Development of a composite map in Vicia faba,breeding applications and future prospects

A composite map of the Vicia faba genome based on morphological markers, isozymes, RAPDs, seed protein genes and microsatellites was constructed. The map incorporates data from 11 F₂ families for a total of 654 individuals all sharing the common female parent Vf 6. The integrated map is arranged in 14 major linkage groups (five of which were located in specific chromosomes). These linkage groups include 192 loci and cover 1,559 cM with an overall average marker interval of 8 cM. By joining data of a new F₂ population segregating for resistance to ascochyta, broomrape and others traits of agronomic interest, have been saturated new areas of the genome. The combination of trisomic segregation, linkage analysis among loci from different families with a recurrent parent, and the analysis of new physically located markers, has allowed the establishment of the present status of the V. faba map with a wide coverage. This map provides an efficient tool in breeding applications such as disease-resistance mapping, QTL analyses and marker-assisted selection.Communicated by J.W. Snape     

Estimation of linkage in trisomic inheritance

 Based on F₂ families derived from selfed F₁ trisomic plants we have developed a genetic model to estimate linkage relationships between pairs of loci located on the extra chromosome. Genotypic frequencies of each class expected in a trisomic F₂ family have been calculated and the maximum-likelihood equations for recombination-fraction estimation have been derived for a variety of genetic situations. Morton’s test of homogeneity was used to compare recombination fractions estimated between loci exhibiting trisomic segregation to those obtained in families where the same loci showed Mendelian segregation. This method has been applied to an analysis of morphological, isozyme and RAPD data from faba bean (Vicia faba L.). Received: 11 October 1996 / Accepted: 21 March 1997     

Development of a genetic composite map of Vicia faba using F2 populations derived from trisomic plants

 Seven F₂ families of faba bean descendent from plants trisomic for chromosomes 3, 4, 5 and 6 were analyzed for isozyme markers and two of these were also studied for morphological and RAPD markers and seed-protein genes. Linkage analysis revealed 14 linkage groups, 8 of which were unambiguously assigned to specific chromosomes. Several QTLs for seed weight were identified, the most important of which, located on chromosome 6, explained approximately 30% of the total phenotypic variation. Comparison of results from Vicia faba with the maps of the related species Pisum sativum L. and Cicer arietinum L. revealed one possible new case of linkage conservation. A composite linkage analysis based on 42 markers analyzed in this and previous studies, where line Vf 6 was also used as the female parental, allowed the new assignment of previously independent linkage groups and/or markers to specific chromosomes. Thus, the number of linkage groups was reduced to 13, each comprising an increased number of markers. No contradictory results were detected, indicating the suitability of the statistical procedure and methodology used so far in the development of the map of this species. Received: 30 April 1998 / Accepted: 24 August 1998     

Molecular mapping of the centromeres of tomato chromosomes 7 and 9

The centromeres of two tomato chromosomes have been precisely localized on the molecular linkage map through dosage analysis of trisomic stocks. To map the centromeres of chromosomes 7 and 9, complementary telo-, secondary, and tertiary trisomic stocks were used to assign DNA markers to their respective chromosome arms and thus to localize the centromere at the junction of the short and long arms. It was found that both centromeres are situated within a cluster of cosegregating markers. In an attempt to order the markers within the centric clusters, genetic maps of the centromeric regions of chromosomes 7 and 9 were constructed from F₂ populations of 1620Lycopersicon esculentum × L. pennellii (E × P) plants and 1640L. esculentum × L. pimpinellifolium (E × PM) plants. Despite the large number of plants analyzed, very few recombination events were detected in the centric regions, indicating a significant suppression of recombination at this region of the chromosome. The fact that recombination suppression is equally strong in crosses between closely related (E × PM) and remotely related (E × P) parents suggests that centromeric suppression is not due to DNA sequence mismatches but to some other mechanism. The greatest number of centromeric markers was resolved in theL. esculentum × L. pennellii F₂ population. The centromere of chromosome 7 is surrounded by eight cosegregating markers: three on the short arm, five on the long arm. Similarly, the centric region of chromosome 9 contains ten cosegregating markers including one short arm marker and nine long arm markers. The localization of centromeres to precise intervals on the molecular linkage map represents the first step towards the characterization and ultimate isolation of tomato centromeres.     

Genetics and mapping of new isozyme loci in Vicia faba L using trisomics

Polymorphism in ten enzyme systems (ACO, ACP, AAT, EST, FK, ME, NAG, PRX, 6PGD, and SOD) in Vicia faba L. was analyzed, revealing 13 loci, six of which have not been reported before. Inheritance, genetics, possible location, and linkage analysis were studied in 13 different F² populations trisomic for four of the six chromosomes (nos. 3, 4, 5, and 6) of the species. Each of these loci exhibited typical Mendelian inheritance except for those involved in the trisomic chromosome. Five loci have been assigned to a specific chromosome: Est-2 to chromosome 3, Fk-2 to chromosome 4, Prx-1 to chromosome 5, and Sod-1 and Pgd-p to chromosome 6. Nag-1 and Pgd-c displayed a linkage of 22.8 cM indicating a clear homology with chromosome 5 of garden pea on which both markers are syntenic.     

Localization of QTLs for tolerance to Cercospora beticola on sugar beet linkage groups

We present a new linkage map for sugar beet (Beta vulgaris) which has been developed using a population segregating for genetic factors that confer tolerance to the leaf spot fungus (Cercospora beticola), the causal factor of leaf spot disease in sugar beet). In the F₂ population studied, a subset of 36 RFLP probes, mapping on eight out of the nine linkage groups of sugar beet, provided the anchor markers to assign chromosomes. A total of 224 markers, including RFLPs, AFLPs, SCARs and microsatellites, were mapped. Estimates of leaf damage in F₂ and test-cross families were repeated at different stages of plant development. Each set of data was analysed as such. An average estimate was also considered. QTLs with highly significant LOD scores revealed both by the F₂ and test-cross analyses were localized on linkage groups 2, 6 and 9. Linkage groups 4 and 5 gave a clear indication of the presence of a QTL only when F₂ data were considered. One highly significant QTL with a LOD of 16.0 was revealed only by the data obtained under conditions of artificial inoculation. This QTL maps at position 90 on chromosome 3. Received: 3 February 1999 / Accepted: 20 February 1999     

Identification and mapping of resistance gene analogs and a white rust resistance locus in<Emphasis Type="Italic"> Brassica rapa</Emphasis> ssp.<Emphasis Type="Italic"> oleifera</Emphasis>

The objective of this investigation was to tag a locus for white rust resistance in a Brassica rapa ssp. oleifera F₂ population segregating for this trait, using bulked segregant analysis with random amplified polymorphic DNA (RAPD) markers, linkage mapping and a candidate gene approach based on resistance gene analogs (RGAs). The resistance source was the Finnish line Bor4109. The reaction against white rust races 7a and 7v was scored in 20 seedlings from each self-pollinated F₂ individual. The proportion of resistant plants among these F₃ families varied from 0 to 67%. Bulked segregant analysis did not reveal any markers linked with resistance and, therefore, a linkage map with 81 markers was created. A locus that accounted for 18.4% of the variation in resistance to white rust was mapped to linkage group (LG) 2 near the RAPD marker Z19a. During the study, a bacterial resistance gene homologous to Arabidopsis RPS2 and six different RGAs were sequenced. RPS2 and five of the RGAs were mapped to linkage groups LG1, LG4 and LG9. Unfortunately, none of the RGAs could be shown to be associated with white rust resistance.Communicated by H.C. BeckerThe nucleotide sequence data reported has been deposited in the Genbank under the accession numbers AF315081–AF315087.     

Assignment of RFLP linkage groups to chromosomes using monosomic F1 analysis in hexaploid oat

The availability of molecular genetic maps in oat (Avena spp.) and improved identification of chromosomes by C-banding are two recent developments that have made locating linkage groups to chromosomes possible in cultivated hexaploid oat, 2n=6x=42. Monosomic series derived from Avena byzantina C. Koch cv Kanota and from Avena sativa L. cv Sun II were used as maternal plants in crosses with the parents, Kanota-1 and Ogle-C, of the oat RFLP mapping population. Monosomic F₁ plants were identified by root-tip cell chromosome counts. For marker analysis, DNAs of eight F₂ plants from a monosomic F₁ were combined to provide a larger source of DNA that mimicked that of the monosomic F₁ plant. Absence of maternal alleles in monosomic F₁s served to associate linkage groups with individual chromosomes. Twenty two linkage groups were associated with 16 chromosomes. In seven instances, linkage groups that were independent of each other in recombination analyses were associated with the same chromosome. Five linkage groups were shown to be associated with translocation differences among oat lines. Additionally, the results better-characterized the oat monosomic series through the detection of duplicates and translocation differences among the various monosomic lines. The F₁ monosomic series represents a powerful cytogenetic tool with the potential to greatly improve understanding of the oat genome. Received: 24 April 2000 / Accepted: 10 May 2000     

Mapping quantitative trait loci (QTLs) for resistance to Gibberella zeae infection in maize

The basic prerequisite for an efficient breeding program to improve levels of resistance to pathogens in plants is the identification of genes controlling the resistance character. If the response to pathogens is under the control of a multilocus system, the utilization of molecular markers becomes essential. Stalk and ear rot caused by Gibberella zeae is a widespread disease of corn: resistance to G. zeae is quantitatively inherited. Our experimental approach to understanding the genetic basis of resistance to Gibberella is to estimate the genetic linkage between available molecular markers and the character, measured as the amount of diseased tissue 40 days after inoculation of a suspension of Fusarium graminearum, the conidial form of G. zeae, into the first stalk internode. Sensitive and resistant parental inbreds were crossed to obtain F₁ and F₂ populations: the analysis of the segregation of 95 RFLP (restriction fragment length polymorphism) clones and 10 RAPD (random amplified polymorphic DNA) markers was performed on a population of 150 F₂ individuals. Analysis of resistance was performed on the F₃ families obtained by selfing the F₂ plants. Quantitative trait loci (QTL) detection was based either on analysis of regression coefficients between family mean value and allele values in the F₂ population, or by means of interval mapping, using MAPMAKER-QTL. A linkage map of maize was obtained, in which four to five genomic regions are shown to carry factors involved in the resistance to G. zeae.     

Characterization and mapping of <Emphasis Type="Italic">Rpi1</Emphasis>, a gene that confers dominant resistance to stalk rot in maize

The maize inbred lines 1145 (resistant) and Y331 (susceptible), and the F₁, F₂ and BC₁F₁ populations derived from them were inoculated with the pathogen Pythium inflatum Matthews, which causes stalk rot in Zea mays. Field data revealed that the ratio of resistant to susceptible plants was 3:1 in the F₂ population, and 1:1 in the BC₁F₁population, indicating that the resistance to P. inflatum Matthews was controlled by a single dominant gene in the 1145×Y331 cross. The gene that confers resistance to P. inflatum Matthews was designated Rpi1 for resistance to P. inflatum) according to the standard nomenclature for plant disease resistance genes. Fifty SSR markers from 10 chromosomes were first screened in the F₂ population to find markers linked to the Rpi1 gene. The results indicated that umc1702 and mmc0371 were both linked to Rpi1, placing the resistance gene on chromosome 4. RAPD (randomly amplified polymorphic DNA) markers were then tested in the F₂population using bulked segregant analysis (BSA). Four RAPD products were found to show linkage to the Rpi1 gene. Then 27 SSR markers and 8 RFLP markers in the region encompassing Rpi1 were used for fine-scale mapping of the resistance gene. Two SSR markers and four RFLP markers were linked to the Rpi1 gene. Finally, the Rpi1 gene was mapped between the SSR markers bnlg1937 and agrr286 on chromosome 4, 1.6 cM away from the former and 4.1 cM distant from the latter. This is the first time that a dominant gene for resistance to maize stalk rot caused by P. inflatum Matthews has been mapped with molecular marker techniques.     

Genetic studies of self-fertility in rye (Secale cereale L.). 1. The identification of genotypes of self-fertile lines for the Sf alleles of self-incompatibility genes

Segregation for self-fertility has been studied in progenies from the crosses of self-sterile (SS) plants with interline hybrids obtained by a diallel scheme of pollinations between seven self-fertile (SF) lines (nos. 2–8) and with F₁ (SS plant x SF line) hybrids. All the offspring families from the SS plant x F₁ (SS plant x SF line) crosses demonstrated a 1SF1SS segregation. The crosses of SS plants with some interline hybrids gave only self-fertile plants, whereas the crosses with other interline hybrids gave a segregation of 3SF:1SS expected in the case of digenic segregation. The data obtained permitted us to identify three different S loci (S1, S2, S5) and to estimate the genotypes of self-fertile lines for their Sf alleles: lines 5, 6, 7 and 8 are S1f/S1f S2n/S2n S5m/S5m, line 4 is S1n/S1n S2f/S2f S5m/S5m, and lines 2 and 3 are S1n/S1n S2m/S2m S5f/S5f(Sn, Sm designate active alleles of the incompatibility genes). The identification of the particular S gene which is presented by the Sf allele in each line has been made on the basis of our data concerning the linkage of the Sf mutation with isozyme markers of particular rye chromosomes, which is reported in an accompanying paper.     

A major locus for submergence tolerance mapped on rice chromosome 9

Submergence stress is a widespread problem in rice-growing environments where drainage is impeded. A few cultivars can tolerate more than 10 days of submergence, but the genes conferring this tolerance have not been identified. We used randon-amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers to map submergence tolerance in 169 F₂ plants and the resulting F₃ families of a cross between a tolerant indica rice line, IR40931-26, and a susceptible japonica line, PI543851. IR40931-26 inherited strong submergence tolerance from the unimproved cultivar FR13A. Eight-day old F₃ seedlings were submerged for 14–16 days in 55-cm deep tanks, and tolerance was scored after 7 days recovery on a scale of 1 (tolerant) to 9 (susceptible). The tolerant and susceptible parents scored 1.5 and 8.4, respectively, and the F₃ means ranged from 1.6 to 8.9. Two bulks were formed with DNA from F₂ plants corresponding to the nine most tolerant and the nine most susceptible F₃ families. Of 624 RAPD primers used to screen the bulks, five produced bands associated with either tolerance or susceptibility. These markers were mapped to a region of chromosome 9 by linkage to RFLP markers. A submergence tolerance quantitative trait locus (QTL), here designatedSub1, was located ca. 4 cM from the RFLP marker C1232 and accounted for 69% of the phenotypic variance for the trait.     

Primary trisomics obtained from autotriploid by diploid reciprocal crosses in cucumber

To promote cytogenetical studies on cucumber (Cucumis sativus L., 2n = 2x = 14), the reciprocal crosses were made between autotriploid and diploid for selecting the primary trisomics. Meanwhile, chromosome behavior during meiosis in autotriploid cucumber was investigated to look for cytological evidences for origin of primary trisomics. Many viable F₁ seeds were obtained from reciprocal crosses between autotriploid and diploid. The number of chromosomes of 56 surviving progenies varied from 14 to 28, with plants having 2n = 15 occurring at the highest frequency (51.8%). Primary trisomics were firstly obtained in this study. Four types of primary trisomics were isolated and they could be distinguished from each other, as well as diploid. Variable chromosome configurations, e.g. univalent, bivalents and trivalents were observed in many pollen mother cells of the autotriploid at metaphase I. Binomial chromosome distribution was observed at anaphase I and frequency of 8/13 was 6.25%. The meiosis of autotriploid, especially the class of gametes with eight chromosomes, gave the cytological evidence of producing 2x + 1 type gamete and could be induced into primary trisomic plants from progeny of autotriploid–diploid crosses. These studies have established a ground work for selecting a series of primary trisomics, and further using them for associating linkage groups with specific chromosomes in cucumber.     

Mapping of a broad-spectrum brown planthopper resistance gene, <Emphasis Type="Italic">Bph3</Emphasis>, on rice chromosome 6

The brown planthopper (BPH) is one of the most destructive insect pests of rice in Thailand. We performed a cluster analysis that revealed the existence of four groups corresponding to the variation of virulence against BPH resistance genes in 45 BPH populations collected in Thailand. Rice cultivars Rathu Heenati and PTB33, which carry Bph3, showed a broad-spectrum resistance against all BPH populations used in this study. The resistant gene Bph3 has been extensively studied and used in rice breeding programs against BPH; however, the chromosomal location of Bph3 in the rice genome has not yet been determined. In this study, a simple sequence repeat (SSR) analysis was performed to identify and localize the Bph3 gene derived from cvs. Rathu Heenati and PTB33. For mapping of the Bph3 locus, we developed two backcross populations, BC₁F₂ and BC₃F₂, from crosses of PTB33 × RD6 and Rathu Heenati × KDML105, respectively, and evaluated these for BPH resistance. Thirty-six polymorphic SSR markers on chromosomes 4, 6 and 10 were used to survey 15 resistant (R) and 15 susceptible (S) individuals from the backcross populations. One SSR marker, RM190, on chromosome 6 was associated with resistance and susceptibility in both backcross populations. Additional SSR markers surrounding the RM190 locus were also examined to define the location of Bph3. Based on the linkage analysis of 208 BC₁F₂ and 333 BC₃F₂ individuals, we were able to map the Bph3 locus between two flanking SSR markers, RM589 and RM588, on the short arm of chromosome 6 within 0.9 and 1.4 cM, respectively. This study confirms both the location of Bph3 and the allelic relationship between Bph3 and bph4 on chromosome 6 that have been previously reported. The tightly linked SSR markers will facilitate marker-assisted gene pyramiding and provide the basis for map-based cloning of the resistant gene.     

A molecular linkage map of rye

A genetic map of six chromosomes of rye, (all of the rye chromosomes except for 2R), was constructed using 77 RFLP and 12 RAPD markers. The map was developed using an F₂ population of 54 plants from a cross between two inbred lines. A rye genomic library was constructed as a source of clones for RFLP mapping. Comparisons were made between the rye map and other rye and wheat maps by including additional probes previously mapped in those species. These comparisons allowed (1) chromosome arm orientation to the linkage groups to be given, (2) the corroboration of several evolutionary translocations between rye chromosomes and homoeologous chromosomes of wheat; (3) an increase in the number of available markers for target regions of rye that show colinearity with wheat. Inconsistencies in the location of markers between the wheat and rye maps were mostly detected by multi-copy probes.     

Use of Random Amplified Polymorphic DNA Markers for Mapping the Chickpea Genome

Three interspecific crosses were developed using Cicer arietinum (ICC 4918) as the female parent and wild Cicer species [C. reticulatum - JM 2100, JM 2106 and C. echinospermum - ICCW 44] as the male parent. Cicer arietinum (ICC 4918) × C. reticulatum (JM 2100) cross produced the largest number of F₂ plants and was chosen for linkage mapping using Random Amplified Polymorphic DNA (RAPD) primers. A partial linkage map was constructed based upon the segregation of 36 RAPD markers obtained by amplification using 35 primers. The linkage map consists of two linkage groups with 17 linked markers covering a total of 464.9 cM. Analyses also revealed association of three morphological traits with linked RAPD markers. Out of seven morphological traits tested for association with linked markers in the segregating plants, four Quantitative trait loci (QTL) were detected for the trait leaf length and three QTLs each for the traits leaf width and erect plant habit.     

Molecular mapping of the ge s gene controlling the super-giant embryo character in rice (Oryza sativa L.)

The giant-embryo character is useful for quality improvement in rice. Three alleles controlling embryo size have been reported at the ge locus. Based on trisomic analysis, this locus is known to reside on chromosome 7. The objective of the present study was to identify linkage between molecular markers and the ge ^s gene using an existing molecular map of rice and an F₂ population derived from Hwacheongbyeo-ge ^s (super-giant embryo)/Milyang 23. The bulked-segregant method was used to screen 38 RFLPs and two microsatellite markers from rice chromosome 7. RZ395 and CDO497 flanked the ge ^s gene, at 2.4 cM and 3.4 cM, respectively. The two microsatellite markers, RM18 and RM10, were linked with ge ^s at 7.7 cM and 9.6 cM, respectively. The availability of molecular markers will facilitate selection of this grain character in a breeding program and provide the foundation for map-based gene isolation.     

RFLP mapping of the sugary enhancer1 gene in maize

RFLP marker data from an F₂₃ population derived from a cross between a sugary1 (su1) and a sugary enhancer1 (su1, sel) inbred were used to construct a genetic linkage map of maize. This map includes 93 segregating marker loci distributed throughout the maize genome, providing a saturated linkage map that is suitable for linkage analysis with quantitative trait loci (QTL). This population, which has been immortalized in the form of sibbed F₂₃ families, was derived from each of the 214 F₂ plants and along with probe data are available to the scientific community. QTL analysis for kernel sucrose (the primary form of sugar) concentration at 20 days after pollination (DAP) uncovered the segregation of seven major QTL influencing sucrose concentration; a locus linked to umc36a described the greatest proportion of the variation (24.7%). Since maltose concentration has previously been reported to be associated with the se1 phenotype, an analysis of probe associations with maltose concentration at 40 DAP was also conducted. The highly significant association of umc36a with maltose and sucrose concentrations provided evidence that this probe is linked to se1. Phenotypic evaluation for the se1 genotype in each F₂₃ family enabled us to map the gene 12.1 cM distal to umc36a. In contrast to previous work where se1 was reported to be located on chromosome four, our data strongly suggest that the sugary enhancer1 locus maps on the the distal portion of the long arm of chromosome 2 in the maize genome.     

Mapping genes essential for embryo development in Arabidopsis thaliana.

Summary We have previously isolated and characterized over 90 recessive mutants of Arabidopsis thaliana defective in embryo development. These emb mutants have been shown to differ in lethal phase, extent of abnormal development, and response in culture. We demonstrate in this report the value and efficiency of mapping emb genes relative to visible and molecular markers. Sixteen genes essential for embryo development were mapped relative to visible markers by analyzing progeny of selfed F₁ plants. Embryonic lethals are now the most common type of visible marker included on the linkage map of Arabidopsis. Backcrosses were used in several cases to orient genes relative to adjacent markers. Three genes were located to chromosome arms with telotrisomics by screening for a reduction in the percentage of aborted seeds produced by F₁ plants. A restriction fragment length polymorphism (RFLP) mapping strategy that utilizes pooled EMB/EMB F₂ plants was devised to increase the efficiency of mapping embryonic lethals relative to molecular markers. This strategy was tested by demonstrating that the biol locus of Arabidopsis is within 0.5 cM of an existing RFLP marker. Mapping embryonic lethals with both visible and molecular markers may therefore help to identify large numbers of genes with essential functions in Arabidopsis.     

RFLP and RAPD mapping of the sunflower Pl1 locus for resistance to Plasmopara halstedii race 1

The Pl1 locus in sunflower, Helianthus annuus L., conferring resistance to downy mildew, Plasmopara halstedii, race 1 has been located in linkage group 1 of the consensus RFLP map of the cultivated sunflower. Bulked segregant analyses were used on 135 plants of an F₂ progeny from a cross between a downy mildew susceptible line, GH, and RHA266, a line carrying Pl1. Two RFLP markers and one RAPD marker linked to the Pl1 locus have been identified. The RFLP markers are located at 5.6 cM and 7.1 cM on either side of Pl1. The RAPD marker is situated at 43.7 cM from Pl1. The significance and applications of these markers in sunflower breeding are discussed.