Induction of somatic embryogenesis and caulogenesis from cotyledon and leaf protoplast-derived colonies of melon (Cucumis melo L.)

Summary A procedure leading to the regeneration of whole plants from protoplasts of melon is described. Protoplasts were isolated from cotyledons and leaves of plants grown in vitro. After 14 days of culture, average viability and division rates were respectively 60% and 30% for the two organs, considering total initial protoplasts plated. The manipulation of the exogenous auxin / cytokinin balance in regeneration media enabled to direct morphogenesis towards somatic embryogenesis (1 mg·l^–1 2,4-dichlorophenoxyacetic acid and 0.1 mg·l^–1 6-benzylaminopurine) or caulogenesis (0.5 mg·l^–1 6-benzylaminopurine and 0.5 mg·l^–1 kinetin). Contrary to division ability, regeneration capacity was genotype-dependent under our conditions, but the two organs expressed similar division and regeneration capacities. Maltose was superior to sucrose for the development of caulogenic nodules into buds. Some plants were transplanted to soil, where they appeared to be fertile and produced seeds.Abbreviations BAP 6-benzylaminopurine - CPW Cell and Protoplast Washing medium - KIN kinetin - MES 2-(N-morpholino) ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA 1 — naphthaleneacetic acid - PAS H (staining), Periodic Acid-Schiff / Hematoxylin - 2,4-D 2,4-dichlorophenoxyacetic acid     

甜瓜(Cucumis melo L.)AFLP标记的多态性及聚类分析研究

利用 AFLP 技术对30个甜瓜材料进行多态性和聚类分析研究。从120对 MseI 和 PstI 引物中筛选出25对扩增效果好的引物,共扩增出262条多态性带。聚类分析结果新疆甜瓜中的夏甜瓜类型,新疆甜瓜中的冬甜瓜类型、美国粗皮甜瓜类型、日本甜瓜类型、梨瓜类型各聚为一组。上述材料的聚类分析结果与依据生态类型和地理起源的分类结果基本吻合,表明 AFLP 用于甜瓜种内不同材料间的遗传变异性分析是可行的。     

An improved method of Agrobacterium tumefaciens-mediated genetic transformation system of melon (Cucumis melo L.)

A reliable Agrobacterium-mediated transformation and shoot regeneration protocol was developed for breeding lines of commercially important melon. Genetic manipulation has been considered a feasible approach for melon improvement; however, melon is considered a crop species difficult to manipulate. Here we proposed meristematic cells from mature embryos as target for gene transfer by Agrobacterium. In vitro meristems proliferation and multiple shoots regeneration were evaluated by sowing melon mature seeds on MS with 1.0 mg/L benzyladenine (BA), and 0.05 mg/L indole acetic acid (IAA) were used for shoot regeneration. The highest number of regenerated shoots was obtained from half mature seeds. A DNA fragment corresponding to selection marker nptII was amplified from genomic DNA extracted from leaves of regenerated plant on hormone free MS medium with 75 mg/L kanamycin, suggesting their transgenic nature. Southern hybridization of transgenic lines revealed random insertion of the transgene in host genome, with insert numbers differing among transformants anthesis, suggesting that ethylene is important for sex determination. Field studies showed that CmACS-7 melons had earlier mature bisexual flowers, increased femaleness as measured by earlier and bisexual buds, and increased number of fruit set on closely spaced nodes on the main stem. Transformation efficiencies of cultivar CM-23 with EHA105 (pBI121-cm) were 4 %, demonstrating that melon meristematic cells are an useful target for genetic manipulation by agroinfection.     

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.     

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.     

A genetic map of melon (Cucumis melo L.) based on amplified fragment length polymorphism (AFLP) markers

 Genetic maps facilitate the study of genome structure and evolution, and the identification of monogenic traits or Mendelian components of quantitative traits. We evaluated 228 RAPD, microsatellite and AFLP markers for linkage analysis in melon (Cucumis melo L.) varieties MR-1 (resistant to Fusarium wilt, powdery and downy mildews) and Ananas Yokneum (AY; susceptible to these diseases) and constructed a detailed genetic map. The mapping population consisted of 66 backcross progenies derived from AY×(MR-1×AY). Despite a relatively low level of polymorphism in the species, AFLP markers were found to be more efficient in mapping the melon genome than RAPD or microsatellite markers. The map contains 197 AFLPs, six RAPDs and one microsatellite marker assigned to 14 major and six minor linkage groups, and covers 1942 cM with the average distance between adjacent markers of approximately 10 cM. The maximum distance allowed between markers is 27.5 cM. About 11% of the intervals (20 out of 173) are over 20 cM (but less than 27.5 cM). The map has immediate utility for identifying markers linked to disease resistance genes that are suitable for marker-assisted breeding. The use of microsatellite markers for integration with other maps is also discussed. Received: 12 March 1997 / Accepted: 20 May 1997     

On the genetic control of heterosis for fruit shape in melon (Cucumis melo L.)

The objective of the present work is to study the genetic basis of heterosis for fruit shape (FS) in melon observed in a cross between the Spanish cultivar "Piel de Sapo" (PS) and the Korean accession PI 161375 (Songwang Charmi [SC]) using a set of near-isogenic lines (NILs) with contrasting phenotypes for FS, each carrying a single chromosomal introgression from SC within the genetic background of PS. We investigated the FS of homozygous NILs, hybrids NIL x PS, and all 2-way crosses between NILs to test the main heterosis hypotheses (dominance, overdominance, and epistatic interactions). Gene action of alleles of quantitative trait loci inducing fruit enlargement was dominance, whereas those inducing rounder fruit were additive or recessive. Only minor epistatic interactions were found. Therefore, the most plausible explanation for FS heterosis in this cross is in agreement with the dominance complementation hypothesis. Over 70% of the hybrid heterosis could be achieved by combining just 2 loci, indicating that the genetic control of FS heterosis in this cross is relatively simple. FS is proposed as a reproductive trait in melon because of the high correlation to the number of seeds produced along the fruit longitudinal axis.     

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.     

Effects of cyanide on the respiration of musk melon (Cucumis melo L.) roots

The characteristics of root respiration of melon were examinedwith an oxygen electrode. The Hofstee plot of root respirationbreaks into two straight lines. The results of cyanide inhibitionexperiments and curve-fitting analysis suggest that one cyanide-insensitiveand two cyanide-sensitive oxidases operate in melon roots. (Received December 24, 1976; )     

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     

中国甜瓜种质资源形态性状遗传多样性分析

对我国各地257份代表性的甜瓜种质资源的20个形态性状进行调查分析,研究其遗传多样性。结果表明,7个质量性状(果实形状、果皮底色、覆纹颜色、覆纹形状、果肉颜色、果肉质地和种子颜色)和6个数量性状(果实横径、果实纵径、单果鲜重、果肉厚度、可溶性固形物含量和种子千粒重)差异明显,其Shannon’s指数分别大于1.00和1.50。所有种质的平均遗传多样性指数为1.09,不同地区种质资源遗传多样性差异明显,多样性指数高低次序分别为：西北、华中、华东、华北、东北和华南。主座标标分析(PCO)将所有种质划分为3个区域,即厚皮种质优势区、厚皮和薄皮种质混合分布区、薄皮种质优势区,不同地区的种质在PCO图上的分布差异明显,西北地区的厚皮甜瓜种质和华中、华东地区的薄皮甜瓜种质广泛分布于3个区域中,其形态遗传多样性比其他地区的种质更加丰富,支持了新疆地区为厚皮甜瓜次级起源中心、黄淮及长江流域为薄皮甜瓜初级起源中心的观点。     

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     

一个获得与缺失变异(PAV)调控甜瓜果实苦味

甜瓜苦味物质严重影响其口感和品质。本研究利用不苦的薄皮甜瓜品系C69和苦的薄皮甜瓜品系C14构建了一个包含100个单株的F2群体。首先利用2b-RAD测序构建一个遗传连锁图谱。其次,结合群体的苦味性状进行全基因组的QTL定位和关联分析。然后,利用2b-RAD测序特有的技术优势进行群体的获得与缺失变异(PAV)的挖掘。最后,利用亲本的重测序信息确定控制苦味性状的关键基因。结果发现,F1的果实表现出强烈的苦味,F2群体中苦与不苦的单株分别为81个和19个,符合3∶1的分离比(χ^2=1.92,P=0.1659),表型表明所用甜瓜材料的苦味主要是由一个显性的基因位点控制。利用477个SNP标记构建一张包含10个连锁群的连锁图谱,总长为337.79 cM,标记间平均间隔0.71 cM。全基因组QTL定位在8号连锁群(对应9号染色体),检测到一个解释表型变异为20%的甜瓜苦味QTL。全基因组关联分析检测到7个SNPs与苦味性状相关,全部位于9号染色体苦味QTL的基因组区域。通过PAV分型分析仅发现一个特有的大片段缺失(21707702~21743072 bp),位于QTL区域,且在所有的不苦株系中存在,而苦的株系中不存在。基于两个亲本材料的深度重测序信息,发现这个PAV的区域更大,约为62 Kb,共涉及到9个连续的基因(MELO3C005601、MELO3C005602、MELO3C005603、MELO3C005604、MELO3C005605、MELO3C005606、MELO3C005607、MELO3C005608和MELO3C005609),其中5个是细胞色素P450基因。构建的系统发育树表明,这5个细胞色素P450基因与参与葫芦素C/B/E合成的细胞色素P450基因簇CYP81Q58、CYP81Q59和CYP712D8在一个进化枝,可能行使类似的功能,为潜在的类似于黄瓜葫芦素C合成的基因簇的一部分。前人通过比较基因组学研究获得的2个控制葫芦素B合成的bHLH转录因子CmBr(MELO3C005610)和CmBt(MELO3C005611)同在9号染色体,与本研究检测到的PAV紧密挨在一起。我们的研究结果为后续不苦甜瓜的育种提供了新的理论支撑和分子辅助育种目标。     

Ploidy of somatic embryos and the ability to regenerate plantlets in melon (Cucumis melo L.)

Summary The number of chromosomes in cells of callus, somatic embryos and regenerated plantlets during somatic embryogenesis were examined in two cultivars of melon (Cucumis melo L.). Somatic embryos were diploid (50.0%/32.1%), tetraploid (38.5%/57.5%) and octoploid (11.5%/10.4%) whereas in callus cells diploidy (41.9%/43.3%), tetraploidy (27.9%/25.8%), octoploidy (11.6%/15.5%) and a low frequency of other types of ploidy and aneuploidy were observed. Mixoploid somatic embryos were not observed. These results suggest that the somatic embryos were selectively differentiated from diploid, tetraploid and octoploid cells, and that endopolyploidization of cultured cells occurred before the start of cell division leading to somatic embryogenesis. The ratio of diploid to tetraploid (1.30/0.55) in somatic embryos was less than that in callus cells (1.50/1.68) while ratios of diploid to octoploid (4.35/3.09) and tetraploid to octoploid (3.35/5.52) in somatic embryos were greater than those in callus cells (3.61/2.80 and 2.40/1.67). Therefore, it appears that the ability of callus cell to differentiate into somatic embryos increases in the following order: octoploid < diploid < tetraploid. Regenerated plantlets were diploid (65.5%/55.1%) and tetraploid (34.5%/44.9%). No octoploid plantlets were observed. The ratio of diploid to tetraploid in regenerated plantlets (1.72/1.23) was greater than that in somatic embryos. Therefore, it appears that the ability of somatic embryos to develop into plantlets increases in the following order: octoploid < tetraploid < diploid.     

Genetic control of fruit shape acts prior to anthesis in melon (Cucumis melo L.)

Genetic control of fruit shape in Cucumis melo was studied using QTL analysis in two Recombinant Inbred (RI) populations consisting of 163 and 63 individuals, respectively, obtained by crossing the same round-fruited parent with two different elongated-fruit lines. Fruit shape is mainly explained by fruit length in these two populations. Most QTLs for fruit shape and ovary shape detected were found to co-segregate, thus demonstrating early control of fruit shape during ovary development. A high level of correlation between fruit shape and ovary shape was also found in 14 unrelated genetic lines, a finding which suggests that control of fruit shape by gene(s) active early in the ovary is a general feature in C. melo. Two major flower genes, a ( monoecious) and p ( pentamerous), were shown to have major effects on fruit shape. Major tightly linked QTLs for fruit and ovary shape were found close to the a and p genes, probably reflecting their pleiotropic effect on fruit shape. Moreover, one of the two QTLs detected in the Védrantais x PI 414723 population was also found in the Védrantais x PI 161375 population. Variation of fruit shape in melon could be due to variations having quantitative effects on a large set of genes that are probably involved in ovary development.     

Characterization of two cDNA clones for mRNAs expressed during ripening of melon (Cucumis melo L.) fruits

In vitro translation of mRNAs and polyacrylamide gel electrophoresis of proteins from melons revealed that several mRNAs increased in amount during ripening, indicating the existence of other ripening genes in addition to those cloned previously. To identify ripening-related genes we have screened a ripe melon cDNA library and isolated two novel cDNA clones (MEL2 and MEL7) encoding unidentified proteins. Southern analysis revealed that MEL2 and MEL7 are encoded by low-copy-number genes. The MEL2 cDNA clone is near full-length, corresponds to a 1600 nucleotide mRNA that accumulates during ripening and encodes a predicted protein rich in hydrophobic amino acids. The MEL7 cDNA clone is full-length, corresponds to a mRNA of 0.7 kb which accumulates during early ripening stages and is also present at low levels in other organs of the melon plant. The MEL7 predicted polypeptide is 17 kDa and shows significant homology with the major latex protein from opium-poppy. Wounding and ethylene treatment of unripe melon fruits 20 days after anthesis showed that MEL2 and MEL7 mRNAs are only induced by ethylene.     

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。