分子标记辅助甜瓜抗蔓枯病基因的聚合及品种改良

doi:10.3864/j.issn.0578-1752.2015.03.12

摘要/Abstract

摘要： 【目的】蔓枯病（gummy stem blight）是一种真菌性土传病害，是危害甜瓜生产的严重病害之一，其存在生理小种的分化，品种即使仅携带单个抗病基因，仍会导致甜瓜抗性逐步降低甚至丢失。建立甜瓜抗蔓枯病聚合育种的分子标记辅助选择体系，选育抗性高且不再分离的聚合抗源自交系和品质优良且高抗甜瓜蔓枯病的改良白皮脆品种（品系），为甜瓜优质、抗病和高产育种提供一种简单、快捷的选择方法和重要的中间材料。【方法】利用5份甜瓜单一抗源材料PI140471、PI157082、PI511890、PI482398和PI420145两两杂交获得8份聚合抗源（082-471、082-890、082-398、145-471、145-082、145-890、145-398和890-398）。利用3个不同浓度的蔓枯病病菌孢子液（5×10⁵个/mL、5×10⁷个/mL、5×10⁹个/mL）对8份聚合抗源的F₁植株幼苗进行接种鉴定。结合春、秋两季的接种鉴定结果，筛选得到2份聚合后抗性显著提高的基因组合145-471和145-398。一方面，以145-471和145-398为材料，利用苗期梯度接种鉴定、分子标记辅助选择以及农艺性状观察，对其自交后代进行逐代筛选至F₇。另一方面，选用组合PI420145和PI140471以及PI420145和PI482398作为蔓枯病抗性基因的供体亲本，综合性状优良的感病甜瓜品种‘白皮脆’为抗性基因受体亲本。先分别单向回交5代，在单个基因回交转移时，利用分子标记检测结合农艺性状观察对回交后代进行筛选。当回交后代的遗传背景得到一定程度恢复后，再将2个方向的回交后代进行杂交，后自交3代稳定。【结果】聚合抗源对不同浓度的蔓枯病菌均表现为抗，而单一抗源对不同浓度蔓枯病菌表现出选择性抗性且抗性水平低于聚合抗源。SSR标记CMCT505和CMTA170a在PI140471和PI482398上可分别扩增出189 bp和121 bp的特异性片段，SCAR标记SGSB₁₈₀₀可以在PI420145上扩增出1 800 bp的特异性片段，而145-471（145-398）的聚合单株可以同时扩增出189 bp和1 800 bp（121 bp和1 800 bp）两条特异片段。聚合抗源145-471（或145-398）的自交F₇世代以及改良白皮脆BC₅F₄世代群体的分子标记鉴定结果显示，各单株已经成功聚合了Gsb-1和Gsb-6（或Gsb-4和Gsb-6）两个抗病基因，抗性不再有分离且农艺性状稳定一致。初步建立了甜瓜抗蔓枯病聚合育种的分子标记辅助选择体系，获得了两份表现高抗且抗性不再分离的聚合抗源自交系F₇和2个以优质品种‘白皮脆’为受体亲本的改良白皮脆BC₅F₄品种（系）。改良白皮脆BC₅F₄世代表现为高抗甜瓜蔓枯病，且在单果质量、果形指数、果实脆度、果肉质地、果肉厚度和可溶性固形物含量等农艺性状方面与白皮脆并无显著差异。【结论】创建的3个分子标记CMCT505、CMTA170a和SGSB₁₈₀₀对抗病基因Gsb-1、Gsb-4和Gsb-6的选择具有较高的准确性。初步建立了甜瓜抗蔓枯病聚合育种的分子标记辅助选择体系，为甜瓜优质、抗病和高产育种提供了一种简单、快捷的选择方法，将大大提高育种的效率。改良‘白皮脆’作为抗病育种的新材料，为甜瓜抗病品种的选育和抗病基因进一步聚合提供了材料。

关键词: 甜瓜, 分子标记, 抗病性, 品种改良

Abstract: 【Objective】Gummy stem blight (GSB) is caused by Didymella bryoniae and is a serious fungal disease of melon (Cucumis melon L.). The resistance of melon varieties carrying a single resistance gene is not enough to the disease because of the isolates variation of Didymella bryoniae. Therefore, this study aims to develop a molecular marker-assisted selection system and provide an important intermediate materials for melon disease-resistance breeding. 【Method】Firstly, the single resistant sources PI140471, PI157082, PI511890, PI482398 and PI420145 were used to obtain polymerization resistant sources (082-471, 082-890, 082-398, 145-471, 145-082, 145-890, 145-398 and 890-398). Secondly, three different gradient spores vaccination identification (5×10⁵ spores/mL, 5×10⁷ spores/mL and 5×10⁹ spores/mL) was employed to distinguish the resistance of F₁ plant of eight different polymerization resistant sources. The gene combinations of 145-471 and 145-398 with their resistance significantly improved were screened by inoculation identification results of spring and autumn. Single resistant sources PI140471, PI482398 and PI420145 were used as the donor parents with the gummy stem blight resistance genes Gsb-1 Gsb-4 and Gsb-6, respectively. Commercial melon cultivar Baipicui was used as the receipt parent to cross and backcross with the donor parents. Marker assisted selection (MAS) and gradient spores vaccination identification (5×10⁵ spores/mL, 5×10⁷ spores/mL and 5×10⁹ spores/mL) were used in each backcross and self-cross progeny. 【Result】The single resistant sources showed lower resistance than polymerization individuals and showed selective resistance to the different gummy stem blight spore concentrations (5×10⁵ spores/mL, 5×10⁷ spores/mL and 5×10⁹ spores/mL). The detection results of molecular markers showed that all the individuals of 145-471 (or 145-398) F₇ and BC₅F₄ generation had two resistance genesof Gsb-1 and Gsb-6 (or Gsb-4 and Gsb-6). The polymorphism of molecular markers was different among the parents. The PCR amplification of SSR marker CMCT505 could augment one specific fragments of 189 bp in single resistant source PI140471. The PCR amplification of SSR marker CMTA170a could augment one specific fragments of 121 bp in single resistant source PI482398. A 1 800 bp specific fragment was obtained with SCAR marker SGSB₁₈₀₀ in resistant source PI420145. The polymerization individual 145-471 could be amplified two fragments of 189 bp and 1 800 bp by SSR marker CMCT505 and SCAR marker SGSB₁₈₀₀. The polymerization individual 145-398 could be amplified two fragments of 121 bp and 1 800 bp by SSR marker CMTA170a and SCAR marker SGSB₁₈₀₀. At F₇ generation, the pyramiding lines with homozygosity at both gene loci were obtained. The polymerization individuals showed higher resistance than single resistant sources PI140471, PI482398 and PI420145. The individuals were resistant to melon gummy stem blight, which was accordant to the expected result of molecular detection. Improved Baipicui BC₅F₄ generation showed high resistance to melon gummy stem blight and had no significant differences in fruit weight, fruit shape index, fruit crispness, flesh texture, flesh thickness and soluble solids content compared with the Baipicui. A molecular marker-assisted selection system was developed and thus providing important intermediate materials for melon disease-resistance breeding. 【Conclusion】The results of disease resistance evaluation indicated that the molecular markers developed in the study were efficient in selecting three resistance genes by MAS. The molecular marker-assisted selection system developed in this study was efficient for pyramiding multiple resistant genes. The resistance improved materials of Baipicui would be used in breeding and further resistance gene pyramiding.

Key words: melon (Cucumis melon L.), marker-assisted selection, disease resistance, variety improvement

毕研飞，徐兵划，钱春桃，郭静，张永兵，伊鸿平，陈劲枫. 分子标记辅助甜瓜抗蔓枯病基因的聚合及品种改良[J]. 中国农业科学, 2015, 48(3): 523-533.

BI Yan-fei, XU Bing-hua, QIAN Chun-tao, GUO Jing, ZHANG Yong-bing, YI Hong-ping, CHEN Jin-feng. Pyramiding Disease Resistance Genes and Variety Improvement by Molecular Marker-Assisted Selection in Melon (Cucumis melon L.)[J]. Scientia Agricultura Sinica, 2015, 48(3): 523-533.

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