QTL analysis of important pith characteristics in a cross between two inbred lines of chicory (Cichorium intybus var. foliosum): a preliminary study

The genetic basis of pith characteristics in chicory (Cichorium intybus L. var. foliosum Hegi) was investigated. Quantitative trait loci (QTL) were mapped in an F2 population (565 F2 plants) derived from a cross between two inbred chicory lines. A molecular marker linkage map of this cross had previously been constructed based on 129 random amplified polymorphic DNA markers. Each F2 plant was selfed and plant characteristics were measured in the F3 populations. Although variation in pith characteristics was largely environmentally influenced, QTL for the characteristics length of pith, browning of the pith, hollow pith and apple pith were detected in many linkage groups. Interactions between QTL were found for the three characteristics: pith length, browning of the pith and hollow pith. The QTL detected confirmed the early forcing suitability of the one parent inbred line and late forcing suitability of the other.     

A new citrus linkage map based on SRAP,SSR, ISSR,POGP, RGA and RAPD markers

Sequence-related amplified polymorphism (SRAP), simple sequence repeats (SSR), inter-simple sequence repeat (ISSR), peroxidase gene polymorphism (POGP), resistant gene analog (RGA), randomly amplified polymorphic DNA (RAPD), and a morphological marker, Alternaria brown spot resistance gene of citrus named as Cabsr caused by (Alternaria alternata f. sp. Citri) were used to establish genetic linkage map of citrus using a population of 164 F₁ individuals derived between ‘Clementine’ mandarin (Citrus reticulata Blanco ‘Clementine) and ‘Orlando’ tangelo’ (C. paradisi Macf. ‘Duncan’ × C. reticulata Blanco ‘Dancy’). A total of 609 markers, including 385 SRAP, 97 RAPD, 95 SSR, 18 ISSR, 12 POGP, and 2 RGA markers were used in linkage analysis. The ‘Clementine’ linkage map has 215 markers, comprising 144 testcross and 71 intercross markers placed in nine linkage groups. The ‘Clementine’ linkage map covered 858 cM with and average map distance of 3.5 cM between adjacent markers. The ‘Orlando’ linkage map has 189 markers, comprising 126 testcross and 61 intercross markers placed in nine linkage groups. The ‘Orlando’ linkage map covered 886 cM with an average map distance of 3.9 cM between adjacent markers. Segregation ratios for Cabsr were not significantly different from 1:1, suggesting that this trait is controlled by a single locus. This locus was placed in ‘Orlando’ linkage group 1. The new map has an improved distribution of markers along the linkage groups with fewer gaps. Combining different marker systems in linkage mapping studies may give better genome coverage due to their chromosomal target site differences, therefore fewer gaps in linkage groups.     

Mapping Co, a gene controlling the columnar phenotype of apple, with molecular markers

The columnar phenotype is a very valuable genetic resource for apple breeding because of its compact growth form determined by the dominant gene Co. Using bulked segregant analysis combined with several DNA molecular marker techniques to screen the F₁ progeny of Spur Fuji × Telamon (heterozygous for Co), 9 new DNA markers (6 RAPD, 1 AFLP and 2 SSRs) linked to the Co gene were identified. A total of 500 10-mer random primers, 56 pairs of selective AFLP primers and 8 SSR primer pairs were screened. One RAPD marker S1142₆₈₂, and the AFLP marker, E-ACT/M-CTA₃₄₆, were converted into SCAR markers designated SCAR₆₈₂ and SCAR₂₁₆, respectively. These markers will enable early selection in progenies where Co is difficult to identify. The Co gene was located between the SSR markers CH03d11 and COL on linkage group 10 of the apple genetic linkage map. Finally, a local genetic map of the region around the Co gene was constructed by linkage analysis of the nine new markers and three markers developed earlier.     

Construction of a linkage map for watermelon (Citrullus lanatus (Thunb.) Matsum & Nakai) using random amplified polymorphic DNA (RAPD)

Summary A linkage map for watermelon (Citrullus lanatus) was constructed on the basis of RADP, ribosomal DNA restriction fragment length polymorphism (RFLP), isozyme, and morphological markers using F₁BC1. A segregating population of 78 individuals was the result of a backcross of a cultivated inbred line (H-7; Citrullus lanatus; 2n=22) and a wild form (SA-1; C. lanatus; 2n=22), in which the latter was the recurrent (male) parent. A total of 69 RAPD, one RFLP, one isozyme, and three morphological markers was found to segregate in the BC1 population. Linkage analysis revealed that 62 loci could be mapped to 11 linkage groups that extended more than 524 centimorgans (cM), while 12 loci segregated independently of all other markers. The locus for exocarp color was linked to two RAPD markers within a region of 5 cM on linkage group 4. The locus for flesh color was linked to a RAPD marker within a region of 30 cM on linkage group 6. The isozyme marker GOT was located on the linkage group 1. Linkage group 2 contained a locus for ribosomal DNA within 5 cM of a RAPD marker. Half of the RAPD markers on the linkage group 7 displayed severely distorted segregation. The construction of linkage map using molecular markers is necessary for the breeding of watermelon to introduce useful gene of wild watermelon efficiently. However the linkage map that was constructed for the most part on the basis of RAPD markers could not cover significant parts of the genome, the linkage map provides breeders of watermelons the possibility of tagging useful agronomic traits, as well as the gene for exocarp color.Abbreviations RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - GOT glutamate oxaloacetate transaminase - MDH malate dehydrogenase - ACP acid phosphatase - 6PGH 6-phosphogluconate dehydrogenase     

The use of bulked segregant analysis to accumulate RAPD markers near a locus for beet cyst nematode resistance in Beta vulgaris

Bulked segregant analysis (BSA) was used to accumulate RAPD markers near the beet cyst nematode resistance locus Hsl^pro-1 of sugar beet (Beta vulgaris L.). Graphical genotypes constructed from RFLP data were utilized to select F₂ individuals in (1) the construction of pools of plants used in the initial screening for polymorphisms, and (2) the selection of individual plants used to confirm the potential linkage. The pooled DNA samples were screened for polymorphisms using 668 RAPD primers. Forty-four candidate markers potentially linked to the region were analysed further using 14 segregating individuals. Close linkage was confirmed for 17 of the markers. Four of the RAPD markers were assigned map coordinates within the RFLP map. Three of these markers extended the RFLP map by 3cM. Altogether, the 8cM target interval contains 10 RFLP and 17 RAPD markers, corresponding to an average marker density of 0.3cM in the Hsl_pro-1 region.     

Genetic analysis of Lolium. I. Identification of linkage groups and the establishment of a genetic map

A genetic map of Lolium has been produced using isozyme, restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers applied to a segregating family derived from an F₁ hybrid plant of L. perenne × L. multiflorum provenance, crossed on to a doubled haploid L. perenne. A total of 106 markers, out of a total of 160 polymorphic loci analysed, have been ascribed to seven linkage groups covering a map distance of 692cM, Two of these groups may be allocated to chromosomes 2 and 6 of the Lolium genome. The remaining unallocated markers, the majority of which showed severe segregation distortion, could be associated into small groups of two or three markers which showed no linkage with the main groups at a LOD of 2.8 or, if associated, could not be mapped in a satisfactory manner. This high incidence of disturbed segregations could be accounted for by the use of an interspecific hybrid between two species of differing genome size, with consequent cytological imbalance.     

Linkage groups of isozymes,RFLP and RAPD markers in carrot (Daucus carota L. sativus)

Summary Genetic and linkage analysis of marker loci were performed with 4 selfed progenies, derived from single plant (I_0/1 lines) of carrot (Daucus carota L. sativus). The analysis of 58 markers included 1 morphological marker, 10 isozyme loci, 14 RFLPs, 28 RAPD markers, and 6 isolated PCR fragments used as RFLP probes. Linkage analysis was carried out with the MAPMAKER program and resulted in the construction of 8 linkage groups containing 55 markers with an average distance of 13.1 cM, 3 marker loci remained unlinked. 24% of the markers deviated significantly from the expected Mendelian ratios (1:2:1 or 3:1) due to gametic or zygotic selection. It was shown that isolated PCR amplification products can be used as RFLP probes to detect polymorphisms for a certain locus in progenies where the corresponding RAPD pattern is monomorphic or no amplification product is observed. Since carrot has a relative small genome the probability of amplifying repetitive DNA sequences is comparatively low. Thus PCR amplification products represent an additional useful source of RFLP probes.     

Random Amplified Polymorphic DNA (RAPD) Markers in Sugar Beet (Beta vulgaris L.): Mapping the Genes for Nematode Resistance and Hypocotyl Colour

The random amplified polymorphic DNA (RAPD) technique was adapted for segregation analysis in sugar beet. 83 I_O/I individuals were scored with a set of 20 arbitrary decamer primers. 4 preliminary linkage groups could be established, enclosing 9 RAPD markers, 2 isozyme loci, a gene for the hypocotyl colour and a gene for resistance to the root knot nematode (Heterodera schachtii Schm.).     

Male and female genetic linkage map of hops, Humulus lupulus

A male and female linkage map of hop has been constructed using 224 DNA polymorphisms (106 amplified fragment length polymorphisms (AFLPs), three random amplified polymorphic DNAs (RAPDs), one RAPD‐sequence‐tagged‐site (STS), and three microsatellite (STSs) segregating in an F₁ population of the English cultivar ‘Wye Target’‐the German male breeding line ‘85/54/15’. Linkage between these loci was estimated using JOINMAP Version 2.0. The final map for the female parent consisted of 110 loci assigned to eight linkage groups covering a distance of 346.7 cM. For the male map, 57 loci could be mapped on nine linkage groups spanning over 227.4 cM. One of these male linkage groups (Gr09‐M) presumably represents the Y chromosome, since all markers assigned (10 AFLPs, three RAPDs and one STS) were closely linked to the male sex (M). Because of their sex‐specific segregation, 10 doubly heterozygous AFLPs spanning a distance of 18.7 cM could be identified as markers describing the X chromosome, which is part of the male and female map. Three STMSs, which had already proved useful in hop genotyping, could be integrated as codominant locus‐specific markers and thus allowed to produce reliable allelic bridges between the female and male counterparts.     

Identification of PCR-based markers linked to the powdery-mildew-resistance gene Pl1 from Malus robusta in cultivated apple

The availability of molecular markers linked to mildew resistance genes would enhance the efficiency of apple-breeding programmes. This investigation focuses on the identification of random amplified polymorphic DNA (RAPD) markers linked to the Pl₁ gene for mildew resistance, which has introgressed from Malus robusta into cultivated apples. The RAPD marker technique was combined with a modified ‘bulked seg-regant analysis’ mapping strategy. About 850 random decamer primers used as single primers or in combinations were tested by PCR analysis on the basis of resistant and susceptible DNA pools. Selected primers producing RAPD fragments were applied in an additional selection step to M. robusta and genotypes representing intermediate breeding stages of the breeding population 93/9, for which a 1:1 segregation could be observed for the resistance trait. Seven RAPD markers, all representing introgressed DNA sequences from M. robusta, were identified and arranged with the Pl₁ locus in a common linkage group. The two most tightly-linked RAPD markers, OPAT20₄₅₀ and OPD2₁₀₀₀ were mapped with a genetic distance of 4.5 and 5 cM, respectively, from the Pl₁ gene. Both markers are suitable for marker-assisted selection in apple breeding. The polymorphic DNA fragment OPAT20₄₅₀ was cloned and sequenced, and longer primers for the generation of a sequence-characterized amplified region (SCAR) marker have been constructed; this marker was easier to score than the original RAPD marker.     

Linkage map construction and mapping QTL for cotton fibre quality using SRAP, SSR and RAPD

Tetraploid cotton is one of the most extensively cultivated species. Two tetraploid species, Gossypium hirsutum L. and G. barbadense L., dominate the world's cotton production. To better understand the genetic basis of cotton fibre traits for the improvement of fibre quality, a genetic linkage map of tetraploid cotton was constructed using sequence‐related amplified polymorphisms (SRAPs), simple sequence repeats (SSRs) and random amplified polymorphic DNAs (RAPDs). A total of 238 SRAP primer combinations, 368 SSR primer pairs and 600 RAPD primers were used to screen polymorphisms between G. hirsutum cv. Handan208 and G. barbadense cv. Pima90 which revealed 749 polymorphic loci in total (205 SSRs, 107 RAPDs and 437 SRAPs). Sixty‐nine F₂ progeny from the interspecific cross of ‘Handan208’בPima90’ were genotyped with the 749 polymorphic markers. A total of 566 loci were assembled into 41 linkage groups with at least three loci in each group. Twenty‐eight linkage groups were assigned to corresponding chromosomes by SSR markers with known chromosome locations. The map covered 5141.8 cM with a mean interlocus space of 9.08 cM. A × test for significance of deviations from the expected ratio (1: 2: 1 or 3: 1) identified 135 loci (18.0%) with skewed segregation, most of which had an excess of maternal parental alleles. In total, 13 QTL associated with fibre traits were detected, among which two QTL were for fibre strength, four for fibre length and seven for micronaire value. These QTL were on nine linkage groups explaining 16.18‐28.92% of the trait variation. Six QTL were located in the A subgenome, six QTL in the D subgenome and one QTL in an unassigned linkage group. There were three QTL for micronaire value clustered on LG1, which would be very useful for improving this trait by molecular marker‐assisted selection.     

Identification of commercial chicory cultivars for hydroponic forcing and their phenetic relationships revealed by random amplified polymorphic DNAs and amplified fragment length polymorphisms

One‐hundred and twenty‐four amplified fragment length polymorphism (AFLP) and 49 random amplified polymorphic DNA (RAPD) markers have been used to distinguish between 20 and 23 commercial chicory cultivars, respectively. These were all Cichorium intybus var. foliosum F₁ hybrids, currently used in hydroponic forcing. Five‐hundred and twenty RAPD primers (OPERON) were tested, of which 156 resulted in reproducible patterns and 26 yielded polymorphisms. Two‐hundred and fifty‐six AFLP primer‐combinations were tested and six combinations were selected for identification purposes. Similarity indices were measured and clustering has been done using pairwise comparison. Both types of marker provide similar conclusions. Two major clusters are formed, representing late and early cultivars. All cultivars were identified using 10 informative RAPD primers or three AFLP primer combinations. A low degree of polymorphism was detected between some early cultivars, suggesting a narrow genetic base in their breeding strategy.     

Construction of a genetic map of melon with molecular markers and horticultural traits, and localization of genes associated with ZYMV resistance

Abstract: A partial linkage map of melon was constructed from a cross between PI414723 and Dulce. Twenty-two SSR, 46RAPD, 2 ISSR markers and four horticultural markers [female flower form (a), Fusarium resistance, striped epicarp (st), and fruit flesh pH (pH)] were analyzed in an F₂/F₃ population to produce a map spanning 14 linkage groups. We report for the first time map positions for the st, a, and pH genes. One SSR marker was tightly linked to pH. Mapping the a gene for the female flower form to molecular linkage group 4 enabled the merging of the map of horticultural traits with the of molecular markers in this region. Using the 22 SSR markers of this map, two of the three postulated ZYMV resistance genes were located using a BC₁ population (PI414723 recurrent parent). One SSR marker was tightly linked to a ZYMV resistance gene, designated Zym-1. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular markers linked to rhizomania resistance in sugar beet, Beta vulgaris, from two different sources map to the same linkage group

Rhizomania, one of the most important diseases of sugar beet, is caused by beet necrotic yellow vein virus, a Furovirus vectored by the fungus Polymyxa betae Keskin. Reduction of the production losses caused by this disease can only be achieved by using tolerant cultivars. The objective of this study was the identification and mapping of random amplified polymorphic DNA (RAPD) markers linked to a rhizomania resistance gene. The RAPD markers were identified using bulked segregant analysis in a segregating population of 62 individuals derived by intercrossing plants of the resistant commercial hybrid GOLF, and the resistance locus was positioned in a molecular marker linkage map made with a different population of 50 GOLF plants. The resistance locus, Rr1, was mapped to linkage group III of our map of Beta vulgaris L. ssp. vulgaris, which consisted of 76 RAPDs, 20 restriction fragment length polymorphisms (RFLPs), three sequence characterized amplified regions (SCARs) and one sequence tagged site (STS). In total, 101 molecular markers were mapped over 14 linkage groups which spanned 688.4 cM with an average interval length of 8.0 cM. In the combined map, Rr1 proved to be flanked by the RAPD loci RA411₁₈₀₀ and AS7₁₁₀₀ at 9.5 and 18.5cM, respectively. Moreover, in our I₂ population, we found that a set of markers shown by Barzen et al. (1997) to be linked to the ‘Holly’ type resistance gene was also linked to the ‘GOLF’-type resistance gene. These results appeared to indicate that the rhizomania resistance gene present in the GOLF hybrid could be the same gene underlying resistance in ‘Holly’-based resistant genotypes. Two other explanations could be applied: first, that two different alleles at the same locus could have been selected; second, that two different genes at two different but clustered loci underwent the selection process.     

Molecular mapping of black rot resistance locus Xca1bo on chromosome 3 in Indian cauliflower (Brassica oleracea var. botrytis L.)

Black rot is the most devastating disease of cauliflower worldwide causing severe damage to crop. The identification of markers linked to loci that control resistance can facilitate selection of plants for breeding programmes. In the present investigation, F₂ population derived from a cross between ‘Pusa Himjyoti’, a susceptible genotype, and ‘BR‐161’, a resistant genotype, was phenotyped by artificial inoculation using Xcc race 1. Segregation analysis of F₂ progeny indicated that a single dominant locus governed resistance to Xcc race 1 in ‘BR‐161’. Bulk segregant analysis in resistant and susceptible bulks of F₂ progeny revealed seven differentiating polymorphic markers (three RAPD, two ISSR and two SSR) of 102 markers screened. Subsequently, these markers were used to genotype the entire F₂ population, and a genetic linkage map covering 74.7 cM distance was developed. The major locus Xca1bo was mapped in 1.6‐cM interval flanked by the markers RAPD 04₈₃₃ and ISSR 11₆₃₅. The Xca1bo locus was located on chromosome 3. The linked markers will be useful for marker‐assisted resistance breeding in cauliflower.     

Identification and mapping of random amplified polymorphic DNA markers linked to a rhizomania resistance gene in sugar beet (Beta vulgaris L.) by bulked segregant analysis

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to a gene that confers rhizomania resistance to a sugar beet line created from a Holly Sugar Company breeding population (USA). Polymorphism revealed with 160 arbitrary 10-mer oligonucleotide primers was screened in two bulks produced by separately pooling the individual DNAs from the six most resistant and the six most susceptible plants of an F₂ population segregating for rhizomania resistance. A study of the F₂ individuals showed that 19 primers generated 44 polymorphic markers which were then grouped into nine linkage groups. By analysis of variance, 12 were shown to have a significant effect upon the level of resistance and were mapped on a segment 22.3 cM long. A quantitative trait locus (QTL) of resistance was identified and located in a 4.6cM interval between two markers. It accounted for 67.4% of the observed variation and almost all the genetic variation. These results suggest that the identified QTL corresponds to a unique major gene conditioning the Holly resistance studied, which we have named Rz-l.     

Genetic mapping of quantitative resistance to race 5 of Pseudomonas syringae pv. phaseolicola in common bean

Halo-blight is an important worldwide bacterial disease of common bean (Phaseolus vulgaris L.) caused by Pseudomonas syringae pv. phaseolicola. Nine races of the pathogen and five race-specific resistance genes have been previously described. However, a quantitative response to this pathogen has also been described. The objective of this study was to identify halo-blight resistance loci linked to molecular markers that could be used in resistance breeding. Chromosomal regions related to race 5 halo-blight resistance were localized on a genetic map of RAPD and AFLP molecular markers and constructed by the analysis of a “Jules” × “Canela” F₂ progeny. “Jules” shows quantitative resistance to halo-blight and “Canela” is a very appreciated but susceptible Spanish bean landrace. Two QTL for resistance to halo-blight were mapped in two linkage groups. There were four large groups, with 14–22 molecular markers each, five with 4–8 markers each, and three with 2 or 3 markers each.     

Stability of the association of molecular markers with common bacterial blight resistance in common bean (Phascolus vulgaris L.)

Common bacterial blight (CBB) caused hy Xanthomonas campestrts pv. phaseoli is an important disease of common bean (Phaseolus vulgaris L.) throughout the world. Two random amplified polymorphic DNA (RAPD) markers (R7313 and R4865) linked to genes for CBB resistance, that were transferred to P- vulgaris by an interspecific cross with Phaseohus acutifoluis. Were identified in a previous study. The current study was conducted to examine the use of these markers for selecting CBB resistant material from 85 F_5,6, lines derived from crosses between two of the resistant lines used previously in the linkage study and susceptible breeding lines. The results showed that these two markers were located on the same linkage group and explained 22% (P = 0.0002) of the variation in response to CBB in the current population. Seventy per cent of the lines that had both markers were classified as resistant in a disease test of the F_5,6, lines, whereas 73% of the lines that had neither of the RAPD markers were susceptible. The results indicated that the marker-disease resistance associations remained stable in a plant breeding programme and that they can be used lor marker-assisted selection of CBB-resistant beans.     

AFLP fingerprinting in Medicago spp.: Its development and application in linkage mapping

Cultivated alfalfa (Medicago sativa L., 2n= 4x= 32) is one of the most important forage crops in temperate climates. The genus Medicago includes diploid species that are a valuable source of wild germplasm for studying the reproductive system of alfalfa and its abnormalities. A linkage map of an apomeiotic mutant of Medicago falcata (L.) Arcang. (2n= 2x= 16) that spanned 368.6 cM and included 29 amplified fragment length polymorphism (AFLP), 35 random amplified polymorphic DNA (RAPD) and three restriction fragment length polymorphism (RFLP) loci was constructed using a one-way pseudo-testcross mapping strategy. The success of such a strategy depends on the presence of sufficiently high levels of heterozygosity in the individual plant which is being mapped and on the informativeness of the marker system that is used. In general: (1) highly informative and reproducible RAPD and AFLP fingerprints were generated and several genome-specific primers selected; (2) of 67 marker loci mapped, 51 were arranged in 11 main linkage groups and eight additional couples of linked marker loci were detected; (3) mapping of an F₁ population theoretically allowed a better estimation of linkage distances since it avoided segregation distortion (x² analyses revealed segregation distortion in only 5.2% of marker loci); (4) the high frequency of unlinked marker loci obtained suggests that, in this alfalfa genotype, DNA markers are distributed throughout the genome. This type of genetic map should find application and prove useful in marker-assisted selection and map-based breeding programmes in meiotic mutants of alfalfa for which there is a lack of suitable genetic markers.     

Construction of high-resolution linkage map of the Ms locus, a restorer-of-fertility gene in onion (Allium cepa L.)

For the purpose of developing closely-linked molecular markers to the Ms locus, a restorer-of-fertility gene in onions (Allium cepa L.), bulked segregant analysis and randomly amplified polymorphic DNA (RAPD) analyses were utilized. Five RAPD markers polymorphic between male-fertile and male-sterile bulks were identified. These RAPD markers were converted into a simple PCR marker or cleaved amplified polymorphic sequence (CAPS) markers after sequencing the RAPD products and obtaining flanking sequences of the RAPD markers by genome walking. A linkage map was constructed with the Ms locus and flanking markers using a F₂ population. There was no recombinant between the Ms locus and two CAPS markers, jnurf05 and jnurf17. To increase resolution among these closely linked molecular markers and the Ms locus, a total of 1,346 F_2:3 and 2,927 F_2:4 plants were analyzed with two flanking markers for detection of recombinants. Segregation of male-fertility phenotypes in large-sized populations confirmed allelic segregation distortion in favor of the recessive Ms allele. Analysis of the recombinants with closely linked markers revealed only two recombinants between the Ms locus and the jnurf05 markers among 4,273 segregating plants, showing very tight linkage between the two loci. However, linkage disequilibrium between the two loci was not too strong among the breeding lines. Despite weak linkage disequilibrium, these tightly linked markers are useful in accurate marker-assisted selection of the Ms alleles and ultimate isolation of the Ms gene by map-based cloning approach.