Cytogenetics, Haploid Breeding And Molecular Markers Linked To Genes For Resistance To Gummy Stem Blight In Melon

Melon is one of the most important horticultural crops worldwide, but melon is a less studied species cytologically because of its small chromosome and poor staining ability, which would go against the studies of genetic and breeding of melon. In this paper, studies on mitotic and meiotic cytology of two main ecological melon in china will be presented, which would be usful for research work of evolution, genes location in chromosome and physical mapping of melon.The main obstacle inhibited thick rind melon production is fungi diseases, especially gummy stem blight (GSB) which is a severely destructive disease worldwide caused by Didymella bryoniae (Auersw.) Rehm. Resistance breeding is laborious, time consuming and low efficiency by the way of traditional approach, otherwise by means of haploid technology and molecular marker assisted selection the approach would be time saving, more accurate and high efficiency. In this paper, technological system of haploid melon induced through pollination with irradiated pollens was proposed, and haploid plants were obtained from two types of melon. Technology of propagation in vitro and chromosome doubling of haploid melon were presented in this paper. After comparison of different GSB, many populations derived from resistant and susceptible melon were developed, then SSR and ISSR marker linked to Gsb-2, AFLP marker linked to Gsb-4 were screened.1 Cytological observation of mitosis and meiosis in melonThe effect of different sampling time and pre-treating chemicals on chromosome preparation of root tip cells of melon was studied. After sampling at 8:00 in morning, which is the only one of sampling time tested, there are up to 14% of cells observed in metaphase. Theefficiency of four pre-treating chemicals were compared, the best one was a mixture of two chemicals, cycloheximide (CHX, 40 mg·L^-1) and p-dichlorobenzene (pDB, saturated), after pretreatment with the mixture clearly morphological and well distributed chromosomes were observed in metaphase which indicated that would be good for karyotype analysis in melon. Metaphase chromosomes of two kinds of melon, thick and thin rind melon, were analyzed by the modified chromosome preparation. The results are as followed: the karyotype formula of two kinds are the same that is 2n=2x=24=14m+10st (2SAT), and the karyotype of them are 2A type, there are pare of satellite chromosomes in both. Otherwise, the total and average length of chromosome, the satellite chromosomes of the two kinds were different.Meiosis behaviors of pollen mother cells (PMC) and microspore development in thin and thick rind melon (Cucumis melo L.) were characterized herein. All melons observed have a simultaneous cytokinesis and a relative high synchronization in nuclear division. Nueleolus disappeared gradually from prophaseâ… to metaphaseâ… , reappeared at prophaseâ…¡and then disappeared. With condensation of chromatins, chromosome became more and more identifiable from prophaseâ… to metaphaseâ… , and illegible at telophaseâ… with de-helixation. Chromosomes were prominent again at both poles in PMC at prophaseâ…¡. At diakinesis, most chromosome configurations were ring bivalents for all melons. No chromosome bridge and lagging chromosomes were observed at anaphaseâ… andâ…¡, and their trtrads were decussate types. In Jiashi melon, nucleolus number was more than the other ecotype melons both at prophaseâ… andâ…¡, and a case of univalent and tetravalent occurred at diakinesis and at metaphaseâ… , respectively, which both demonstrating the speciality of Jiashi melon. During the development of mierospore, unicleate central-located and bicleate stage were observed in both types melon.2 Technological system of haploid induction in melonIn order to get the technique on induction of haploid melon, haploid melon (Cucumis melo L.) plants were obtained from two among fourteen genotypes tested through pollination with y-irradiated pollens and subsequent embryo culture in vitro in 2 seasons, with 0.3% average induction rate. These two genotypes represent thick (C. melo. ssp. melo) and thin (C. melo. ssp. conomon (Thunb) Greb) rind melon. Average fruit settings of donor plants were 50% and 10%, when theγ-ray doses were 300 Gy and 600 Gy respectively. Haploid were induced from thick and thin rind melon, line '4810' and cultivar 'Xinfuyu', to which the induction rate of haploid were 0.55% and 0.63% respectively, when the dose was 300 Gy. When the dose was 600 Gy, however, no haploid plant was produced from any tested material.In order to discuss the evolution of melon, meiosis behaviors of pollen mother cells (PMC) in haploid melon were studied. PMC of haploid melon had very low synchronization in nuclear division and had simultaneous or successive cytokinesis both. At diakinesis, most chromosome configurations were univalents for all haploid melons, except that only one bivalent occured. At metaphaseâ… , univalents scattered in cytoplasm mostly, but aligned in metophase plate in a few PMC. At anaphaseâ… , 12 univalents divided in two or three direction randomly, chromosomes distributed in form of 6+6 and 7+5 to two poles in most PMC. Centomere divided in advance, and chromosome bridge and lagging chromosomes were observed at anaphaseâ… . Nucleolus reappeared at prophaseâ…¡. At telohaseâ…¡, diads, triads, tetrads and polyads were developed, and about 10% pollen grains were produced.Using shoot-tips as explants, in order to setup the propagation system of melon haploid in vitro effect of different plant growth regulation (PGR), its concentration and combination on regeneration of haploid were investigated. The results indicated that the optimal media for shoot was MS+6-BA 0.5mg·L^-1, with multiplication coefficient of 3.57, the best media for root proliferation was 1/2MS+IBA 0.5mg·L^-1, with rooting ratio of 100%. More than 80% regenerated haploid plantlets were transplanted in field after acclimatization. The ploidy level of acclimatized plantlets from melon haploid was stable after cytological identification, and no ploidy variation occurred among these plantlets. Morphological comparison between haploid and diploid donor plants showed that the haploid had smaller leaves and sterile male and female flower, grew less vigorously. Meanwhile, induction on chromosome doubling of melon haploid was conducted by the way of colchicines soaking, the results showed that the optimal concentration and treat time of colchicines was 250 mg·L-1 and 8 hours respectively, with doubling ratio of 29.4%. The chromosome of melon haploid treated by eolchicines was 2n=24 after cytological observation.3 Molecular markers linked to genes for resistance to GSB in melonPure cultures of gummy stem blight were obtained from isolates collected from diseased plant in different production area of melon, under 26℃and 16h/8h photoperiod, colony, aerial and substrate mycelium, speed of colony, and sporulation of 4 isolates were observed after culture in PDA. The results showed that no differences were observed between the colony, color of mycelium, development speed of colony, and shape of pycnidia spore of 4 isolates, but the sporulation of its showed difference, a lot of pycnidias were found in HN-1 isolates. Except for NJ-1, abundant pycnldia and ascospores were found in 4 isolates after induction under UV light. After pathogenicity test, the results showed that no differentiate significance presented between 4 isolates.Gummy stem blight, caused by the fungal pathogen Didymella bryoniae (Auersw.) Rehm, is one of the most serious diseases of melon, molecular marker linked to gene resistance to GSB is important to breeding of melon. Parents, F₁ and 142 F₂ individuals derived from the cross of the resistant germplasm PI 482398 and the susceptible inbred line 'Baipicui' were identified for their resistance to GSB in seedling stage. Using bulked segregant analysis (BSA), amplified fragment length polymorphism (AFLP) technique with 64 primer combinations were employed to fred the polymorphism between resistant and susceptible bulks, and in resistant DNA pool a 285bp specific fragment was amplified in the primer combination of E-TA+M-CTT. This marker was testified with parents, F₁ and resistant and susceptible F₂ individuals and the specific band could be amplified in the resistant plants.In order to obtain molecular markers linked to gene resistance to GSB in melon, Parents, F₁ and 134 F₂ individuals derived from the cross of the resistant germplasm PI 157082 (Gsb-2) and the susceptible inbred line 'Baipicui' were identified for their resistance to GSB in seedling stage. Using bulked segregant analysis (BSA), 15 SSR and 72 ISSR primers were screened between resistant and susceptible bulks. 220bp and 560bp fragments specifically amplified by primer CMTC160a+b and ISSR-57 were present in PI 157082 and the resistant gene bulk but absent in 'Baipicui' and in the susceptible bulk. It was confirmed that markers CMTC160a+b₂₂₀ and ISSR-57₅₆₀ were linked to the resistant gene Gsb-2 in PI 157082 by screening 134 F₂ individuals, and the linkage distance was 26.4cM and 11.3cM, respectively. Two markers could be useful for marker-assisted selection in melon breeding for GSB resistance.