Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon

Three different types of molecular markers, RAPD, AFLP and RFLP were used to measure genetic diversity among six genotypes of Cucumis melo L. Each line represented a different melon genotype: Piel de Sapo, Ogen, PI161375, PI414723, Agrestis and C105. A number of polymorphic RAPD, AFLP and RFLP bands were scored on all materials and the genetic similarity measured. Clustering analysis performed with the three types of markers separated the genotypes into two main groups: (1) the sweet type, cultivated melons and (2) the exotic type, not cultivated melons. While the data obtained suggest that all three types of markers are equally informative, AFLPs showed the highest efficiency in detecting polymorphism. Received: 30 December 1999 / Accepted: 24 January 2000     

新疆甜瓜地方品种资源核心种质构建

新疆甜瓜地方种质资源具有丰富的遗传多样性,是新疆哈密瓜遗传改良的重要基因库。以121份新疆甜瓜地方品种为研究对象,结合按来源分组和系统聚类选择的方法,通过多重比较29个表型性状数据确定适宜的取样比例,筛选出25份地方品种为初选核心种质。在初选核心种质取样量上,人工定向补充5份优异种质和极值材料确定了核心种质,约占地方品种总数量的25%。对表型保留比例、遗传多样性指数、变异系数、表型频率方差、极差符合率、均值符合率、标准差符合率等检验参数进行了检验和评价。结果表明:调整后的核心种质除标准差符合率降低外,其余参数均优于或等于初选核心种质,更能代表原始样品;所构建的核心种质很好地保留了所有地方品种资源的遗传多样性和变异幅度。     

Genetic diversity and interrelationship among Indian and exotic melons based on fruit morphology,quality components and microsatellite markers

Physiology and Molecular Biology of Plants - Seventy melon (Cucumis melo L.) accessions comprising of landraces, inbred lines, cultivars, wild relatives and exotic populations were characterized...     

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.     

Population structure,morphological and genetic diversity within and among melon (Cucumis melo L.) landraces in Iran

ISSR markers were applied to evaluate the genetic diversity and differentiation of 270 individuals of 27 Iranian C. melo landraces of various varietal groups include vars. inodorous, cantalupensis, reticulatus, ameri, dudaim. Genetic diversity among the studied genotypes obtained by GeneAlex analysis (H?=?0.08, I?=?0.12, Na?=?0.77, PPL?=?22.6%). Cluster analysis divided Iranian melon landraces into two main cluster. Non-sweet genotype (dudaim group) was well separated from sweet genotypes (inodorous, ameri, reticulatus, cantalupensis). The most similar genotypes were BANI and TONI (0.95) and the most dissimilar ones were GER and TS (0.58). AMOVA result showed that the percentage of genetic variation among and within Iranian melon is 69% and 31%, respectively. All landraces evaluated based on 10 morphological traits which revealed the diversity of melon varietal groups. Bayesian analysis assigned ten landraces to Pop 1, eight landraces to Pop 2 and nine melon landraces to Pop 3. Bayesian and UPGMA cluster analyses demonstrated the almost related results. Our results indicated that ISSR markers technique alongside polyacrylamide gel analysis could be helpful to discriminate varieties of melon.     

Dissection of the oligogenic resistance to Cucumber mosaic virus in the melon accession PI 161375

Resistance to Cucumber mosaic virus (CMV) in the exotic melon accession PI 161375, cultivar "Sonwang Charmi" (SC) had previously been described as oligogenic, recessive and quantitative, with a major QTL residing in linkage group XII (LGXII). We have used a collection of near isogenic lines (NILs) with introgressions of SC into the genome of the susceptible accession Piel de Sapo (PS) to further characterise this resistance. Infection of NILs carrying introgressions on LGXII showed that only NIL SC12-1 was resistant to CMV strains P9 and P104.82, but not to strains M6 and TL. Further mapping of this region showed that the resistance, named cmv1 maps in an area of 2.2 cM, between markers CMN61_44 and CMN21_55. Moreover, cmv1 confers total resistance to strains P9 and P104.82, indicating that in these cases it is not quantitative and that cmv1 is sufficient to confer full resistance to these CMV strains. Candidate gene mapping of ten translation initiation factors in the melon genome failed to find any of them in the interval between markers CMN61_44 and CMN21_55. All these results suggest that the resistance to CMV present in SC is oligogenic, where different loci confer resistance to different CMV strains, but not necessarily quantitative, since at least one of these genes (cmv1) confers total resistance, similar to that of the parental SC, and does not need the contribution of other loci.     

Characterization of an abscisic acid responsive gene homologue from Cucumis melo

A cDNA and genomic DNA encoding an abscisic acid responsive gene (ASR) homologue (Asr1) was isolated from an inodorus melon, Cucumis melo var. kuwata, cDNA and genomic library. The Asr1 gene showed the strongest fruit-specific expression and differential expression profiles during fruit development, which were expressed from a low copy gene. The promoter region of the Asr1 gene contained several putative functional cis-elements, which may be involved in the response to plant hormones and environmental stresses. These results suggest that Asr1 may play an important role in the regulation of melon fruit ripening.     

Genetic diversity and population structure of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) landraces from the East African highlands

The East African highlands are a region of important common bean production and high varietal diversity for the crop. The objective of this study was to uncover the diversity and population structure of 192 landraces from Ethiopia and Kenya together with four genepool control genotypes using morphological phenotyping and microsatellite marker genotyping. The germplasm represented different common bean production ecologies and seed types common in these countries. The landraces showed considerable diversity that corresponded well to the two recognized genepools (Andean and Mesoamerican) with little introgression between these groups. Mesoamerican genotypes were predominant in Ethiopia while Andean genotypes were predominant in Kenya. Within each country, landraces from different collection sites were clustered together indicating potential gene flow between regions within Kenya or within Ethiopia. Across countries, landraces from the same country of origin tended to cluster together indicating distinct germplasm at the national level and limited gene flow between the two countries highlighting divided social networks within the regions and a weak trans-national bean seed exchange especially for landrace varieties. One exception to this may be the case of small red-seeded beans where informal cross-border grain trade occurs. We also observed that genetic divergence was slightly higher for the Ethiopian landraces compared to Kenyan landraces and that Mesoamerican genotypes were more diverse than the Andean genotypes. Common beans in eastern Africa are often cultivated in marginal, risk-prone farming systems and the observed landrace diversity should provide valuable alleles for adaptation to stressful environments in future breeding programs in the region.     

Genetic diversity, inter-gene pool introgression and nutritional quality of common beans (Phaseolus vulgaris L.) from Central Africa

The Great Lakes region of Central Africa is a major producer of common beans in Africa. The region is known for high population density and small average farm size. The common bean represents the most important legume crop of the region, grown on over a third of the cultivated land area, and the per capita consumption is among the highest in the world for the food crop. The objective of this study was to evaluate the genetic diversity in a collection of 365 genotypes from the Great Lakes region of Central Africa, including a large group of landraces from Rwanda as well as varieties from primary centers of diversity and from neighboring countries of Central Africa, such as the Democratic Republic of Congo and Uganda, using 30 fluorescently labeled microsatellite markers and automated allele detection. In addition, the landraces were evaluated for their seed iron and zinc concentration to determine if genetic diversity influenced nutritional quality. Principal coordinate and neighbor-joining analyses allowed the separation of the landraces into 132 Andean and 195 Mesoamerican (or Middle American) genotypes with 32 landraces and 6 varieties intermediate between the gene pools and representing inter-gene pool introgression in terms of seed characteristics and alleles. Genetic diversity and the number of alleles were high for the collection, reflecting the preference for a wide range of seed types in the region and no strong commercial class preference, although red, red mottled and brown seeded beans were common. Observed heterozygosity was also high and may be explained by the common practice of maintaining seed and plant mixtures, a coping strategy practiced by Central African farmers to reduce the effects of abiotic and biotic stresses. Finally, nutritional quality differed between the gene pools with respect to seed iron and zinc concentration, while genotypes from the intermediate group were notably high in both minerals. In conclusion, this study has shown that Central African varieties of common bean are a source of wide genetic diversity with variable nutritional quality that can be used in crop improvement programs for the region.     

Introgression from modern hybrid varieties into landrace populations of maize (Zea mays ssp. mays L.) in central Italy

Landraces are domesticated local plant varieties that did not experience a deliberate and intensive selection during a formal breeding programme. In Europe, maize landraces are still cultivated, particularly in marginal areas where traditional farming is often practiced. Here, we have studied the evolution of flint maize landraces from central Italy over 50 years of on-farm cultivation, when dent hybrid varieties were introduced and their use was widespread. We have compared an 'old' collection, obtained during the 1950s, before the introduction of hybrids, and a recent collection of maize landraces. For comparison, a sample of maize landraces from north Italy, and of improved germplasm, including hybrids and inbred lines were also used. A total of 296 genotypes were analysed using 21 microsatellites. Our results show that the maize landraces collected in the last 5–10 years have evolved directly from the flint landrace gene pool cultivated in central Italy before the introduction of modern hybrids. The population structure, diversity and linkage disequilibrium analyses indicate a significant amount of introgression from hybrid varieties into the recent landrace populations. No evidence of genetic erosion of the maize landraces was seen, suggesting that in situ conservation of landraces is an efficient strategy for preserving genetic diversity. Finally, the level of introgression detected was very variable among recent landraces, with most of them showing a low level of introgression; this suggests that coexistence between different types of agriculture is possible, with the adoption of correct practices that are aimed at avoiding introgression from undesired genetic sources.     

浙江省薄皮甜瓜地方品种的表型遗传多样性

利用表型性状探讨了浙江省沿海地区27份薄皮甜瓜地方品种资源的遗传多样性。结果表明48个质量性状遗传多样性指数在0.17–1.98之间;33个数量性状的变异系数在4.56%–83.50%,表明其丰富的遗传多样性。形态学聚类分析表明,在相似系数0.30处可以将27个资源分成两大类,这与按生育期长短分类结果完全一致;亚类的划分与果实形状、种子形状、叶片颜色、果皮颜色、覆纹颜色等质量性状具有一定的相关性,但其划分依据相对独立。本研究结果进一步丰富了甜瓜的评价体系,并为今后优异基因资源的挖掘与利用提供重要依据。     

采用SSR和RAPD标记研究黄瓜属（葫芦科）的系统发育关系

野黄瓜Cucumis hystrix（2n=24）是在亚洲发现的第一个染色体基数为12的黄瓜属物种。这一发现对现行的以染色体基数和地理分布为基础的黄瓜属分类系统提出了质疑。采用SSR和RAPD两种分子标记对黄瓜属22份不同类型材料的亲缘关系进行了研究。结果表明,野黄瓜C.hystrix与黄瓜C.sativus var.sativus（2n=14）间的遗传距离（SSR:0.59,RAPD:0.57）小于其与甜瓜C.melo var.melo（2n=24）间的距离（SSR:0.87,RAPD:0.70）。SS     

Interaction between QTLs induces an advance in ethylene biosynthesis during melon fruit ripening

The coexistence of both climacteric and non-climacteric genotypes and the availability of a set of genetic and genomic resources make melon a suitable model for genetic studies of fruit ripening. We have previously described a QTL, ETHQB3.5, which induces climacteric fruit ripening in the near-isogenic line (NIL) SC3-5 that harbors an introgression on linkage group (LG) III from the non-climacteric melon accession PI 161375 in the, also non-climacteric cultivar, “Piel de Sapo” genetic background. In the current study, a new major QTL, ETHQV6.3, on LG VI was detected on an additional introgression in the same NIL. These QTLs are capable, individually, of inducing climacteric ripening in the non-climacteric background, the effects of ETHQV6.3 being greater than that of ETHQB3.5. The QTLs interact epistatically, advancing the timing of ethylene biosynthesis during ripening and, therefore, the climacteric responses. ETHQV6.3 was fine-mapped to a 4.5 Mb physical region of the melon genome, probably in the centromeric region of LG VI. The results presented will be of value in the molecular identification of the gene underlying ETHQV6.3     

Analysis of a diverse global Pisum sp. collection and comparison to a Chinese local P. sativum collection with microsatellite markers

Twenty-one informative microsatellite loci were used to assess and compare the genetic diversity among Pisum genotypes sourced from within and outside China. The Chinese germplasm comprised 1243 P. sativum genotypes from 28 provinces and this was compared to 774 P. sativum genotypes that represented a globally diverse germplasm collection, as well as 103 genotypes from related Pisum species. The Chinese P. sativum germplasm was found to contain genotypes genetically distinct from the global gene pool sourced outside China. The Chinese spring type genotypes were separate from the global gene pool and from the other main Chinese gene pool of winter types. The distinct Chinese spring gene pool comprised genotypes from Inner Mongolia and Sha'anxi provinces, with those from Sha'anxi showing the greatest diversity. The other main gene pool within China included both spring types from other northern provinces and winter types from central and southern China, plus some accessions from Inner Mongolia and Sha'anxi. A core collection of Chinese landraces chosen to represent molecular diversity was compared both to the wider Chinese collection and to a geographically diverse core collection of Chinese landraces. The average gene diversity and allelic richness per locus of both the micro-satellite based core and the wider collection were similar, and greater than the geographically diverse core. The genetic diversity of P. sativum within China appears to be quite different to that detected in the global gene pool, including the presence of several rare alleles, and may be a useful source of allelic variation for both major gene and quantitative traits.     

Melon fruits: genetic diversity, physiology, and biotechnology features

Among Cucurbitaceae, Cucumis melo is one of the most important cultivated cucurbits. They are grown primarily for their fruit, which generally have a sweet aromatic flavor, with great diversity and size (50 g to 15 kg), flesh color (orange, green, white, and pink), rind color (green, yellow, white, orange, red, and gray), form (round, flat, and elongated), and dimension (4 to 200 cm). C. melo can be broken down into seven distinct types based on the previously discussed variations in the species. The melon fruits can be either climacteric or nonclimacteric, and as such, fruit can adhere to the stem or have an abscission layer where they will fall from the plant naturally at maturity. Traditional plant breeding of melons has been done for 100 years wherein plants were primarily developed as open-pollinated cultivars. More recently, in the past 30 years, melon improvement has been done by more traditional hybridization techniques. An improvement in germplasm is relatively slow and is limited by a restricted gene pool. Strong sexual incompatibility at the interspecific and intergeneric levels has restricted rapid development of new cultivars with high levels of disease resistance, insect resistance, flavor, and sweetness. In order to increase the rate and diversity of new traits in melon it would be advantageous to introduce new genes needed to enhance both melon productivity and melon fruit quality. This requires plant tissue and plant transformation techniques to introduce new or foreign genes into C. melo germplasm. In order to achieve a successful commercial application from biotechnology, a competent plant regeneration system of in vitro cultures for melon is required. More than 40 in vitro melon regeneration programs have been reported; however, regeneration of the various melon types has been highly variable and in some cases impossible. The reasons for this are still unknown, but this plays a heavy negative role on trying to use plant transformation technology to improve melon germplasm. In vitro manipulation of melon is difficult; genotypic responses to the culture method (i.e., organogenesis, somatic embryogenesis, etc.) as well as conditions for environmental and hormonal requirements for plant growth and regeneration continue to be poorly understood for developing simple in vitro procedures to culture and transform all C. melo genotypes. In many cases, this has to be done on an individual line basis. The present paper describes the various research findings related to successful approaches to plant regeneration and transgenic transformation of C. melo. It also describes potential improvement of melon to improve fruit quality characteristics and postharvest handling. Despite more than 140 transgenic melon field trials in the United States in 1996, there are still no commercial transgenic melon cultivars on the market. This may be a combination of technical or performance factors, intellectual property rights concerns, and, most likely, a lack of public acceptance. Regardless, the future for improvement of melon germplasm is bright when considering the knowledge base for both techniques and gene pools potentially useable for melon improvement.     

The complex resistance to cucumber mosaic cucumovirus (CMV) in the melon accession PI161375 is governed by one gene and at least two quantitative trait loci

The complex resistance to cucumber mosaic virus (CMV) present in the exotic melon accession Sonwang Charmi PI161375 (SC) has been studied using two populations, a near-isogenic line (NIL) collection and a doubled haploid line (DHL) collection, both generated from a cross between SC and the cultivar Piel de Sapo as resistant and susceptible parents, respectively. The NIL collection had previously allowed us to describe a single recessive gene, cmv1, which conferred full resistance to CMV strains P9 and P104.82. Screening of the whole DHL population followed by quantitative trait locus (QTL) analysis revealed that resistance to the strains M6 and TL, both non-responsive to cmv1, was quantitative and governed by at least three QTLs. One of them, cmvqw12.1, co-located with cmv1 in linkage group (LG) XII. The QTL analysis mapped another two QTLs in LGIII (cmvqw3.1) and LGX (cmvqw10.1) and showed interaction between cmvqw12.1 and cmvqw3.1. Progeny of crosses between resistant DHLs carrying the three main QTLs showed complete resistance to the strain M6, validating the accuracy of the QTL analysis. However, in our screening, there were resistant DHLs carrying only two QTLs, suggesting that there are other regions involved in resistance to M6 and required when one of the main QTLs is missing. Therefore, resistance to CMV in melon SC is qualitative for some strains and quantitative for the rest. For this late resistance, cmv1 is necessary and explains most of the phenotypic variance, but it is not sufficient, and needs the interaction with other loci.     

摘心时期和留蔓数对甜瓜苗期叶面积扩展和功能叶寿命的影响

以2个厚皮甜瓜和1个薄皮甜瓜品种为材料,研究了摘心时期和留蔓数对苗期叶面积扩展和功能叶寿命的影响.结果表明,厚皮甜瓜苗期叶面积发育快于薄皮甜瓜,早熟厚皮甜瓜品种的叶面积发育快于中晚熟品种;甜瓜真叶净光合速率显著大于子叶,厚皮甜瓜的子叶光合速率高于薄皮甜瓜;摘心时预留子蔓数越多,甜瓜幼苗期叶面积扩展速度越快,但留蔓数增加会促进主蔓叶片衰老.从叶面积扩展速度和叶片功能寿命看,五叶期是甜瓜幼苗主蔓摘心的最佳时期,适宜留蔓数则因品种而异.     

Plastome data analysis of Cucumis melo subsp.agrestis

It is of interest to refine the taxonomic status of C. melo ssp. agrestis using its plastome data. The chloroplast size and GC% was found to be 1,56,016 bp and 36.92% respectively in Cucumis melo subsp. agrestis. The plastome of C. melo subsp. agrestis comprises of two inverted repeat (IR) regions of 25,797 bp each. It consisted of 133 genes with 88 protein-coding genes, 8 rRNA genes and 37 tRNA genes. Analysis of the C. melo ssp. agrestis plastome data will help breeders to improve the yield the crop.     

Resistance in melon to Monosporascus cannonballus and M. eutypoides: Fungal pathogens associated with Monosporascus root rot and vine decline

The fungal species Monosporascus cannonballus and M. eutypoides have been described as the causal agents of Monosporascus root rot and vine decline disease (MRRVD), which mainly affects melon and watermelon crops. Resistance to M. cannonballus has been reported in some melon cultivars (ssp. melo). Moreover, melon ssp. agrestis accessions have proven to be better resistance sources. This is the case of the Korean accession ‘Pat 81’, highly resistant under field and artificial inoculation. The objective of the work here presented was the evaluation of the resistance to MRRVD of different accessions representing the variability of Cucumis melo ssp. agrestis, against both, M. cannonballus and M. eutypoides, in a multiyear assay under different infection conditions. In general, M. eutypoides was less aggressive than M. cannonballus in the different environmental conditions. There was a strong influence of temperature on MRRVD, with more severe symptoms with higher temperatures and with variable effect of infection on plant development depending on the fungal species considered. Resistance to MRRVD has been confirmed in ‘Pat 81’ and in its derived F1 with a susceptible Piel de Sapo melon. Among the new germplasm explored, African accessions (both wild agrestis and exotic cultivated acidulus) showed good performance in artificial inoculation assays and in field conditions. These sources do not present compatibility problems with commercial melons, so they can be introduced in backcrossing programs. The accession assayed of the wild relative Cucumis metuliferus, also resistant to Fusarium wilt and to root-knot nematode, was highly resistant to MRRVD. The interest of this accession mainly relies in its advantages as a rootstock for melon.