摘要
甜瓜是一种重要的园艺作物，由于染色体小、染色能力弱等原因，甜瓜的细胞遗传学研究非常薄弱，这必将会影响甜瓜遗传育种的深入研究．本文以我国两种主要生态类型的甜瓜为试验材料，进行有丝分裂和减数分裂的细胞遗传学研究，为甜瓜的系统进化、基因染色体定位和物理图谱构建等研究提供必要的细胞遗传学依据．
厚皮甜瓜生产面临的主要障碍是病害，尤其是蔓枯病的发生会导致毁灭性的减产．常规方法选育抗病品种的工作量大、周期长、效率低，而通过单倍体技术和分子标记辅助选择的方法，则可以增加选择的准确性、缩短育种年限、提高育种效率．本文建立了辐射花粉授粉诱导甜瓜单倍体技术体系，掌握单倍体甜瓜离体繁殖及其染色体加倍的方法．此外，在比较不同地区蔓枯病菌致病力的基础上，以抗／感联合世代群体为试材，筛选与甜瓜抗蔓枯病基因Gsb-2连锁的SSR和ISSR分子标记、与抗性基因Gsb-4连锁的AFLP标记。
1甜瓜有丝分裂和减数分裂的细胞遗传学观察
以萌发种子的根尖为试验材料，研究了不同预处理取样时间和不同预处理药剂对甜瓜染色体制片的影响。结果表明：上午8：00左右为最佳取样时间和预处理时间，该时间取样进行预处理后可以观察到近14％的中期分裂相；在4种药剂预处理活体根尖中，以添加对二氯苯(饱和)的放线菌酮(40 mg·L~(-1))水溶液的预处理效果最佳，可以观察到形态清晰、分散良好的中期染色体，便于进行核型分析．用此方法对厚皮和薄皮两种类型甜瓜进行核型分析，结果表明：两类甜瓜核型相似，都具有一对随体，核型均为2A型，核型公式均为2n=2x=24=14m+10st(2SAT)。但两类甜瓜的染色体总长、平均长度以及随体在染色体上的分布都不相同．
研究了薄皮类型和厚皮类型甜瓜花粉母细胞的减数分裂行为和小孢子发育过程，发现甜瓜细胞核减数分裂的同步性较高，细胞质是同时型分裂．在细胞核分裂的过程中，核仁在前期Ⅰ到中期Ⅰ逐渐消失，在前期Ⅱ再次出现，随后消失；染色体在前期Ⅰ到中期Ⅰ逐渐收缩，变得清晰，至末期Ⅰ解螺旋，变得模糊，在前期Ⅱ再次清晰．两种生态类型甜瓜终变期的染色体构型均以环状二价体为主．在后期Ⅰ和后期Ⅱ，未出现染色体桥及落后染色体现象。四分体为十字交叉型。在前期Ⅰ和前期Ⅱ，伽师瓜能够形成多个核仁，在终变期偶尔出现了单价体，表现了伽师瓜甜瓜的特殊性。两种类型甜瓜小孢子发育过程包括单核期和双核期，成熟花粉粒具三个萌发孔。
2甜瓜单倍体诱导技术体系
为了掌握辐射花粉授粉诱导甜瓜单倍体技术，以14种基因型甜瓜为材料，连续两个栽培季节通过应用丫射线辐射的花粉进行授粉结合胚胎培养，从2种基因型中获得了单倍体植株，单倍体的平均诱导频率为0．3％。这2种基因型分别属于厚皮和薄皮甜瓜类型。花粉的辐射剂量为300 Gy和600 Gy时，供试材料的平均坐果率分别为50％和10％。辐射剂量为300 Gy时，诱导厚皮甜瓜‘4810’和薄皮甜瓜‘新富玉’形成了单倍体植株，单倍体植株的诱导频率分别为0．55％和0．63％；剂量为600 Gy时未能诱导供试材料形成单倍体。
为了探讨甜瓜的进化途径对单倍体甜瓜花粉母细胞减数分裂进行了细胞遗传学研究，结果表明单倍体甜瓜细胞核减数分裂的同步性较差，细胞质分裂既有连续型也有同时型分裂。终变期的染色体构型均以单价体为主，仅发现一例具有二价体的细胞．中期Ⅰ单价体散落在细胞质中，少数细胞中的单价体排列在赤道板上。单价体在后期Ⅰ随机向两极或三极方向分离，两极方向主要以6Ⅰ+6Ⅰ和7Ⅰ+5Ⅰ的方式分离．后期Ⅰ部分染色体的着丝粒提前分裂，还观察到染色体桥和滞后染色体。前期Ⅱ，核仁再次出现。末期Ⅱ，可观察到二分体、三分体、四分体和多分体．形成了约10％的正常花粉粒。
以单倍体甜瓜无菌苗的顶芽或腋芽为外植体，研究不同激素及其不同浓度配比对单倍体甜瓜外植体再生的影响，以建立单倍体甜瓜离体繁殖技术体系。结果表明，单倍体甜瓜最佳增殖培养基为MS+6-BA 0．5 mg·L~(-1)，增殖系数为3．57。最适生根培养基为1／2MS+IBA 0．5 mg·L~(-1)，生根率达100％，单倍体再生植株驯化后的成活率达80％。细胞学鉴定表明，单倍体再生植株的倍性稳定，未发生染色体数目的变异．单倍体植株生长势弱，叶片较小，雄花和雌花均败育。利用秋水仙碱溶液浸泡法诱导单倍体甜瓜染色体加倍，结果表明：浓度为250 mg·L~(-1)的秋水仙碱浸泡8 h，染色体加倍效果最佳，加倍率达29．4％。细胞学观察表明，加倍后的单倍体染色体数为2n=24。
3甜瓜抗蔓枯病基因的分子标记
从来源于不同种植区的甜瓜蔓枯病病株上分离得到蔓枯病病原菌，在26℃、16h／8h光周期、PDA培养基上培养，对它们的菌落、气生菌丝、基内菌丝、菌丝生长速度及产孢能力等生物学特性进行观察。结果表明：4种病原菌的菌落形态、菌丝颜色、菌丝生长速度以及分生孢子形态差异不明显；产孢能力略有差异，其中HN-1直接产生分生孢子；在紫外灯照射诱导下，除NJ-1不能产生有性的子囊孢子外，4种病原菌都形成大量的分生孢子和子囊孢子。致病力测定结果表明4种病原菌的致病力差异不显著。
甜瓜蔓枯病(Didymella bryoniae)是为害我国甜瓜的一种主要病害．以抗蔓枯病PI 482398(Gsb-4)与感病甜瓜自交系‘白皮脆’为亲本，建立了抗感F_2代群体，对亲本、F_1及142个F_2代群体进行了苗期抗蔓枯病接种鉴定。利用集团分离分析法．(Bulked Segregant Analysis，BSA)在F_2代建立抗感基因池，以抗感基因池为模板，筛选了64对AFLP选择性引物EcoRI-NN+MseI-NNN组合，发现EcoRI-TA+MseI-CTT引物组合在抗感基因池间扩增出一条分子量为285bp的特异条带．经双亲、F_1和F_2代抗感单株验证，该特异条带与抗病基因Gsb-4不连锁．
为了筛选抗性基因分子标记，以抗蔓枯病种质资源PI 157082(Gsb-2)与感病的甜瓜自交系‘白皮脆’为亲本，建立了抗感F_2代群体，对亲本、F_1及134个F_2代群体进行了苗期抗蔓枯病接种鉴定．利用集团分离分析法(Bulked segregant analysis，BSA)在F_2代建立抗感基因池，以抗感基因池为模板，用15对SSR引物和72条ISSR引物进行PCR扩增筛选．其中引物CMTC160a+b和ISSR-57在亲本和F_2代抗感基因池之间分别扩增出一条多态性片段，大小约为220bp和560bp．经F_2代群体单株验证后，这两条多态性条带与抗性基因Gsb-2表现连锁关系，遗传连锁距离分别为26．4cM和11．3cM，定名为CMTC160a+b_(220)和ISSR-57_(560)．可以作为甜瓜抗蔓枯病辅助选择的分子标记。
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 melon
The 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 prophaseⅠto metaphaseⅠ, reappeared at prophaseⅡand then disappeared. With condensation of chromatins, chromosome became more and more identifiable from prophaseⅠto metaphaseⅠ, and illegible at telophaseⅠwith de-helixation. Chromosomes were prominent again at both poles in PMC at prophaseⅡ. At diakinesis, most chromosome configurations were ring bivalents for all melons. No chromosome bridge and lagging chromosomes were observed at anaphaseⅠandⅡ, and their trtrads were decussate types. In Jiashi melon, nucleolus number was more than the other ecotype melons both at prophaseⅠandⅡ, and a case of univalent and tetravalent occurred at diakinesis and at metaphaseⅠ, 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 melon
In 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 metaphaseⅠ, univalents scattered in cytoplasm mostly, but aligned in metophase plate in a few PMC. At anaphaseⅠ, 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 anaphaseⅠ. Nucleolus reappeared at prophaseⅡ. At telohaseⅡ, 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 melon
Pure 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, F_1 and 142 F_2 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, F_1 and resistant and susceptible F_2 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, F_1 and 134 F_2 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+b_(220) and ISSR-57_(560) were linked to the resistant gene Gsb-2 in PI 157082 by screening 134 F_2 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.
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