pisum sativum L.) consisting of 174 plants was analysed by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) techniques. Ascochyta pisi race C resistance, plant height, flowering earliness and number of nodes were measured in order to map the genes responsible for their variation. We have constructed a partial linkage map including 3 morphological character genes, 4 disease resistance genes, 56 RFLP loci, 4 microsatellite loci and 2 RAPD loci. Molecular markers linked to each resistance gene were found: Fusarium wilt (6 cM from Fw), powdery mildew (11 cM from er) and pea common Mosaic virus (15 cM from mo). QTLs (quantitative traits loci) for Ascochyta pisi race C resistance were mapped, with most of the variation explained by only three chromosomal regions. The QTL with the largest effect, on chromosome 4, was also mapped using a qualitative, Mendelian approach. Another QTL displayed a transgressive segregation, i.e. the parental line that was susceptible to Ascochyta blight had a resistance allele at this QTL. Analysis of correlations between developmental traits in terms of QTL effects and positions suggested a common genetic control of the number of nodes and earliness, and a loose relationship between these traits and height.     

An expanded genetic linkage map of Prunus based on an interspecific cross between almond and peach.

The genetic linkage map of Prunus constructed earlier and based on an interspecific F2 population resulting from a cross between almond (Prunus dulcis D.A. Webb) and peach (Prunus persica L. Batsch) was extended to include 8 isozyme loci, 102 peach mesocarp cDNAs, 11 plum genomic clones, 19 almond genomic clones, 7 resistance gene analogs (RGAs), 1 RGA-related sequence marker, 4 morphological trait loci, 3 genes with known function, 4 simple sequence repeat (SSR) loci, 1 RAPD, and 1 cleaved amplified polymorphic sequence (CAP) marker. This map contains 161 markers placed in eight linkage groups that correspond to the basic chromosome number of the genus (x = n = 8) with a map distance of 1144 centimorgans (cM) and an average marker density of 6.8 cM. Four more trait loci (Y, Pcp, D, and SK) and one isozyme locus (Mdh1) were assigned to linkage groups based on known associations with linked markers. The linkage group identification numbers correspond to those for maps published by the Arús group in Spain and the Dirlewanger group in France. Forty-five percent of the loci showed segregation distortion most likely owing to the interspecific nature of the cross and mating system differences between almond (obligate outcrosser) and peach (selfer). The Cat1 locus, known to be linked to the D locus controlling fruit acidity, was mapped to linkage group 5. A gene or genes controlling polycarpel fruit development was placed on linkage group 3, and control of senesced leaf color (in late fall season) (LFCLR) was mapped to linkage group 1 at a putative location similar to where the Y locus has also been placed.     

Comparative mapping in grasses. Oat relationships

The development of RFLP linkage maps in hexaploid and diploid oat allows us to study genetic relationships of these species at the DNA level. In this report, we present the extension of a previously developed diploid oat map (Avena atlantica x A. hirtula) and its molecular-genetic relationships with wheat, rice and maize. Examination of 92–99% of the length of the oat genome map with probes common to Triticeae species, rice or maize showed that 84, 79 and 71%, respectively, was conserved between these species and oat. Generally, the orders of loci among chromosomes homoeologous to oat chromosomes A and D were the most conserved and those of chromosomes homoeologous to oat chromosome G were the least conserved. Conservation was observed for blocks ranging from whole chromosomes 101 cM long to small segments 2.5 cM long containing two loci. Comparison of the homoeologous segments of Triticeae, rice and maize relative to oat indicated that certain regions have been maintained in all four species. The relative positions of major genes governing traits such as seed storage proteins and resistance to leaf rusts have been conserved between cultivated oat and Triticeae species. Also, the locations of three vernalization/or photo-period response genes identified in hexaploid oat correspond to the locations of similar genes in homoeologous chromosomes of wheat, rice or maize. The locations of the centromeres for six of the seven oat chromosomes were estimated based on the homoeologous segments between oat and Triticeae chromosomes.     

RFLP genetic linkage maps from four F(2.3) populations and a joinmap of Gossypium hirsutum L

An RFLP genetic linkage joinmap was constructed from four different mapping populations of cotton (Gossypium hirsutum L.). Genetic maps from two of the four populations have been previously reported. The third genetic map was constructed from 199 bulk-sampled plots of an F_2.3 (HQ95–6×’MD51ne’) population. The map comprises 83 loci mapped to 24 linkage groups with an average distance between markers of 10.0 centiMorgan (cM), covering 830.1 cM or approximately 18% of the genome. The fourth genetic map was developed from 155 bulk-sampled plots of an F_2.3 (119– 5 sub-okra×’MD51ne’) population. This map comprises 56 loci mapped to 16 linkage groups with an average distance between markers of 9.3 cM, covering 520.4 cM or approximately 11% of the cotton genome. A core of 104 cDNA probes was shared between populations, yielding 111 RFLP loci. The constructed genetic linkage joinmap from the above four populations comprises 284 loci mapped to 47 linkage groups with the average distance between markers of 5.3 cM, covering 1,502.6 cM or approximately 31% of the total recombinational length of the cotton genome. The linkage groups contained from 2 to 54 loci each and ranged in distance from 1.0 to 142.6 cM. The joinmap provided further knowledge of competitive chromosome arrangement, parental relationships, gene order, and increased the potential to map genes for the improvement of the cotton crop. This is the first genetic linkage joinmap assembled in G. hirsutum with a core of RFLP markers assayed on different genetic backgrounds of cotton populations (Acala, Delta, and Texas plain). Research is ongoing for the identification of quantitative trait loci for agronomic, physiological and fiber quality traits on these maps, and the identification of RFLP loci lineage for G. hirsutum from its diploid progenitors (the A and D genomes). Received: 23 February 2001 / Accepted: 8 June 2001     

Characterization and linkage mapping of R-gene analogous DNA sequences in pea (Pisum sativum L.)

Pea (Pisum sativum L.) sequences that are analogous to the conserved nucleotide binding site (NBS) domain found in a number of plant disease resistance genes (R-genes) were cloned. Using redundant oligonucleotide primers and the polymerase chain reaction (PCR), we amplified nine pea sequences and characterised their sequences. The pea R-gene analog (RGA)- deduced amino acid sequences demonstrated significant sequence similarity with known R-gene sequences lodged in public databases. The genomic locations of eight of the pea RGAs were determined by linkage mapping. The eight RGAs identified ten loci that mapped to six linkage groups. In addition, the genomic organization of the RGAs was inferred. Both single-copy and multicopy sequence families were present among the RGAs, and the multicopy families occurred most often as tightly linked clusters of related sequences. Intraspecific copy number variability was observed in three of the RGA sequence families, suggesting that these sequence families are evolving rapidly. The genomic locations of the pea RGAs were compared with the locations of known pea R-genes and sym genes involved in the pea-rhizobia symbiosis. Two pea RGAs mapped in the genomic region containing a pea R-gene, Fw, and four pea RGAs mapped in regions of the genome containing sym genes. Received: 4 August 1999 / Accepted: 11 November 1999     

A linkage map of hexaploid oat based on grass anchor DNA clones and its relationship to other oat maps.

A cultivated oat linkage map was developed using a recombinant inbred population of 136 F6:7 lines from the cross 'Ogle' x 'TAM O-301'. A total of 441 marker loci, including 355 restriction fragment length polymorphism (RFLP) markers, 40 amplified fragment length polymorphisms (AFLPs), 22 random amplified polymorphic DNAs (RAPDs), 7 sequence-tagged sites (STSs), 1 simple sequence repeat (SSR), 12 isozyme loci, and 4 discrete morphological traits, was mapped. Fifteen loci remained unlinked, and 426 loci produced 34 linkage groups (with 2-43 loci each) spanning 2049 cM of the oat genome (from 4.2 to 174.0 cM per group). Comparisons with other Avena maps revealed 35 genome regions syntenic between hexaploid maps and 16-34 regions conserved between diploid and hexaploid maps. Those portions of hexaploid oat maps that could be compared were completely conserved. Considerable conservation of diploid genome regions on the hexaploid map also was observed (89-95%); however, at the whole-chromosome level, colinearity was much lower. Comparisons among linkage groups, both within and among Avena mapping populations, revealed several putative homoeologous linkage group sets as well as some linkage groups composed of segments from different homoeologous groups. The relationships between many Avena linkage groups remain uncertain, however, due to incomplete coverage by comparative markers and to complications introduced by genomic duplications and rearrangements.     

A genetic linkage map for Eucalyptus globulus with candidate loci for wood,fibre, and floral traits

A genetic linkage map containing potential candidate loci for wood, fibre and floral traits has been constructed for Eucalyptus globulus (Labill.) based on the segregation of 249 codominant loci in an outbred F₁ population of 148 individuals. The map contains 204 RFLP loci, including 31 cambium-specific expressed sequence tags (ESTs) and 14 known function genes, and 40 microsatellite and five isozyme loci. Independent male and female maps were constructed, and the 98 loci (39%) that segregated in both parents were used to combine the parental maps into an integrated map. The 249 loci mapped to 11 major linkage groups (n=11 in eucalypts) and a 12th small linkage group containing three loci that segregated in the male parent only. Total map distance is 1375 cM with an average interval of 6 cM. Forty one of the mapped loci identify known proteins (five isozymes) or sequences with known function (14 genes and 22 ESTs). The mapped genes include enzymes involved in lignin and cell-wall polysaccharide biosynthesis, and floral-development genes. This map will be used to locate quantitative trait loci for wood, fibre, and other traits in Eucalyptus. Received: 30 August 2000 / Accepted: 23 March 2001     

A consensus map for <Emphasis Type="Italic">Cucurbita pepo</Emphasis>

Using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), and morphological traits, the first genetic maps for Cucurbita pepo (2n=2x=40) were constructed and compared. The two mapping populations consisted of 92 F₂ individuals each. One map was developed from a cross between an oil-seed pumpkin breeding line and a zucchini accession, into which genes for resistance to Zucchini Yellow Mosaic Virus (ZYMV) from a related species, C. moschata, had been introgressed. The other map was developed from a cross between an oil-seed pumpkin and a crookneck variety. A total of 332 and 323 markers were mapped in the two populations. Markers were distributed in each map over 21 linkage groups and covered an average of 2,200 cM of the C. pepo genome. The two maps had 62 loci in common, which enabled identification of 14 homologous linkage groups. Polyacrylamide gel analyses allowed detection of a high number of markers suitable for mapping, 10% of which were co-dominant RAPD loci. In the Pumpkin-Zucchini population, bulked segregant analysis (BSA) identified seven markers less than 7 cM distant from the locus n, affecting lignification of the seed coat. One of these markers, linked to the recessive hull-less allele (AW11-420), was also found in the Pumpkin-Crookneck population, 4 cM from n. In the Pumpkin-Zucchini population, 24 RAPD markers, previously introduced into C. pepo from C. moschata, were mapped in two linkage groups (13 and 11 markers in LGpz1 and LGpz2, respectively), together with two sequence characterized amplified region (SCAR) markers linked to genes for resistance to ZYMV.     

An improved genetic linkage map for cowpea (Vigna unguiculata L.) combining AFLP, RFLP, RAPD, biochemical markers, and biological resistance traits.

An improved genetic linkage map has been constructed for cowpea (Vigna unguiculata L. Walp.) based on the segregation of various molecular markers and biological resistance traits in a population of 94 recombinant inbred lines (RILs) derived from the cross between 'IT84S-2049' and '524B'. A set of 242 molecular markers, mostly amplified fragment length polymorphism (AFLP), linked to 17 biological resistance traits, resistance genes, and resistance gene analogs (RGAs) were scored for segregation within the parental and recombinant inbred lines. These data were used in conjunction with the 181 random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), AFLP, and biochemical markers previously mapped to construct an integrated linkage map for cowpea. The new genetic map of cowpea consists of 11 linkage groups (LGs) spanning a total of 2670 cM, with an average distance of 6.43 cM between markers. Astonishingly, a large, contiguous portion of LG1 that had been undetected in previous mapping work was discovered. This region, spanning about 580 cM, is composed entirely of AFLP markers (54 in total). In addition to the construction of a new map, molecular markers associated with various biological resistance and (or) tolerance traits, resistance genes, and RGAs were also placed on the map, including markers for resistance to Striga gesnerioides races 1 and 3, CPMV, CPSMV, B1CMV, SBMV, Fusarium wilt, and root-knot nematodes. These markers will be useful for the development of tools for marker-assisted selection in cowpea breeding, as well as for subsequent map-based cloning of the various resistance genes.