QTL analysis of powdery mildew resistance in cucumber (Cucumis sativus L.)

A population of F₇ recombinant inbred lines (RILs) was made from a cross between susceptible (‘Santou’) and resistant (PI197088-1) lines of cucumber in order to study powdery mildew resistance loci. Susceptibility to powdery mildew in the F₇ RIL individuals showed a continuous distribution from susceptible to resistant, suggesting that powdery mildew resistance is controlled by quantitative trait loci (QTLs). A QTL analysis identified two and three loci for powdery mildew resistance under 26 and 20°C conditions, respectively. One QTL was found in the same position under both temperature conditions. Therefore, it is more likely that one major QTL acts under both temperature conditions and that other QTLs are specific to the two temperature conditions. The above results suggest that the four QTLs are controlled in a different temperature manner, and that their combination played an important role in expressing a high level of resistance to powdery mildew in this cucumber population. Sequence-tagged site (STS) markers associated with each QTL were developed and would be useful for breeding a cucumber line with a high level of powdery mildew resistance. Y. Sakata and N. Kubo contributed equally to this work and are considered as first authors.     

Codominant PCR-based markers and candidate genes for powdery mildew resistance in melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

Powdery mildew caused by Podosphaera xanthii is a major disease in melon crops, and races 1, 2, and 5 of this fungus are those that occur most frequently in southern Europe. The genotype TGR-1551 bears a dominant gene that provides resistance to these three races of P. xanthii. By combining bulked segregant analysis and amplified fragment length polymorphisms (AFLP), we identified eight markers linked to this dominant gene. Cloning and sequencing of the selected AFLP fragments allowed the development of six codominant PCR-based markers which mapped on the linkage group (LG) V. Sequence analysis of these markers led to the identification of two resistance-like genes, MRGH5 and MRGH63, belonging to the nucleotide binding site (NBS)-leucine-rich repeat (LRR) gene family. Quantitative trait loci (QTL) analysis detected two QTLs, Pm-R1-2 and Pm-R5, the former significantly associated with the resistance to races 1 and 2 (LOD score of 26.5 and 33.3; 53.6 and 61.9% of phenotypic variation, respectively), and the latter with resistance to race 5 (LOD score of 36.8; 65.5% of phenotypic variation), which have been found to be colocalized with the MRGH5 and MRGH63 genes, respectively. The results suggest that the cluster of NBS-LRR genes identified in LG V harbours candidate genes for resistance to races 1, 2, and 5 of P. xanthii. The evaluation of other resistant germplasm showed that the codominant markers here reported are also linked to the Pm-w resistance gene carried by the accession ‘WMR-29’ proving their usefulness as genotyping tools in melon breeding programmes.     

Simple-sequence repeat markers used in merging linkage maps of melon (Cucumis melo L.)

A set of 118 simple sequence repeat (SSR) markers has been developed in melon from two different sources: genomic libraries (gSSR) and expressed sequence-tag (EST) databases (EST-SSR). Forty-nine percent of the markers showed polymorphism between the Piel de Sapo (PS) and PI161375 melon genotypes used as parents for the mapping populations. Similar polymorphism levels were found in gSSR (51.2%) and EST-SSR (45.5%). Two populations, F₂ and a set of double haploid lines (DHLs), developed from the same parent genotypes were used for map construction. Twenty-three SSRs and 79 restriction fragment length polymorphisms (RFLPs), evenly distributed through the melon genome, were used to anchor the maps of both populations. Ten cucumber SSRs, 41 gSSRs, 16 EST-SSR, three single nucleotide polymorphism (SNP) markers, and the Nsv locus were added in the DHL population. The maps developed in the F₂ and DHL populations were co-linear, with similar lengths, except in linkage groups G1, G9, and G10. There was segregation distortion in a higher proportion of markers in the DHL population compared with the F₂, probably caused by selection during the construction of DHLs through in vitro culture. After map merging, a composite genetic map was obtained including 327 transferable markers: 226 RFLPs, 97 SSRs, three SNPs, and the Nsv locus. The map length is 1,021 cM, distributed in 12 linkage groups, and map density is 3.11 cM/marker. SSR markers alone cover nearly 80% of the map length. This map is proposed as a basis for a framework melon map to be merged with other maps and as an anchor point for map comparison between species of the Cucurbitaceae family.Electronic Supplementary Material Supplementary material is available for this article at     

Microsatellite markers for powdery mildew resistance in pea (Pisum sativum L.)

Powdery mildew is a common disease of field pea, Pisum sativum L., and is caused by the ascomycete fungus Erysiphe pisi. It can cause severe damage in areas where pea is cultivated. Today breeders want to develop new pea lines that are resistant to the disease. To make the breeding process more efficient, it is desirable to find genetic markers for use in a marker-assisted selection (MAS) strategy. In this study, microsatellites (SSR) were used to find markers linked to powdery mildew resistance. The resistant pea cultivar '955180' and the susceptible pea cultivar 'Majoret' were crossed and F2 plants were screened with SSR markers, using bulked segregant analysis. A total of 315 SSR markers were screened out of which five showed linkage to the powdery mildew resistance gene. No single marker was considered optimal for inclusion in a MAS program. Instead, two of the markers can be used in combination, which would result in only 1.6% incorrectly identified plants. Thus SSR markers can be successfully used in marker-assisted selection for powdery mildew resistance breeding in pea.     

Comparative mapping of ZYMV resistances in cucumber (Cucumis sativus L.) and melon (Cucumis melo L.)

Zucchini yellow mosaic virus (ZYMV) routinely causes significant losses in cucumber (Cucumis sativus L.) and melon (Cucumis melo L.). ZYMV resistances from the cucumber population TMG1 and the melon plant introduction (PI) 414723 show different modes of inheritance and their genetic relationships are unknown. We used molecular markers tightly linked to ZYMV resistances from cucumber and melon for comparative mapping. A 5-kb genomic region (YCZ-5) cosegregating with the zym locus of cucumber was cloned and sequenced to reveal single nucleotide polymorphisms and indels distinguishing alleles from ZYMV-resistant (TMG1) and susceptible (Straight 8) cucumbers. A low-copy region of the YCZ-5 clone was hybridized to bacterial artificial chromosome (BAC) clones of melon and a 180-kb contig assembled. One end of this melon contig was mapped in cucumber and cosegregated with ZYMV resistance, demonstrating that physically linked regions in melon show genetic linkage in cucumber. However the YCZ-5 region segregated independently of ZYMV resistance loci in two melon families. These results establish that these sources of ZYMV resistances from cucumber TMG1 and melon PI414723 are likely non-syntenic.     

Co-mapping studies of QTLs for fruit acidity and candidate genes of organic acid metabolism and proton transport in sweet melon (Cucumis melo L.)

Sweet melon cultivars contain a low level of organic acids and, therefore, the quality and flavor of sweet melon fruit is determined almost exclusively by fruit sugar content. However, genetic variability for fruit acid levels in the Cucumis melo species exists and sour fruit accessions are characterized by acidic fruit pH of <5, compared to the sweet cultivars that are generally characterized by mature fruit pH values of >6. In this paper, we report results from a mapping population based on recombinant inbred lines (RILs) derived from the cross between the non-sour 'Dulce' variety and the sour PI 414323 accession. Results show that a single major QTL for pH co-localizes with major QTLs for the two predominant organic acids in melon fruit, citric and malic, together with an additional metabolite which we identified as uridine. While the acidic recombinants were characterized by higher citric and malic acid levels, the non-acidic recombinants had a higher uridine content than did the acidic recombinants. Additional minor QTLs for pH, citric acid and malic acid were also identified and for these the increased acidity was unexpectedly contributed by the non-sour parent. To test for co-localization of these QTLs with genes encoding organic acid metabolism and transport, we mapped the genes encoding structural enzymes and proteins involved in organic acid metabolism, transport and vacuolar H+ pumps. None of these genes co-localized with the major pH QTL, indicating that the gene determining melon fruit pH is not one of the candidate genes encoding this primary metabolic pathway. Linked markers were tested in two additional inter-varietal populations and shown to be linked to the pH trait. The presence of the same QTL in such diverse segregating populations suggests that the trait is determined throughout the species by variability in the same gene and is indicative of a major role of the evolution of this gene in determining the important domestication trait of fruit acidity within the species.     

QTL molecular marker location of powdery mildew resistance in cucumber (Cucumis sativus L.)

The cucumber lines, S94 (Northern China open-field type, powdery mildew (PM) susceptible) and S06 (European greenhouse type, PM resistant), and their F6:7 populations were used to investigate PM re-sistance under seedling spray inoculation in 2005/Autumn and 2006/Spring. QTL analysis was under-taken based on a constructed molecular linkage map of the corresponding F6 population using com-posite interval mapping. A total of four QTLs (pm1.1, pm2.1, pm4.1 and pm6.1) for PM resistance were identified and located on LG 1, 2, 4 and 6, respectively, explaining 5.2%-21.0% of the phenotypic variation. Three consistent QTLs (pm1.1, pm2.1 and pm4.1) were detected under the two test conditions. The QTL pm6.1 was only identified in 2005/Autumn. The total phenotypic variation explained by the QTLs was 52.0% and 42.0% in 2005/Autumn and 2006/Spring, respectively. Anchor markers tightly linked to those loci (<5 cM) could lay a basis for both molecular marker-assisted breeding and map-based gene cloning of the PM-resistance gene in cucumber.     

新疆甜瓜地方种质资源遗传多样性的SRAP分析

为研究我国新疆甜瓜地方种质资源亲缘关系及其分类,充分高效的利用种质资源,利用SRAP(sequence-related amplified polymorphism technique)标记对117份中国新疆甜瓜地方品种和28份国内外对照材料进行亲缘关系和遗传多样性分析。结果表明,20对SRAP引物共扩增出224个带,其中多态性谱带216个,多态性比率达96%,平均每对引物扩增的带数和多态性带数分别为11.2个和10.8个,每对引物的多态性信息含量PIC值为0.73~0.94,平均为0.85;不同生态区域供试材料的Nei's基因多样性指数(H)和Shannon's信息指数(I)分别为0.1075~0.2560和0.1569~0.4061,中国新疆的南疆、东疆和北疆均高于其他生态区域供试材料,且以南疆最高,具有非常丰富的遗传多样性;不同生态区域甜瓜种质资源的遗传一致度和遗传距离分别为0.6384~0.9919和0.0081~0.4488,其中南疆、东疆和北疆两两之间的遗传一致度均在0.95以上,遗传距离均在0.04以下,三者之间遗传分化较小;中国新疆甜瓜与印度、西亚、西班牙的甜瓜种质资源亲缘关系较近,与韩国、日本、美国和前苏联的甜瓜种质资源亲缘关系较远。聚类分析结果表明,以遗传相似系数0.548为阈值,145份种质材料可分为3大类群;厚皮甜瓜与薄皮甜瓜间在分子水平上没有严格的界限,两者之间亲缘关系的远近在不同的种质材料间差异很大;117份中国新疆甜瓜地方种质资源可分为A(Ⅰ-1)、B(Ⅰ-2、Ⅰ-3、Ⅰ-5)、C(Ⅰ-6)、D(Ⅱ)等4大类6个亚类群,与传统4个变种10个品种群分类结果不同,但在每个大类或亚类群中属于同一变种或品种群的材料倾向于聚在一起。     

Development of loop-mediated isothermal amplification (LAMP)-based SNP markers for shelf-life in melon (Cucumis melo L.)

In this study, LAMP markers linked to shelf-life in melon (Cucumis melo L.) were developed by converting a cleaved amplified polymorphic sequences (CAPS) marker (C2). The CAPS-PCR fragments from the long-shelf-life melon (O-3) and short-shelf-life melon (Nat-2) were cloned and sequenced to construct LAMP primers. A single nucleotide polymorphism (SNP) was identified between O-3 and Nat-2. LAMP primers were designed to detect the SNP. In the LAMP reaction to detect long-shelf-life melon, the turbidity of the templates using O-3, F1, homozygous long-shelf-life F2 lines and heterozygous long-shelf-life F2 lines started to increase after 40 min. In contrast, the turbidity of Nat-2 and homozygous short-shelf-life F2 lines did not increase even after 90 min. In the LAMP reaction to detect short-shelf-life melon, the turbidity of the templates using Nat-2, F1, homozygous short-shelf-life F2 lines and heterozygous long-shelf-life F2 lines started to increase after 40 min. But the turbidity of O-3 and homozygous long-shelf-life F2 lines did not increase after 90 min. This attests to the high reliability and usefulness of LAMP for marker-assisted selection.     

Bin mapping of genomic and EST-derived SSRs in melon (Cucumis melo L.)

We report the development of 158 primer pairs flanking SSR motifs in genomic (gSSR) and EST (EST-SSR) melon sequences, all yielding polymorphic bands in melon germplasm, except one that was polymorphic only in Cucurbita species. A similar polymorphism level was found among EST-SSRs and gSSRs, between dimeric and trimeric EST-SSRs, and between EST-SSRs placed in the open reading frame or any of the 5′- or 3′-untranslated regions. Correlation between SSR length and polymorphism was only found for dinucleotide EST-SSRs located within the untranslated regions, but not for trinucleotide EST-SSRs. Transferability of EST-SSRs to Cucurbita species was assayed and 12.7% of the primer pairs amplified at least in one species, although only 5.4% were polymorphic. A set of 14 double haploid lines from the cross between the cultivar “Piel de Sapo” and the accession PI161375 were selected for the bin mapping approach in melon. One hundred and twenty-one SSR markers were newly mapped. The position of 46 SSR loci was also verified by genotyping the complete population. A final bin-map was constructed including 80 RFLPs, 212 SSRs, 3 SNPs and the Nsv locus, distributed in 122 bins with an average bin length of 10.2 cM and a maximum bin length of 33 cM. Map density was 4.2 cM/marker or 5.9 cM/SSR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ethylene production, shelf-life and evidence of RFLP polymorphisms linked to ethylene genes in melon (Cucumis melo L.)

Sixty three cultigens from eight market types of the melon (Cucumis melo L. subsp. melo) groups Cantaloupensis and Inodorus were evaluated for ethylene production rate, shelf-life (postharvest decay), and RFLP polymorphisms. The ethylene production rates of melon fruits at maturity and (after) postharvest decay were measured on individual genotypes. The ethylene production rates of individual genotypes ranged from undetectable to 103 nl/g per h. The mean ethylene production rates of the eight market types, ranked from highest to lowest, were Eastern U.S. type, Charentais, Western U.S. type, Long Shelf-Life cantaloupes (LSL), Galia, Ananas, Honeydew, and Casaba. Ethylene production and postharvest decay rating were positively significantly correlated (r ²=0.87, P=0.05). Orange-fleshed melon fruits produced significantly (P=0.05) more ethylene than did green- or white-fleshed types. Melon fruits with a netted rind had significantly (P=0.05 for orange-flesh fruits and 0.01 for green- or white-flesh fruits) higher ethylene production than did smooth-type fruits. Using probes made from cDNAs encoding ACC oxidase (MEL1) or ACC synthase (MEACS1) genes, RFLPs were detected melon cultigens of the eight marker types showing varying ethylene production rates and different flesh colors. Low ethylene production and green- and white-flesh color were associated (r ²=0.91; P=0.05) with the presence of a putative RFLP-MEL1 allele A ₀ (15-kb), whereas high ethylene production and orange-flesh color were associated with allele B ₀ (8.5-kb) in the homozygous condition, after probing MEL1 with EcoRV-digested genomic DNA. Also, after probing MEACS1 with NdeI-digested genomic DNA, RFLP polymorphism revealed five fragments denoted as A, B, C, D and E, with molecular sizes of 5.2-, 4.2-, 3.8-, 3.0- and 1.0-kb, respectively. A two-fragment pattern, AB, and a three-fragment pattern, ACE, the two predominant RFLP patterns, were also associated with low and high ethylene production, respectively. The ACE fragment pattern was also associated with orange-flesh melons. Scoring of both probes allowed for the unique classification of most melon market types consistent with ethylene production and the postharvest decay phenotypes. Therefore, these RFLPs might have utility in marker-assisted selection for the development of melons with enhanced postharvest keeping ability. Received: 26 March 1998 / Accepted: 12 January 2000     

A genetic map of melon (Cucumis melo L.) with RFLP, RAPD, isozyme, disease resistance and morphological markers

One hundred and ten markers were analysed for linkage in 218 F₂ plants derived from two divergent cultivars (Védrantais and Songwhan Charmi) of Cucumis melo (L.). Thirty-four RFLPs, 64 RAPDs, one isozyme, four disease resistance markers and one morphological marker were used to construct a genetic map spanning 14 linkage groups covering 1390 cM of the melon genome. RAPD and RFLP markers detected similar polymorphism levels. RFLPs were largely due to base substitutions rather than insertion/deletions. Twelve percent of markers showed distorted segregation. Phenotypic markers consisted of two resistance genes against Fusarium wilt (Fom-1 and Fom-2), one gene (nsv) controlling the resistance to melon necrotic spot virus, one gene (Vat) conferring resistance to Aphis gossypii, and a recessive gene for carpel numbers (3 vs 5 carpels: p).     

Selection of somaclonal variants with low-temperature germinability in melon (Cucumis melo L.)

Summary Plants were regenerated from adventitious buds and somatic embryos (R₀) of melon (Cucumis melo L.), the cultivar Andes. Somaclonal variants of melon with low temperature germinability were selected from the progenies (R₁) of R₀ plants. Among 5,618 R₁ seeds harvested from 23 R₀ plants that were regenerated from adventitious buds 4 seeds germinated after 5 days of culture at 15 °C (selection rate; 0.07%). However, among 374 R₂ seeds harvested from 2 R₁ plants no seed germinated after 7 days of culture at 14 °C. Among 9,181 R₁ seeds harvested from 50 R₀ plants regenerated from somatic embryos 110 seeds germinated after 5 days of culture at 15 °C (selection rate; 1.20%). Among 3,717 R₂ seeds harvested from 17 R₁ plants 113 seeds germinated after 7 days of culture at 14 °C (selection rate; 3.04%). R₃ seeds were collected from these R₂ plants following self-pollination. Forty-five of the 47 lines (R₃) originated from 10 R₀ plants showed higher germination rates than that of the original cultivar. Selected lines with low-temperature germinability showed greater fruit growth rate than the original cultivar during the middle stage when they were cultivated in a greenhouse under low-temperature conditions. Of fruits harvested from 31 lines, 15 lines showed greater fruit volume than the original cultivar.     

QTLs for tomato powdery mildew resistance (Oidium lycopersici) in Lycopersicon parviflorum G1.1601 co-localize with two qualitative powdery mildew resistance genes

Tomato (Lycopersicon esculentum) is susceptible to the powdery mildew Oidium lycopersici, but several wild relatives such as Lycopersicon parviflorum G1.1601 are completely resistant. An F2 population from a cross of Lycopersicon esculentum cv. Moneymaker x Lycopersicon parviflorum G1.1601 was used to map the O. lycopersici resistance by using amplified fragment length polymorphism markers. The resistance was controlled by three quantitative trait loci (QTLs). Ol-qtl1 is on chromosome 6 in the same region as the Ol-1 locus, which is involved in a hypersensitive resistance response to O. lycopersici. Ol-qtl2 and Ol-qtl3 are located on chromosome 12, separated by 25 cM, in the vicinity of the Lv locus conferring resistance to another powdery mildew species, Leveillula taurica. The three QTLs, jointly explaining 68% of the phenotypic variation, were confirmed by testing F3 progenies. A set of polymerase chain reaction-based cleaved amplified polymorphic sequence and sequence characterized amplified region markers was generated for efficient monitoring of the target QTL genomic regions in marker assisted selection. The possible relationship between genes underlying major and partial resistance for tomato powdery mildew is discussed.     

Genetic difference in somatic embryogenesis from seeds in melon (Cucumis melo L.)

Significant differences in somatic embryogenesis from melon seeds were observed among 18 cultivars; especially, cultivars Earl's Favorite and Barnett which produced a large number of somatic embryos. F₁ seeds were obtained by reciprocal crosses between cultivars. Some lines produced a large number of somatic embryos whereas others showed no or poor embryogenic response. Most of the F₁ seeds formed somatic embryos. The frequency of somatic embryogenesis decreased as compared to the parents with the highest potential. Transfer of the frequency of somatic embryogenesis from superior responding cultivars to inferior cultivars was proved. It was difficult to determine the mode of inheritance of somatic embryogenesis because there was a large variation in the range of somatic embryogenesis from F₂ seeds, and cytoplasmic effect was recognized in certain combinations.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Genetic mapping of a fusarium wilt resistance gene (Fom-2) in melon (Cucumis melo L.)

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis is one of the most devastating diseases in melon production worldwide. The most effective control measure available is the use of resistant varieties. Identifying molecular markers linked to resistance genes can serve as a valuable tool for the selection of resistant genotypes. Bulked segregant analysis was used to identify markers linked to the Fom-2 genes, which confers resistance to races 0 and 1 of the fungal pathogen. Pooled DNA from homozygous resistant or homozygous susceptible progeny of F₂ cross between MR-1 and AY was screened using 240 PstI/MseI and 200 EcoRI/MseI primer combinations to identify AFLP markers linked to Fom-2. Fifteen markers potentially linked to Fom-2 were identified, all with EcoRI/MseI primer pairs. These were mapped relative to Fom-2 in a backcross (BC) population of 60 progeny derived from MR-1 × AY with AY as recurrent parent. Two AFLP markers (ACT/CAT1 and AAC/CAT1) flanked the gene at 1.7 and 3.3 cM, respectively. Moreover, AFLP marker AGG/CCC and the previously identified RAPD marker 596-1 cosegregated with Fom-2. These two dominant markers were converted to co-dominant markers by designing specific PCR primers that produced product length polymorphisms between the parents. A survey of 45 melon genotypes from diverse geographic origins with the co-dominant markers demonstrated a high correlation between fragment size and the resistance phenotype. These markers may therefore be useful in marker-assisted breeding programs.