Tools For Marker-assisted Selection Research Articles

Gene action analysis by inheritance and quantitative trait loci mapping of resistance to root-knot nematodes in cotton

With 2 figures and 5 tables Abstract Host–plant resistance is highly effective in controlling crop loss from nematode infection. In addition, molecular markers can be powerful tools for marker-assisted selection (MAS), where they reduce laborious greenhouse phenotype evaluation to identify root-knot nematode (RKN) Meloidogyne incognita Kofoid and White (Chitwood) resistant genotypes. To better understand host–plant interactions, we investigated gene action and genomic locations of RKN resistance genes in cotton (Gossypium hirsutum L). Twelve parents, 17 intraspecific (G. hirsutum × G. hirsutum) and four interspecific (G. hirsutum ×G. barbadense L.) F1, and 11 F2 populations were investigated by examining different RKN resistance backgrounds. The F1 and F2 generation means and distributions, and differences between F1 and mid-parent values for Galling Index (GI) indicated that allelic interaction, epistasis and heterosis operated in these crosses. Genetic and quantitative trait loci (QTL) analyses in crosses NemX × SJ-2 and SJ-2 × Clevewilt revealed resistance because of at least one major recessive QTL with strong additive effect, while in PS 7 × NemXresistance was because of at least one major QTL with strong dominant effect. Simple sequence repeat (SSR) markers CIR316 and MUCS088 on chromosomes 11 and 21 showed the association of two different chromosomes with the RKN resistance. This also supported the two-gene model of observed resistant : susceptible ratios in F2 and F3 (PS 7 × NemX) populations. Selection success rate for MAS was >85% for identifying resistant genotypes with GI < 3 from these crosses with SSRS CIR316, BNL1231 and MUCS088, and CAPS (GHACC1). QTL analyses validated the importance of a major genome-telomeric region on chromosome 11, harbouring RKN resistance genes from different backgrounds. We conclude that breeding for optimal resistance must be based on selection of progeny with combinations of genes homozygous for resistance.

Genetic mapping for resistance to gray leaf spot in maize

The molecular marker technology has been used on mapping of quantitative trait loci (QTLs) associated with plant resistance. The objectives of this research were to estimate genetic parameters and to map genomic regions involved in the resistance to gray leaf spot in maize. Ninety F3 families from the BS03 (susceptible) and BS04 (resistant) cross were used. Field trials were performed using a 10 × 10 square lattice design with three replications. Data from 62 SSR markers were used for linkage analysis. The locations of the QTLs on the linkage groups were determined by composite interval mapping method and the phenotypic variance explained by each marker was determined by regression analysis. Several QTLs associated to disease resistance were identified in the population BS03 × BS04. Some QTLs showed significant effects over the different environments studied. The existence of significant QTLs in common among different environments indicates these genomic regions as possible new tools for marker-assisted selection in maize breeding programs.

Fine genetic mapping of xa24, a recessive gene for resistance against Xanthomonas oryzae pv. oryzae in rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating plant bacterial disease worldwide. Different bacterial blight resistance (R) genes confer race-specific resistance to different strains of Xoo. We fine mapped a fully recessive gene, xa24, for bacterial blight resistance to a 71-kb DNA fragment in the long arm of rice chromosome 2 using polymerase chain reaction-based molecular markers. The xa24 gene confers disease resistance at the seedling and adult stages. It mediates resistance to at least the Philippine Xoo races 4, 6 and 10 and Chinese Xoo strains Zhe173, JL691 and KS-1-21. Sequence analysis of the DNA fragment harboring the dominant (susceptible) allele of xa24 suggests that this gene should encode a novel protein that is not homologous to any known R proteins. These results will greatly facilitate the isolation and characterization of xa24. The markers will be convenient tools for marker-assisted selection of xa24 in breeding programs.

Functional markers for selection of potyvirus resistance alleles at the pvr2-eIF4E locus in pepper using tetra-primer ARMS–PCR

Functional markers targeted on single nucleotide polymorphisms (SNPs) responsible for phenotypic variation constitute optimal tools for marker-assisted selection (MAS) of resistance alleles with different specificities. Here, we used the tetra-primer ARMS-PCR procedure to assay SNP signatures of four distinct alleles at the pvr2-eIF4E locus, which controls pepper resistance to several potyviruses. These simple, economical, and codominant markers open the way for MAS of pepper genotypes resistant to the potyviral strains and species that are prevalent in distinct cultivation areas across the world.

A new PCR-based marker on chromosome 4AL for resistance to pre-harvest sprouting in wheat (Triticum aestivum L.)

Pre-harvest sprouting (PHS) is a complex trait controlled by multiple genes with strong interaction between environment and genotype that makes it difficult to select breeding materials by phenotypic assessment. One of the most important genes for pre-harvest sprouting resistance is consistently identified on the long arm of chromosome 4A. The 4AL PHS tolerance gene has therefore been targeted by Australian white-grained wheat breeders. A new robust PCR marker for the PHS QTL on wheat chromosome 4AL based on candidate genes search was developed in this study. The new marker was mapped on 4AL deletion bin 13-0.59-0.66 using 4AL deletion lines derived from Chinese Spring. This marker is located on 4AL between molecular markers Xbarc170 and Xwg622 in the doubled-haploid wheat population Cranbrook × Halberd. It was mapped between molecular markers Xbarc170 and Xgwm269 that have been previously shown to be closely linked to grain dormancy in the doubled haploid wheat population SW95-50213 × Cunningham and was co-located with Xgwm269 in population Janz × AUS1408. This marker offers an additional efficient tool for marker-assisted selection of dormancy for white-grained wheat breeding. Comparative analysis indicated that the wheat chromosome 4AL QTL for seed dormancy and PHS resistance is homologous with the barley QTL on chromosome 5HL controlling seed dormancy and PHS resistance. This marker will facilitate identification of the gene associated with the 4A QTL that controls a major component of grain dormancy and PHS resistance.

Molecular Characterization of a Stripe Rust Resistance Gene from Wheat Line S2199 and Its Allelism with Yr5

Yellow rust, caused by Puccinia striiformis f. sp. tritici (PST), is one of the most devastating diseases in common wheat ( Triticum aestivum L.) worldwide. Molecular markers are powerful tools in marker-assisted selection, gene pyramiding, and gene cloning of important crop traits, especially for disease resistance. The objectives of this study were to develop tightly linked molecular marker of a yellow rust resistance gene against the prevalent Chinese races of PST in an improved wheat line S2199 and to characterize its allelism with Yr5. Genetic analysis indicated that a single dominant gene was responsible for the yellow rust resistance in S2199, which was temporarily designated as YrS2199. By screening 1,856 pairs of SSR primers, 2 markers, Xdp269 and Xgwm120, were linked to the yellow rust resistance gene with genetic distance of 0.7 and 11.0 cM, respectively. The SSR marker Xgwm120 has been genetically and physically mapped on 2BL chromosome arm in wheat. Using Chinese Spring nullisomic-tetrasomics, ditelosomics, and deletion lines of homoeologous group 2, Xdp269 was physically mapped on the terminal bin (0.89–1.0) of chromosome arm 2BL. Both allelism test of 700 F 2 plants from the cross YrS2199/Yr5 and seedling tests of YrS2199 and Yr5 on 14 PST isolates indicated that YrS2199 and Yr5 were likely to be the same gene or allelic genes. The YrS2199 tightly linked to SSR marker Xdp269 can be used as a potential tool for cloning the yellow rust resistance gene or for marker assisted breeding program.

1H NMR‐based metabolite profiling as a potential selection tool for breeding passive resistance against Fusarium head blight (FHB) in wheat

(1)H NMR measurements on protein-free extracts from wheat leaf and stem tissue were used to investigate the biochemical correlates of partial resistance to fungal species implicated in the Fusarium head blight (FHB) disease complex. The wheat genotypes included five commercial wheat cultivars and 116 wheat genotypes, from the CIMMYT international FHB breeding programme in Mexico, that had been bred for FHB disease resistance, utilizing exotic, typically Chinese, resistance sources. Principal component analysis of the nuclear magnetic resonance (NMR)-derived metabolite profiles revealed distinct separation of the commercial wheat cultivars from the majority of the CIMMYT wheat genotypes with the commercial cultivars exhibiting markedly higher carbohydrate concentrations. A cross-validated partial least squares (PLS) regression model of the metabolite profile against the partial disease resistance component latent period (delay in sporulation of the fungus on the plant tissue) predicted latent periods that were significantly correlated with the experimentally determined values (R(2) = 0.34, P < 0.001). Identified metabolites that were found in plants with shorter latent periods (higher disease susceptibility) included choline, the single most influential metabolite in the PLS model, betaine, the amino acids glutamine, glutamate and alanine, trans-aconitate and sucrose. Metabolites related to increased disease resistance included glucose and unassigned resonances in the carbohydrate and aromatic regions of the NMR spectra. The current study has demonstrated the potential of metabolite profiling as a tool for marker-assisted selection in commercial breeding for resistance to FHB in wheat.

Identification of quantitative trait loci controlling winter hardiness in an annual × perennial ryegrass interspecific hybrid population

Winter hardiness is a quantitative trait and the lack of it limits geographic distribution of ryegrass. Improving winter hardiness is an important breeding goal in ryegrass breeding programs. An understanding of the genetic basis for the component traits of winter hardiness would allow more efficient selection. A three-generation interspecific population of an annual × perennial ryegrass consisting of 152 progenies was used to map quantitative trait loci (QTL) that control winter hardiness-related traits including fall growth (FG), freezing tolerance (FT), and winter survival (WS) over 2 years. A total of 39 QTL were identified for the three traits from both the female parental (MFA) and the male parental (MFB) maps, of which 13 were for FG, 6 for FT, and 20 for WS. The proportion of phenotypic variation explained by individual QTL ranged from 10.4 to 22.1%. Both FG and FT were positively correlated with WS. Common QTL were detected between FG, FT, and WS. The QTL associated with WS on linkage groups (LGs) 4 and 5, and the QTL for FT on LG 5 were consistently identified over years and maps. These consistent QTL might serve as potential tools for marker-assisted selection to improve ryegrass winter hardiness.

A quantitative-trait locus for resistance to ascochyta blight [Ascochyta lentis] maps close to a gene for resistance to anthracnose [Colletotrichum truncatum] in lentil

Ascochyta blight, caused by Ascochyta lentis, is an important disease of lentil causing serious yield and quality losses. The objectives of the present research were to investigate the genetics of resistance to ascochyta blight, using a population of recombinant inbred lines (RILs) developed from a cross between the susceptible Lens culinaris 'Eston' and a resistant germplasm line (PI 320937), and to identify markers associated with resistance to ascochyta blight, using a quantitative-trait loci (QTLs) analysis. The RILs were spray-inoculated with an isolate of A. lentis (3C-2) in a replicated greenhouse test. The RILs scores for reaction to ascochyta blight showed a continuous distribution, suggesting a polygenic inheritance of resistance to ascochyta blight in lentil. A QTL that explained 41% of the variation in the reaction to ascochyta blight was identified on the linkage-group 6. This QTL was localized between an amplified fragment length polymorphism (AFLP) marker (ctcaccB) and LCt2, a gene for resistance to anthracnose [Colletotrichum truncatum]. This is the first evidence in lentil for linkage between genes conferring resistance to two different foliar pathogens. The result was further verified by cosegregation of a marker locus (OP-P4400) in our mapping population, which was also previously identified to be linked to a major QTL for resistance to ascochyta blight in the lentil germplasm line ILL-5588. The AFLP and random amplified polymorphic DNA (RAPD) markers localized around the resistance region will be useful tools in marker-assisted selection.

Haplotype characterization and markers at the barley Mlo powdery mildew resistance locus as tools for marker-assisted selection

Recessive mlo alleles of the barley Mlo gene confer resistance to almost all known isolates of the powdery mildew fungal pathogen targeting barley (Hordeum vulgare). To characterize haplotypes present in the Mlo chromosomal region of cultivated Mlo and mlo barley genotypes, we conducted a polymorphism search in 3 predicted low-copy sequence regions adjacent to the Mlo gene by examining a sample of 4 Mlo and 3 mlo cultivars. Eight single-nucleotide polymorphisms (SNPs) and 1 insertion-deletion (indel) were detected, and easy to use PCR-based markers were developed for typing the SNPs. The PCR markers were used to characterize a collection of 46 Mlo and 25 mlo barley cultivars, identifying 3 distinct mlo-11 haplotypes, 1 mlo-9 haplotype, and 4 Mlo haplotypes. We summarized the haplotype and marker information obtained here and in a previous study to help breeders identify strategies for mlo marker-assisted selection. The ability of the markers to identify mlo-resistant genotypes in segregating populations was demonstrated using 2 resistance-characterized F2 populations derived by 3-way crosses.

Genetics and Marker-assisted Selection of the Chemotype in Cannabis sativa L.

Cannabis sativa is an interesting crop for several industrial uses, but the legislations in Europe and USA require a tight control of cannabinoid type and content for cultivation and subsidies release. Therefore, cannabinoid survey by gas chromatography of materials under selection is an important step in hemp breeding. In this paper, a number of Cannabis accessions were examined for their cannabinoid composition. Their absolute and relative content was examined, and results are discussed in the light of both the current genetic model for cannabinoid’s inheritance, and the legislation’s requirements. In addition, the effectiveness of two different types of markers associated to the locus determining the chemotype in Cannabis was evaluated and discussed, as possible tools in marker-assisted selection in hemp, but also for possible applications in the forensic and pharmaceutical fields.

A Class IV Chitinase Is Up-Regulated by Fungal Infection and Abiotic Stresses and Associated with Slow-Canker-Growth Resistance to Cronartium ribicola in Western White Pine (Pinus monticola)

ABSTRACT In the present study, in a candidate gene approach, a class IV chitinase gene (PmCh4A) of pathogenesis-related family three was cloned and characterized in western white pine (Pinus monticola). PmCh4A chitinase expression in the different organs of healthy seedlings was below levels detectable by western immunoblot analysis using an antibody raised against PmCh4A protein. However, a 27-kDa isozyme of PmCh4A accu mulated in both susceptible and slow-canker-growth (SCG) resistant seedlings after infection by Cronartium ribicola. As with fungal infection, the application of a signal chemical (methyl jasmonate) and a protein phosphatase 1 and 2A inhibitor (okadaic acid) increased the PmCh4A protein accumulation. Furthermore, another 26-kDa isozyme was expressed specifically in SCG resistant seedlings, providing a potential tool for marker-assisted selection in forest breeding. Wounding treatment also induced expression of the protein. These data suggest that the class IV chitinase PmCh4A is involved in the defense response of western white pine to infection and abiotic stresses.

Resistance gene analogues identified through the NBS-profiling method map close to major genes and QTL for disease resistance in apple

We used a new method called nucleotide-binding site (NBS) profiling to identify and map resistance gene analogues (RGAs) in apple. This method simultaneously allows the amplification and the mapping of genetic markers anchored in the conserved NBS-encoding domain of plant disease resistance genes. Ninety-four individuals belonging to an F1 progeny derived from a cross between the apple cultivars 'Discovery' and 'TN10-8' were studied. Two degenerate primers designed from the highly conserved P-loop motif within the NBS domain were used together with adapter primers. Forty-three markers generated with NBS profiling could be mapped in this progeny. After sequencing, 23 markers were identified as RGAs, based on their homologies with known resistance genes or NBS/leucine-rich-repeat-like genes. Markers were mapped on 10 of the 17 linkage groups of the apple genetic map used. Most of these markers were organized in clusters. Twenty-five markers mapped close to major genes or quantitative trait loci for resistance to scab and mildew previously identified in different apple progenies. Several markers could become efficient tools for marker-assisted selection once converted into breeder-friendly markers. This study demonstrates the efficiency of the NBS-profiling method for generating RGA markers for resistance loci in apple.

Microsatellite markers associated with two Aegilops tauschii-derived greenbug resistance loci in wheat

A new source of greenbug (Schizaphis graminum Rondani) resistance derived from Aegilops tauschii (Coss.) Schmal was identified in W7984, a synthetic hexaploid wheat line and one parent of the International Triticeae Mapping Initiative (ITMI) mapping population. Segregation analysis of responses to greenbug feeding in a set of recombinant inbred lines (RILs) identified a single, dominant gene governing the greenbug resistance in W7984, which was placed in chromosome arm 7DL by linkage analysis with molecular markers in the ITMI population. Allelism tests based on the segregation of responses to greenbug feeding in F2 and testcross plants revealed that the greenbug resistance in W7984 and Largo, another synthetic line carrying the greenbug resistance gene Gb3, was controlled by different but linked loci. Using the ITMI reference map and a target mapping strategy, we have constructed a microsatellite map of Gb3 in a mapping population of 130 F7 RILs from Largo x TAM 107 and identified one marker (Xwmc634) co-segregating with Gb3 and four markers (Xbarc76, Xgwm037, Xgwm428 and Xwmc824) closely linked with Gb3. Deletion mapping of selected microsatellite markers flanking the Gb3 locus placed this resistance gene into the distal 18% region of 7DL. Comparative mapping in the ITMI and Largo x TAM 107 populations using the same set of microsatellite markers provided further evidence that greenbug resistance in W7984 and Largo is conditioned by two different loci. We suggest that the greenbug resistance gene in W7984 be designated Gb7. The microsatellite map of Gb3 constructed from this study should be a valuable tool for marker-assisted selection of Gb3-conferred greenbug resistance in wheat breeding.

Fine-mapping of the BaMMV, BaYMV-1 and BaYMV-2 resistance of barley (Hordeum vulgare) accession PI1963

Barley yellow mosaic disease caused by the bymoviruses barley mild mosaic virus (BaMMV) and barley yellow mosaic virus (BaYMV) is one of the economically most important diseases of winter barley in Europe. In European barley breeding programmes, resistance is currently due to only two genes-rym4, which is effective against viruses BaMMV and BaYMV-1, and rym5, which is effective against BaYMV-2. Diversification of resistance is therefore an important task. Because the accession PI1963 confers immunity against all European strains of barley yellow mosaic disease and is not allelic to rym5, we have attempted to develop closely linked markers in order to facilitate the efficient introgression of this resistance into adapted germplasm. By means of restriction fragment length polymorphism analysis, we located a gene locus for resistance to BaMMV, BaYMV-1 and BaYMV-2 of PI1963 on chromosome 4HL using a mapping population (W757) comprising 57 doubled haploid (DH) lines. Subsequent tests for allelism indicated that the BaMMV resistance gene in PI1963 is allelic to rym11. Two DH populations, IPK1 and IPK2, comprising 191 and 161 DH lines, respectively, were derived from the initial mapping population W757 and used for further analysis. As random amplified polymorphic DNA development did not facilitate the identification of more closely linked markers, simple sequence repeat (SSR) analyses were conducted. For population IPK1, the closest SSRs detected were Bmac181 and Bmag353, which flank the gene at 2.1 cM and 2.7 cM, respectively. For the IPK2 population, the SSR markers HVM3 and Bmag353 are located proximally at 2.5 cM and distally at 8.2 cM, respectively. In order to develop markers more tightly linked to rym11, a targeted amplified fragment length polymorphism (AFLP) marker identification approach was adopted using bulks comprising lines carrying recombination events proximal and distal to the target interval. Using this approach we identified six AFLP markers closely linked to rym11, with the two markers, E56M32 and E49M33, co-segregating with rym11 in both populations. The SSRs and AFLPs identified in this study represent useful tools for marker-assisted selection.

Acetohydroxyacid synthase mutations conferring resistance to imidazolinone or sulfonylurea herbicides in sunflower.

Wild biotypes of cultivated sunflower ( Helianthus annuus L.) are weeds in corn ( Zea mays L.), soybean ( Glycine max L.), and other crops in North America, and are commonly controlled by applying acetohydroxyacid synthase (AHAS)-inhibiting herbicides. Biotypes resistant to two classes of AHAS-inhibiting herbicides-imidazolinones (IMIs) or sulfonylureas (SUs)-have been discovered in wild sunflower populations (ANN-PUR and ANN-KAN) treated with imazethapyr or chlorsulfuron, respectively. The goals of the present study were to isolate AHAS genes from sunflower, identify mutations in AHAS genes conferring herbicide resistance in ANN-PUR and ANN-KAN, and develop tools for marker-assisted selection (MAS) of herbicide resistance genes in sunflower. Three AHAS genes ( AHAS1, AHAS2, and AHAS3) were identified, cloned, and sequenced from herbicide-resistant (mutant) and -susceptible (wild type) genotypes. We identified 48 single-nucleotide polymorphisms (SNPs) in AHAS1, a single six-base pair insertion-deletion in AHAS2, and a single SNP in AHAS3. No DNA polymorphisms were found in AHAS2 among elite inbred lines. AHAS1 from imazethapyr-resistant inbreds harbored a C-to-T mutation in codon 205 ( Arabidopsis thaliana codon nomenclature), conferring resistance to IMI herbicides, whereas AHAS1 from chlorsulfuron-resistant inbreds harbored a C-to-T mutation in codon 197, conferring resistance to SU herbicides. SNP and single-strand conformational polymorphism markers for AHAS1, AHAS2, and AHAS3 were developed and genetically mapped. AHAS1, AHAS2, and AHAS3 mapped to linkage groups 2 ( AHAS3), 6 ( AHAS2), and 9 ( AHAS1). The C/T SNP in codon 205 of AHAS1 cosegregated with a partially dominant gene for resistance to IMI herbicides in two mutant x wild-type populations. The molecular breeding tools described herein create the basis for rapidly identifying new mutations in AHAS and performing MAS for herbicide resistance genes in sunflower.

Discovery of single nucleotide polymorphisms in Lycopersicon esculentum by computer aided analysis of expressed sequence tags

Single nucleotide polymorphisms SNPs are useful for characterizing allelic variation, for genome-wide mapping, and as a tool for marker-assisted selection. Discovery of SNPs through de novo sequencing is inefficient within cultivated tomato Lycopersicon esculentum Mill. because the polymorphism rate is more than ten-fold lower than the sequencing error rate. The availability of expressed sequence tag EST data has made it feasible to discover putative SNPs “in silico” prior to experimental verification. By exploiting redundancy among EST data available for different varieties among 148,373 tomato ESTs, we have identified candidate SNPs for use within cultivated germplasm pools. 1,245 contigs having three EST sequences of Rio Grande and three EST sequences of TA496 were used for SNP discovery. We detected 1 SNP for every 8,500 bases analyzed, with 101 candidate SNPs in 44 genes identified. Sixty-six SNPs could be recognized by restriction enzymes, and subsequent experimental verification using restriction digestion or CEL I digestion confirmed 83% of the putative polymorphisms tested. SNPs between TA496 and Rio Grande have a high probability 53% of detecting polymorphisms between other L. esculentum varieties. Twenty-six SNPs in 18 unigenes were mapped to specific chromosomes. Two SNPs, LEOH23 and LEOH37, were shown to be linked to quantitative trait loci contributing to fruit color within elite breeding populations. These results suggest that the growing databases of DNA sequence will yield information that facilitates improvement within the germplasm pools that have contributed to productive modern varieties.

Molecular mapping of a gene responsible for Al-activated secretion of citrate in barley.

Aluminium (Al) toxicity is an important limitation to barley (Hordeum vulgare L.) on acid soil. Al-resistant cultivars of barley detoxify Al externally by secreting citrate from the roots. To link the genetics and physiology of Al resistance in barley, genes controlling Al resistance and Al-activated secretion of citrate were mapped. An analysis of Al-induced root growth inhibition from 100 F2 seedlings derived from an Al-resistant cultivar (Murasakimochi) and an Al-sensitive cultivar (Morex) showed that a gene associated with Al resistance is localized on chromosome 4H, tightly linked to microsatellite marker Bmag353. Quantitative trait locus (QTL) analysis from 59 F4 seedlings derived from an F3 plant heterozygous at the region of Al resistance on chromosome 4H showed that a gene responsible for the Al-activated secretion of citrate was also tightly linked to microsatellite marker Bmag353. This QTL explained more than 50% of the phenotypic variation in citrate secretion in this population. These results indicate that the gene controlling Al resistance on barley chromosome 4H is identical to that for Al-activated secretion of citrate and that the secretion of citrate is one of the mechanisms of Al resistance in barley. The identification of the microsatellite marker associated with both Al resistance and citrate secretion provides a valuable tool for marker-assisted selection of Al-resistant lines.

Efficient construction of high-density linkage map and its application to QTL analysis in barley.

Using a High Efficiency Genome Scanning (HEGS) system and recombinant inbred (RI) lines derived from the cross of Russia 6 and H.E.S. 4, a high-density genetic map was constructed in barley. The resulting 1,595.7-cM map encompassed 1,172 loci distributed on the seven linkage groups comprising 1,134 AFLP, 34 SSR, three STS and vrs1 (kernel row type) loci. Including PCR reactions, gel electrophoresis and data processing, 6 months of work by a single person was sufficient for the whole mapping procedure under a reasonable cost. To make an appraisal of the resolution of genetic analysis for the 95 RI lines based on the constructed linkage map, we measured three agronomic traits: plant height, spike exsertion length and 1,000-kernel weight, and the analyzed quantitative trait loci (QTLs) associated with these traits. The results were compared on the number of detected QTLs and their effects between a high-density map and a skeleton map constructed by selected AFLP and anchor markers. The composite interval mapping on the high-density map detected more QTLs than the other analyses. Closely linked markers with QTLs on the high-density map could be powerful tools for marker-assisted selection in barley breeding programs and further genetic analyses including an advanced backcross analysis or a map-based cloning of QTL.

APPLICATION OF RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS FOR DETECTION OF RESISTANT CULTIVARS OF MELON (CUCUMIS MELO) AGAINST CUCURBITACEAE VIRUSES

Twenty melon cultivars collected mostly in Asia were tested their resistance against an Indonesian isolate of cucumber mosaic virus (CMV-B2) and a Kagoshima-Japan isolate of cucumber green mottle mosaic virus (CGMMV-K) by manual inoculation and scored as resistant or susceptible using DAS ELISA analysis. Since no cultivars showed resistance to CGMMV, 4 cultivars, ‘Shinjong’ (Inner Mongolian cultivar), ‘Mawatauri’, ‘Yamatour’i, ‘Miyamauri’ (three cultivars from Japan) were shown to be CMV resistant. Forty-six RAPD markers were evaluated to detect DNA bands specific to resistant cultivars. Two specific bands (550 bp and 1050 bp, respectively) by OPE-E14 primer (Operon Technologies Inc.) and APB-05 primer (Amersham Pharmacia Biotech Inc.) were obtained in the 4 resistant cultivars. Among them, bulked segregant analysis of ‘Yamatouri’ (a resistant cultivar) and its family were selected and constructed by crossing between ‘Yamatouri’ and ‘Vakharman’ (a susceptible cultivar from Turkmenistan) to identify RAPD markers linked to CMV-B2 resistance. Specific DNA bands which linked to CMV-B2 resistance were obtained in P1 ‘Yamatouri’, F1, some F2 and BCs. On the other hand, the result of reciprocal crossing showed that all of the tested F1 plants had one specific DNA band derived from both parents, respectively. Thus, we concluded that the resistance to CMV-B2 in ‘Yamatouri’ family plants depends on the existence of the DNA locus that segregated in a single dominant manner. The results also showed that RAPD markers could be used as a tool for marker-assisted selection (MAS) linked to CMV resistance in ‘Yamatouri’ family.