Construction of a reference linkage map for melon.

A map of melon (Cucumis melo L.) with 411 markers (234 RFLPs, 94 AFLPs, 47 RAPDs, 29 SSRs, five inter-SSRs, and two isozymes) and one morphological trait (carpel number) was constructed using the F2 progeny of a cross between the Korean accession P1161375 and the Spanish melon type 'Pinyonet Piel de Sapo'. RFLPs were obtained using 212 probes from different genomic and cDNA melon libraries, including 16 Arabidopsis ESTs, 13 Cucumis known genes, and three resistant gene homologues. Most loci (391) mapped to 12 major linkage groups, spanning a total genetic distance of 1197 cM, with an average map interval of 3 cM/marker. The remaining 21 loci (six RAPDs and 15 AFLPs) were not linked. A majority (66%) of the markers were codominant (RFLPs, SSRs, and isozymes), making them easily transferable to other melon crosses. Such markers can be used as a reference, to merge other melon and cucumber maps already constructed. Indeed, some of them (23 SSRs, 14 RFLPs, one isozyme, and one morphological trait) could act as anchor points with other published cucurbit maps.     

A set of EST-SNPs for map saturation and cultivar identification in melon

Background

There are few genomic tools available in melon (Cucumis melo L.), a member of the Cucurbitaceae, despite its importance as a crop. Among these tools, genetic maps have been constructed mainly using marker types such as simple sequence repeats (SSR), restriction fragment length polymorphisms (RFLP) and amplified fragment length polymorphisms (AFLP) in different mapping populations. There is a growing need for saturating the genetic map with single nucleotide polymorphisms (SNP), more amenable for high throughput analysis, especially if these markers are located in gene coding regions, to provide functional markers. Expressed sequence tags (ESTs) from melon are available in public databases, and resequencing ESTs or validating SNPs detected in silico are excellent ways to discover SNPs.

Results

EST-based SNPs were discovered after resequencing ESTs between the parental lines of the PI 161375 (SC) × 'Piel de sapo' (PS) genetic map or using in silico SNP information from EST databases. In total 200 EST-based SNPs were mapped in the melon genetic map using a bin-mapping strategy, increasing the map density to 2.35 cM/marker. A subset of 45 SNPs was used to study variation in a panel of 48 melon accessions covering a wide range of the genetic diversity of the species. SNP analysis correctly reflected the genetic relationships compared with other marker systems, being able to distinguish all the accessions and cultivars.

Conclusion

This is the first example of a genetic map in a cucurbit species that includes a major set of SNP markers discovered using ESTs. The PI 161375 × 'Piel de sapo' melon genetic map has around 700 markers, of which more than 500 are gene-based markers (SNP, RFLP and SSR). This genetic map will be a central tool for the construction of the melon physical map, the step prior to sequencing the complete genome. Using the set of SNP markers, it was possible to define the genetic relationships within a collection of forty-eight melon accessions as efficiently as with SSR markers, and these markers may also be useful for cultivar identification in Occidental melon varieties.     

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     

Development and characterization of microsatellite markers in Cucumis

This study provides a set of useful SSR markers and describes their development, characterization and application for diversity studies.Sixty one Cucumis SSR markers were developed, most of them (46) from melon (Cucumis melo L.) genomic libraries. Forty of the markers (30 melon and 10 cucumber SSRs) were evaluated for length polymorphism in a sample of 13 melon genotypes and 11 cucumber (Cucumis sativus L.) genotypes. PCR-amplification revealed up to six size alleles among the melon genotypes and up to five alleles among the cucumber genotypes, with mean gene-diversity values of 0.52 and 0.28 for melon and cucumber, respectively. These differences are in accordance with the known narrower genetic background of the cucumber. SSR data were applied to phylogenetic analysis among the melon and cucumber genotypes. A clear distinction between the ’exotic’ groups and the sweet cultivated groups was demonstrated in melon. In cucumber, separation between the two sub-species, C.sativus var. sativus and C.sativus var. hardwickii,was obtained. Conservation of SSR loci between melon and cucumber was proven by sequence comparisons. Received: 17 April 2000 / Accepted: 16 May 2000     

A reference map of Cucumis melo based on two recombinant inbred line populations

A composite genetic melon map was generated based on two recombinant inbred line (RI) populations. By analyzing the segregation of 346 AFLPs, 113 IMAs and phenotypic characters on a RI population of 163 individuals derived from the cross Védrantais x PI 161375, a first map was constructed. About 20% of the molecular markers were skewed, and the residual heterozygosity was estimated at 4.43% which was not significantly different from the theoretical value of 4.2%. The genome distribution of molecular markers among the 12 linkage groups was not different from a random distribution with the exception of linkage group XII which was found significantly less populated. The genome distributions of IMAs and AFLPs were complementary. AFLPs were found mainly in the middle of each linkage group and sometimes clustered, whereas IMAs were found mainly at the end. A total of 318 molecular markers, mainly AFLP and IMA markers, were mapped on 63 RIs of the second population, Védrantais x PI 414723. Comparison of the maps enables one to conclude that AFLPs and IMAs of like molecular size, amplified with the same primer combination, correspond to the same genetic locus. Both maps were joined through 116 common markers comprising 106 comigrating AFLPs/IMAs, plus five SSRs and five phenotypic markers. The integrated melon map contained 668 loci issuing from the segregation of 1,093 molecular markers in the two RI populations. The composite map spanned 1,654 cM on 12 linkage groups which is the haploid number of chromosomes in melon. Thirty two known-function probes, i.e. known-function genes (9) and morphological traits (23), were included in this map. In addition, the composite map was anchored to previously published maps through SSRs, RFLPs and phenotypic characters.     

Length polymorphism and homologies of microsatellites in several Cucurbitaceae species

The objectives of this research were to assess (1) the degree of Simple Sequence Repeats (SSR) DNA length polymorphism in melon (Cucumis melo L.) and other species within the Cucurbitaceae family and (2) the possibility of utilizing SSRs flanking primers from single species to other genera or species of Cucurbitaceae. Five melon (CT/GA) _n SSRs were isolated from a genomic library. Two cucumber (Cucumis sativus L.) SSRs were detected through a search of DNA sequence databases, one contained a (CT)₈ repeat, the other a (AT)₁₃ repeat. The seven SSRs were used to test a diverse sample of Cucurbitaceae, including 8 melon, 11 cucumber, 5 squash, 1 pumpkin, and 3 watermelon genotypes. Five of the seven SSRs detected length polymorphism among the 8 melon genotypes. PCR amplification revealed between three and five length variants (alleles) for each SSR locus, with gene diversity values ranging from 0.53 to 0.75. Codominant segregation of the alleles among F₂ progeny was demonstrated for each of the five SSR loci. Four of the seven SSRs detected polymorphism among the 11 cucumber genotypes, with gene diversity values ranging between 0.18 and 0.64. Primers specific to SSRs of C. melo and C. sativus also amplified DNA extracted from genotypes belonging to other genera of the Cucurbitaceae family.     

Development of genome-wide SSR markers in melon with their cross-species transferability analysis and utilization in genetic diversity study

Simple sequence repeats (SSRs) are one of the most informative and widely used molecular markers in plant research. The melon draft genome has provided a powerful tool for SSR marker development in this species in which there are still not enough SSR markers. We therefore developed genome-wide SSR markers from melon, which were used for genetic diversity analysis in melon accessions and comparative mapping with cucumber and watermelon. A total of 44,265 microsatellites from the melon genome were characterized, of which 28,570 SSR markers were developed. In silico PCR analysis with these SSR markers identified 4002 and 1085 with one amplicon in cucumber and watermelon genome, respectively. With these cross-species transferable melon SSR markers, the chromosome synteny between melon and cucumber as well as watermelon was established, which revealed complicated mosaic patterns of syntenic blocks among them. We experimentally validated 384 SSR markers, from which 42 highly informative SSR ones were selected for genetic diversity and population structure analysis among 118 melon accessions. The large number of melon SSR markers developed in this study provides a valuable resource for genetic linkage map construction, molecular mapping, and marker-assisted selection (MAS) in melon. Furthermore, the cross-species transferable SSR markers could also be useful in various molecular marker-related studies in other closely related species in Cucurbitaceae family in which draft genomes are not yet available.     

Linkage map of Cucumis melo including phenotypic traits and sequence-characterized genes.

A new linkage map of Cucumis melo, derived from the F2 progeny of a cross between PI 414723 and C. melo 'TopMark' is presented. The map spans a total of 1421 cM and includes 179 points consisting of random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSRs), simple sequence repeats (SSRs), and restriction fragment length polymorphism (RFLP) markers. The map also includes an aphid resistance trait (Vat) and the sex type gene, andromonoecious (a), the two of which are important in resistance breeding and the control of hybrid seed production, as well as a seed-color gene, Wt-2. Most RFLPs represent sequence-characterized cDNA probes from C. melo and Cucumis sativus. These include resistance gene homologues and genes involved in various aspects of plant development and metabolism. A sub-set of our SSR and RFLP markers were also mapped, as part of this study, on additional mapping populations that were published for this species. This provides important reference points ("anchors"), enabling us to identify several linkage groups with respect to other melon maps.     

Single-nucleotide polymorphisms detected in expressed sequence tags of melon (Cucumis melo L.).

A search was performed for single-nucleotide polymorphisms (SNP) and short insertions-deletions (indels) in 34 melon (Cucumis melo L.) expressed sequence tag (EST) fragments between two distantly related melon genotypes, a group Inodorus 'Piel de sapo' market class breeding line T111 and the Korean accession PI 161375. In total, we studied 15 kb of melon sequence. The average frequency of SNPs between the two genotypes was one every 441 bp. One indel was also found every 1666 bp. Seventy-five percent of the polymorphisms were located in introns and the 3'untranslated regions. On average, there were 1.26 SNPs plus indels per amplicon. We explored three different SNP detection systems to position five of the SNPs in a melon genetic map. Three of the SNPs were mapped using cleaved amplified polymorphic sequence (CAPS) markers, one SNP was mapped using the single primer extension reaction with fluorescent-labelled dideoxynucleotides, and one indel was mapped using polyacrilamide gel electrophoresis separation. The discovery of SNPs based on ESTs and a suitable system for SNP detection has broad potential utility in melon genome mapping.     

Molecular and genetic characterization of a non-climacteric phenotype in melon reveals two loci conferring altered ethylene response in fruit

Fruit ripening and abscission are associated with an ethylene burst in several melon (Cucumis melo) genotypes. In cantaloupe as in other climacteric fruit, exogenous ethylene can prematurely induce abscission, ethylene production, and ripening. Melon genotypes without fruit abscission or without ethylene burst also exist and are, therefore, non-climacteric. In the nonabscising melon fruit PI 161375, exogenous ethylene failed to stimulate abscission, loss of firmness, ethylene production, and expression of all target genes tested. However, the PI 161375 etiolated seedlings displayed the usual ethylene-induced triple response. Genetic analysis on a population of recombinant cantaloupe Charentais x PI 161375 inbred lines in segregation for fruit abscission and ethylene production indicated that both characters are controlled by two independent loci, abscission layer (Al)-3 and Al-4. The non-climacteric phenotype in fruit tissues is attributable to ethylene insensitivity conferred by the recessive allelic forms from PI 161375. Five candidate genes (two ACO, two ACS, and ERS) that were localized on the melon genetic map did not exhibit colocalization with Al-3 or Al-4.     

An Integrated Genetic and Cytogenetic Map of the Cucumber Genome

The Cucurbitaceae includes important crops such as cucumber, melon, watermelon, squash and pumpkin. However, few genetic and genomic resources are available for plant improvement. Some cucurbit species such as cucumber have a narrow genetic base, which impedes construction of saturated molecular linkage maps. We report herein the development of highly polymorphic simple sequence repeat (SSR) markers originated from whole genome shotgun sequencing and the subsequent construction of a high-density genetic linkage map. This map includes 995 SSRs in seven linkage groups which spans in total 573 cM, and defines ∼680 recombination breakpoints with an average of 0.58 cM between two markers. These linkage groups were then assigned to seven corresponding chromosomes using fluorescent in situ hybridization (FISH). FISH assays also revealed a chromosomal inversion between Cucumis subspecies [C. sativus var. sativus L. and var. hardwickii (R.) Alef], which resulted in marker clustering on the genetic map. A quarter of the mapped markers showed relatively high polymorphism levels among 11 inbred lines of cucumber. Among the 995 markers, 49%, 26% and 22% were conserved in melon, watermelon and pumpkin, respectively. This map will facilitate whole genome sequencing, positional cloning, and molecular breeding in cucumber, and enable the integration of knowledge of gene and trait in cucurbits.     

DNA Fingerprinting of Chinese Melon Provides Evidentiary Support of Seed Quality Appraisal

Melon, Cucumis melo L. is an important vegetable crop worldwide. At present, there are phenomena of homonyms and synonyms present in the melon seed markets of China, which could cause variety authenticity issues influencing the process of melon breeding, production, marketing and other aspects. Molecular markers, especially microsatellites or simple sequence repeats (SSRs) are playing increasingly important roles for cultivar identification. The aim of this study was to construct a DNA fingerprinting database of major melon cultivars, which could provide a possibility for the establishment of a technical standard system for purity and authenticity identification of melon seeds. In this study, to develop the core set SSR markers, 470 polymorphic SSRs were selected as the candidate markers from 1219 SSRs using 20 representative melon varieties (lines). Eighteen SSR markers, evenly distributed across the genome and with the highest contents of polymorphism information (PIC) were identified as the core marker set for melon DNA fingerprinting analysis. Fingerprint codes for 471 melon varieties (lines) were established. There were 51 materials which were classified into17 groups based on sharing the same fingerprint code, while field traits survey results showed that these plants in the same group were synonyms because of the same or similar field characters. Furthermore, DNA fingerprinting quick response (QR) codes of 471 melon varieties (lines) were constructed. Due to its fast readability and large storage capacity, QR coding melon DNA fingerprinting is in favor of read convenience and commercial applications.     

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.     

Construction of a high density integrated genetic map for cucumber (Cucumis sativus L.)

The high-density consensus map was constructed based on the GY14 × PI 183967 map from an inter-subspecific cross and the extended S94 × S06 map from an intra-subspecific cross. The consensus map was composed of 1,369 loci, including 1,152 SSR loci, 192 SRAP loci, 21 SCAR loci and one STS locus as well as three gene loci of fruit external quality traits in seven chromosomes, and spanned 700.5 cM, of which 682.7 cM (97.5%) were covered by SSR markers. The average genetic distance and physical interval between loci were 0.51 cM and ~268 kbp, respectively. Additionally, the physical position of the sequence-associated markers aligned along the assembled cucumber genome sequence established a relationship between genetic maps and cucumber genome sequence and to a great extent validated the order of markers in individual maps and consensus map. This consensus map with a high marker density and well-ordered markers is a saturated and reliable linkage map for genetic analysis of cucumber or the Cucurbitaceae family of plants.     

Impact of melon accessions resistant to aphids on the demographic potential of silverleaf whitefly

Bemisia tabaci (Gennadius) and Aphis gossypii Glover are devastating melon, Cucumis melo L., pests. The geographic areas where they occur overlap, and the same chemicals are used to control both of them. Therefore, to reduce pesticide use, it would be necessary to breed melon lines that simultaneously express a resistance to both insects. Female survival; the time when reproduction starts, peaks, and ends; the number of female offspring at the reproductive peak; and total reproduction (S) were determined under semicontrolled conditions for B. tabaci kept in clip-cages on a susceptible melon genotype Vedrantais, and 12 potential resistant accessions, particularly genotypes expressing the Vat gene controlling resistance to A. gossypii. By using the Lewontin triangular reproductive function and Bootstrapping, the intrinsic rate of increase (r) and its variance were calculated. Statistical analysis showed that the parameter S was as relevant as r for discriminating between the melon accessions. Three genotypes were potential genitors of resistance to the whitefly: PI 161375, PI 414723, and PI 532841. Those possessing the Vat gene were either resistant (PI 161375 and PI 414723) or susceptible (Margot, IsoVat R, and AR 5). This demonstrated the ineffectiveness of Vat against B. tabaci. In this article, we propose a strategy to breed lines that express resistance to aphids and whiteflies on the short-term.     

Identification of QTLs for resistance to powdery mildew and SSR markers diagnostic for powdery mildew resistance genes in melon (Cucumis melo L.)

Powdery mildew caused by Podosphaera xanthii is an important foliar disease in melon. To find molecular markers for marker-assisted selection, we constructed a genetic linkage map of melon based on a population of 93 recombinant inbred lines derived from crosses between highly resistant AR 5 and susceptible ‘Earl’s Favourite (Harukei 3)’. The map spans 877 cM and consists of 167 markers, comprising 157 simple sequence repeats (SSRs), 7 sequence characterized amplified region/cleavage amplified polymorphic sequence markers and 3 phenotypic markers segregating into 20 linkage groups. Among them, 37 SSRs and 6 other markers were common to previous maps. Quantitative trait locus (QTL) analysis identified two loci for resistance to powdery mildew. The effects of these QTLs varied depending on strain and plant stage. The percentage of phenotypic variance explained for resistance to the pxA strain was similar between QTLs (R ² = 22–28%). For resistance to pxB strain, the QTL on linkage group (LG) XII was responsible for much more of the variance (41–46%) than that on LG IIA (12–13%). The QTL on LG IIA was located between two SSR markers. Using an independent population, we demonstrated the effectiveness of these markers. This is the first report of universal and effective markers linked to a gene for powdery mildew resistance in melon.     

Genetic linkage maps of rose constructed with new microsatellite markers and locating QTL controlling flowering traits

New microsatellites markers [simple sequence repeat (SSR)] have been isolated from rose and integrated into an existing amplified fragment-length polymorphism genetic map. This new map was used to identify quantitative trait locus (QTL) controlling date of flowering and number of petals. From a rose bud expressed sequence tag (EST) database of 2,556 unigenes and a rose genomic library, 44 EST-SSRs and 20 genomic-SSR markers were developed, respectively. These new rose SSRs were used to expand genetic maps of the rose interspecific F₁ progeny. In addition, SSRs from other Rosaceae genera were also tested in the mapping progeny. Genetic maps for the two parents of the progeny were constructed using pseudo-testcross mapping strategy. The maps consist of seven linkage groups of 105 markers covering 432 cM for the maternal map and 136 markers covering 438 cM for the paternal map. Homologous relationships among linkage groups between the maternal and paternal maps were established using SSR markers. Loci controlling flowering traits were localised on genetic maps as a major gene and QTL for the number of petals and a QTL for the blooming date. New SSR markers developed in this study will provide tools for the establishment of a consensus linkage map for roses that combine traits and markers in various rose genetic maps.     

An SSR genetic map of Sorghum bicolor (L.) Moench and its comparison to a published genetic map.

Sorghum bicolor (L.) Moench is an important grain and forage crop grown worldwide. We developed a simple sequence repeat (SSR) linkage map for sorghum using 352 publicly available SSR primer pairs and a population of 277 F2 individuals derived from a cross between the Westland A line and PI 550610. A total of 132 SSR loci appeared polymorphic in the mapping population, and 118 SSRs were mapped to 16 linkage groups. These mapped SSR loci were distributed throughout 10 chromosomes of sorghum, and spanned a distance of 997.5 cM. More important, 38 new SSR loci were added to the sorghum genetic map in this study. The mapping result also showed that chromosomes SBI-01, SBI-02, SBI-05, and SBI-06 each had 1 linkage group; the other 6 chromosomes were composed of 2 linkage groups each. Except for 5 closely linked marker flips and 1 locus (Sb6_34), the marker order of this map was collinear to a published sorghum map, and the genetic distances of common marker intervals were similar, with a difference ratio 相似文献   

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.     

梨遗传连锁图谱的构建及其与苹果图谱的比较

以‘丰水’为母本、‘砀山酥梨’为父本杂交所得的F1代104株单体为作图群体,利用SSR分子标记进行遗传连锁分析,应用Jionmap 3.0作图软件,构建了一张包含104个SSR分子标记,分属于18个连锁群的梨遗传连锁图谱,覆盖梨基因组总长831.8cM,平均图距为8.0cM。根据定位到该图谱上的SSR标记与苹果‘Fiesta’图谱进行比较,25个共有的SSR标记将该图谱和苹果图谱各连锁群连接起来,这些标记不仅呈现良好的共线性而且它们之间的相对遗传距离也很相近。研究认为,SSR标记作为锚定引物,可以与不同物种的遗传图谱相比较整合,为不同物种之间遗传信息的转移提供参考依据;同时该研究为梨树相关性状的基因定位、分离以及克隆奠定了基础。