南京农业大学学报  2015, Vol. 38 Issue (3): 375-380   PDF    
http://dx.doi.org/10.7685/j.issn.1000-2030.2015.03.004
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文章信息

毕研飞, 徐兵划, 郭静, 张永兵, 伊鸿平, 钱春桃, 陈劲枫. 2015.
BI Yanfei, XU Binghua, GUO Jing, ZHANG Yongbing, YI Hongping, QIAN Chuntao, CHEN Jinfeng. 2015.
分子标记辅助甜瓜抗蔓枯病基因聚合及‘白皮脆’品种改良
Pyramiding disease resistance genes by marker-assisted selection in melon(Cucumis melo L.)and'Baipicui’breed improvement
南京农业大学学报, 38(3): 375-380
Journal of Nanjing Agricultural University, 38(3): 375-380.
http://dx.doi.org/10.7685/j.issn.1000-2030.2015.03.004

文章历史

收稿日期：2014-08-20
引用本文
毕研飞, 徐兵划, 郭静, 张永兵, 伊鸿平, 钱春桃, 陈劲枫. 2015. 分子标记辅助甜瓜抗蔓枯病基因聚合及‘白皮脆’品种改良[J]. 南京农业大学学报, 38(3): 375-380.
BI Yanfei, XU Binghua, GUO Jing, ZHANG Yongbing, YI Hongping, QIAN Chuntao, CHEN Jinfeng. Pyramiding disease resistance genes by marker-assisted selection in melon(Cucumis melo L.)and'Baipicui’breed improvement[J]. Journal of Nanjing Agricultural University, 38(3): 375-380.  DOI: 10.7685/j.issn.1000-2030.2015.03.004
分子标记辅助甜瓜抗蔓枯病基因聚合及‘白皮脆’品种改良
毕研飞1, 徐兵划1, 郭静1, 张永兵2, 伊鸿平2, 钱春桃1 , 陈劲枫1    
1. 南京农业大学园艺学院/作物遗传与种质创新国家重点实验室, 江苏 南京 210095;
2. 新疆农业科学院哈密瓜研究中心, 新疆 乌鲁木齐 830091
收稿日期：2014-08-20
基金项目：国家自然科学基金新疆联合基金项目(U1178307)
作者简介：毕研飞,硕士研究生。
通迅作者：钱春桃,副教授,主要从事园艺作物遗传育种与分子生物技术研究,Tel:025-84396279，E-mail:chuntaoq@njau.edu.cn
摘要：[目的] 为甜瓜抗蔓枯病聚合育种探索一种简单、快捷的选择方法,同时获得品质优良且高抗甜瓜蔓枯病的改良‘白皮脆’品种(系)。[方法] 利用单一抗源PI140471(含抗病基因Gsb-1)与PI420145(含抗病基因Gsb-6)杂交,结合苗期蔓枯病菌A和A1梯度接种(5×105 mL-1、5×107 mL-1和5×109 mL-1)鉴定与SSR标记CMCT505及SCAR标记SGSB1800筛选,获得聚合2个抗蔓枯病基因Gsb-1和Gsb-6的聚合抗源471-145。以聚合抗源471-145为父本,品质优异的感病品种‘白皮脆’为母本进行杂交获得F1,选取含有聚合抗源且表现高抗的F1单株,再以‘白皮脆’为轮回亲本进行品种改良。[结果] SSR标记CMCT505可以在‘白皮脆’和PI140471上分别扩增出219和190 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,而聚合单株可以同时扩增出190和1 800 bp两条特异性片段。BC3F3群体筛选结果显示一些单株已经成功聚合了Gsb-1和Gsb-6两个抗病基因。单基因抗源PI140471和PI420145对不同菌株表现出选择性抗性且抗性水平低于聚合基因抗源。田间抗性观察显示,含有2个抗性基因的植株表现为高抗甜瓜蔓枯病,与分子标记检测结果一致。本课题组创建的分子标记CMCT505和SGSB1800对抗病基因Gsb-1和Gsb-6的选择具有较高的准确性,可以很好地应用于分子标记辅助选择甜瓜抗蔓枯病聚合育种。另外,抗性观察与果实品质测定表明改良‘白皮脆’品种(品系)高抗甜瓜蔓枯病且商品性优良。[结论] 本研究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,为甜瓜抗蔓枯病聚合育种探索了一种简单、快捷的选择方法,同时也为甜瓜优质、抗病和高产育种提供新的遗传资源。
关键词： 甜瓜     蔓枯病     分子标记     抗病性     聚合育种    
Pyramiding disease resistance genes by marker-assisted selection in melon(Cucumis melo L.)and'Baipicui’breed improvement
BI Yanfei1, XU Binghua1, GUO Jing1, ZHANG Yongbing2, YI Hongping2, QIAN Chuntao1 , CHEN Jinfeng1    
1. College of Horticulture/State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China;
2. Center of Hami Melon, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China
Abstract: [Objectives] The aim of this study is to provide a rapid selection method for gummy stem blight resistance in melon pyramiding breeding and improve the resistance of ‘Baipicui’. [Methods] Single resistance source PI140471 and PI420145 were used as the donor parents with the gummy stem blight resistance genes Gsb-1 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 mL-1, 5×107 mL-1 and 5×109 mL-1)were used in each backcross and self-cross progeny. In the first place, polymerization resistance source 471-145 was used as the male parent to cross with ‘Baipicui’ to get the hybrid F1. Then, the ‘Baipicui’ was used as the female parent to cross and backcross with the F1 individuals which were resistant to gummy stem blight highly and contain two resistant genes. [Results] The results showed that the polymorphism of molecular markers were different among the parents. The PCR amplification of SSR marker CMCT505 could augment two specific fragments of 219 and 190 bp in susceptible melon cultivar ‘Baipicui’ and single resistance source PI140471, respectively. A 1 800 bp specific fragment was obtained with SCAR marker SGSB1800 in resistance source PI420145. The polymerization individuals could be amplified to produce both fragments(190 and 1 800 bp)by CMCT505 and SGSB1800. From different kinds of molecular markers' detection results to the BC3F3 generation, it could be found that some individuals had two resistance genes(Gsb-1 and Gsb-6), while few individuals had one resistance gene. The single resistance source PI140471 and PI420145 showed lower resistance than polymerization individuals and selectively resistant to the different gummy stem blight biological strains(A and A1). The performance of each plant in the field estimated that all of the individuals containing two resistance genes were resistant to melon gummy stem blight, which was accordant to the expected result of molecular detection. Results of disease resistance evaluation indicated that the markers developed in this study were efficient in selecting two genes by MAS. In addition, the improved ‘Baipicui’ showed high resistantce to gummy stem blight and had better agricultural characteristics. [Conclusions] This study developed a molecular marker-assisted selection system for pyramiding breeding of melon. It also provided important intermediate materials and rapid selection method for good quality, disease resistance and high yield in melon breeding.
Keywords: melon(Cucumis melo L.)     gummy stem blight     marker-assisted selection     disease resistance     pyramiding breeding    

甜瓜(Cucumis melo L.)果实香甜,口感优良,深受广大消费者的喜爱。然而,蔓枯病(gummy stem blight)的发生已成为限制甜瓜优质高产的主要因素之一[1, 2, 3, 4]。目前,甜瓜生产上防治蔓枯病的常用方法主要是化学防治,但其效率低而且易引起环境污染。因此,选育甜瓜抗病品种(品系)具有重要的经济价值和生态价值。

国际上已经鉴定并报道了甜瓜蔓枯病抗源PI140471,其抗性基因为Gsb-1 [5],抗源PI420145是由本课题组Joseph等[6]筛选得到的,并命名为Gsb-6 。本课题组前期通过对抗病基因的研究,开发了分别与抗蔓枯病基因Gsb-1和Gsb-6连锁的SSR标记CMCT505和SCAR标记SGSB1800。刘龙洲等[7]以甜瓜JGD-3为抗源鉴定蔓枯病抗性属于多基因控制的数量遗传性状,并发现了与基因位点紧密连锁的3个分子标记CMTCN66、E3S7-1和TJ27。这些分子标记的开发为甜瓜抗蔓枯病育种奠定了良好的基础。

目前抗蔓枯病的甜瓜品种仅携带单个抗病基因(Gsb-1,Gsb-2,Gsb-3,Gsb-4,Gsb-5或Gsb-6),但随着栽培环境的变化,品种的抗性逐渐降低甚至丢失[8, 9, 10]。国内外研究表明,不同抗性基因的聚合有助于提高作物的抗性和抗谱[11, 12, 13],因此,选育高抗聚合基因材料是提高甜瓜蔓枯病抗性的有效途径之一。本文结合苗期人工接种鉴定、分子标记选择和田间抗性统计,初步探究了不同基因聚合后的抗性差异,并选育获得了高抗蔓枯病且商品性优异的改良‘白皮脆’品种(系),为甜瓜优质、抗病和高产育种提供新的遗传资源。 1 材料与方法 1.1 甜瓜抗源材料和接种菌株

甜瓜单基因抗源材料PI140471(含有抗病基因Gsb-1 )和PI420145(含有抗病基因Gsb-6 ),其中PI140471由美国康乃尔大学Molly John教授提供,PI420145是由本课题组的Joseph等筛选得到。聚合基因材料471-145由PI140471与PI420145杂交获得。优质、感病品种‘白皮脆’由新疆农业科学院提供。

蔓枯病菌为本实验室分离纯化并保存的A和A1型菌株,菌株的分生孢子在PDA培养基上经25℃黑暗培养7 d后,再进行4 d间歇紫外灯(12 h紫外/12 h黑暗)处理产生,在显微镜下利用血球计数板将分生孢子悬浮液分别配成5×105、5×107和5×109 mL-1备用。 1.2 苗期接种鉴定

参照Zhang等[9]的方法在苗期接种(3~4片真叶),用微型喷雾器喷洒孢子悬浮液,喷到植株叶片开始滴水为止。接种后用塑料拱棚保湿,相对湿度90%以上,3 d后揭开小拱棚,于7和10 d调查统计病情。叶片侵染分级标准为:0级,无可见侵染;1级,老叶上边缘坏死或斑点<10 mm,新叶无病;2级,老叶同上,新叶边缘坏死;3级,所有叶均有感染,叶坏死面积<25%;4级,25%≤叶坏死面积≤50%;5级,叶坏死面积>50%。根据平均病级(RI)确定蔓枯病抗性级别,高抗(HR):RI<1.0;抗(R):1.0≤RI<2.0;中抗(MR):2.0≤RI<3.0;感(S):3.0≤RI<4.0;高感(HS):RI≥4.0。平均病级计算公式:RI=∑(级值×株数)/总株数。 1.3 DNA提取与分子标记检测

甜瓜DNA的提取参照CTAB法。用于检测抗病基因Gsb-1和Gsb-6的分子标记为本课题组设计,由上海英潍捷基贸易有限公司合成(表 1),扩增产物大小分别为190和1 800 bp。PCR总反应体积为20 μL:ddH2O 12.1 μL,10×PCR Buffer 2.0 μL,2 mmol · L-1 dNTP 1.5 μL,25 mmol · L-1MgCL2 1.2 μL,10 μmol · L-1 3′和5′引物各1.0 μL,5 U · μL-1Taq聚合酶0.2 μL,30 ng · μL-1模板DNA 1.0 μL。扩增反应条件为:94℃ 5 min;94℃ 30 s,55℃ 30 s,72℃ 80 s,35个循环;72℃ 5 min,保持4℃。抗病基因Gsb-1的PCR产物在90 g · L-1聚丙烯酰胺凝胶上电泳,采用改良的银染方法检测。抗病基因Gsb-6的PCR产物用10 g · L-1琼脂糖凝胶电泳检测。

表 1 用于分子标记辅助选择的引物序列 Table 1 Primer sequences used for marker-assisted selection
基因Gene引物Primer引物序列Primer sequence连锁距离/cMDistance
Gsb-1CMCT505 F:5′-GACAGTAATCACCTCATCAAC-3′ R:5′-GGGAATGTAAATTGGATATG-3′5.2
Gsb-6SGSB1800 F:5′-GTTGCGTTCTCTGCTTGGA-3′ R:5′-AGGTAATTGAGGTGTCGTCTTA-3′2.0
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1.4 抗蔓枯病聚合育种过程

通过单一抗源PI140471(Gsb-1 )与PI420145(Gsb-6 )杂交,并结合苗期蔓枯病菌梯度接种鉴定与分子标记(CMCT505、SGSB1800)进行筛选,获得聚合2个抗蔓枯病基因的聚合抗源471-145。以表现高抗的聚合抗源471-145为父本,品质优异的感病品种‘白皮脆’为母本进行杂交获得F1,再以‘白皮脆’为轮回亲本进行连续回交3代自交2代,结合苗期接种鉴定与分子标记辅助选择筛选表现高抗且含有2个抗病基因的各世代进行品种改良。 1.5 田间抗性观察与果实品质测定

2014年春季对回交世代BC3F3进行田间蔓枯病抗性观察与果实品质测定。材料种植于南京农业大学江浦实验基地塑料大棚内(甜瓜重茬地,蔓枯病发严重),株距60 cm,行距120 cm,采用吊蔓栽培的方式,单蔓整枝,施肥、排灌、除草等按常规管理。果实品质测定以‘白皮脆’和聚合抗源471-145的果实性状为对照,选择9个果实进行品质测定。单果质量为9个果实的平均值;果实脆度测定采用马庆华等[14]的方法,用英国Stable Micro Systems公司生产的TAXT plus质构仪,采用P/2n针状探头(直径2 mm),测前速度2 mm · s-1,贯入速度1 mm · s-1,测后速度5 mm · s-1,最小感知力为10 g;果肉颜色利用CR-400型色差仪测定;将果实打成汁后,利用糖度计测果实可溶性固形物含量;采用分光光度计法测定维生素C含量。 1.6 数据分析

采用SPSS 19.0软件对数据进行统计分析。 2 结果与分析 2.1 聚合材料抗性基因分子标记的鉴定

利用本课题组创建的与抗病基因Gsb-1和Gsb-6紧密连锁的SSR标记CMCT505和SCAR标记SGSB1800筛选471-145聚合单株,含有基因Gsb-1的材料可以扩增出190 bp的特异性片段(图 1-A),而含有基因Gsb-6的材料可以扩增出1 800 bp的特异性片段(图 1-B)。因此,聚合基因471-145单株可以同时扩增出190和1 800 bp 2条特异性条带。

图 1 PCR检测BC3F3部分单株的抗病基因 Fig. 1 PCR detection of resistance genes in parts of BC3F3 individuals 1)A.CMCT505标记检测BC3F3部分单株含抗病基因Gsb-1的结果The result of detection about resistance gene Gsb-1 in parts of BC3F3 individuals by marker CMCT505;B.SGSB1800标记检测BC3F3部分单株含抗病基因Gsb-6的结果The result of detection about resistance gene Gsb-6 in parts of BC3F3 individuals by marker SGSB1800.
2)M:标准Marker;S:白皮脆Baipicui;R1:PI140471;R2:PI420145;1~30:BC3F3
3)编号2、4、8、17、18、24、25和28可扩增出190和1 800 bp两条特异性条带,为聚合单株。Number 2,4,8,17,18,24,25 and 28 were pyramided individuals which could amplify two bands of 190 and 1 800 bp.
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2.2 聚合基因材料和抗、感材料苗期接种蔓枯病菌的抗性鉴定

由表 2可见:A型菌株接种浓度为5×105 mL-1时,回交亲本‘白皮脆’的平均病级RI为3.60,表现为感病,单基因抗源材料和聚合基因材料BC3F3均表现为高抗;当接种浓度为5×107 mL-1时,‘白皮脆’平均病级RI为4.50,表现为高感,单基因抗源PI140471和聚合基因材料BC3F3的平均病级RI分别为1.90和1.66,表现为抗,PI420145的RI为2.30,表现中抗;当接种浓度为5×109 mL-1时,‘白皮脆’平均病级RI为4.90,表现高感,单基因抗源PI140471和PI420145均表现为感病,而聚合基因材料BC3F3的平均病级RI为2.70,表现中抗。

表 2 不同基因型甜瓜材料接种鉴定结果统计 Table 2 Disease ratings for gummy stem blight of different genotype materials
菌株Strain材料Material5×105 mL-15×107 mL-15×109 mL-1
RI抗性ResistanceRI抗性ResistanceRI抗性Resistance
A白皮脆Baipicui3.60aS4.50aHS4.90aHS
PI1404710.00dHR1.90dR3.10cS
PI4201450.12dHR2.30cMR3.30bS
BC3F30.75cHR1.66eR2.70dMR
A1白皮脆Baipicui3.40bS4.20bHS4.80aHS
PI1404710.00dHR1.84dR2.81dMR
PI4201450.00dHR1.92dR3.10cS
BC3F30.69cHR1.58eR2.60dMR
    注:1)同一列内不同小写字母表示差异性显著(P<0.05)。2)HR:高抗;R:抗;MR:中抗;S:感病;HS:高感。
Note: 1)Values followed by different lowercase letters are significant differentce at 0.05 level.2)HR:High resistance;R:Resistance;MR:Moderate resistance;S:Susceptible;HS:High sensitive.The same as follows.
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A1型菌株接种浓度为5×105 mL-1时,‘白皮脆’平均病级RI为3.40,表现为感病,单基因抗源和聚合基因材料BC3F3均表现为高抗;当接种浓度为5×107 mL-1时,‘白皮脆’平均病级RI为4.20,表现为高感,单基因抗源和聚合基因材料均表现为抗;当接种浓度为5×109 mL-1时,‘白皮脆’平均病级RI为4.80,表现为高感,单基因抗源PI140471的RI为2.81,表现中抗,PI420145的RI为3.10,表现感病,而聚合基因材料BC3F3的平均病级RI为2.60,表现为中抗。 2.3 田间抗性观察与果实品质测定

由表 3可以看出:改良‘白皮脆’聚合了双亲的优良性状,田间表现高抗甜瓜蔓枯病,除果实维生素C含量高于‘白皮脆’外,在果型、单果质量、果实脆度、果肉厚度及可溶性固形物含量方面与回交亲本‘白皮脆’并无显著差异。

表 3 不同基因型甜瓜蔓枯病抗性及果实品质的测定结果 Table 3 Resistance and fruit quality statistics of different genotype materials
材料Material抗性Resistance果型Fruitshape单果质量/kgFruit weight果实脆度/(kg·cm-2)Fruit crispness果肉颜色Fleshcolor果肉厚度/cmFleshthickness果肉质地Fleshtexture可溶性固形物含量/%Soluble solidcontent维生素C含量/(mg·100 g-1)Vitamin C content
白皮脆BaipicuiS橄榄型Oval1.50±0.2161.14±0.082橙色Orange3.21±0.135脆Fragile12.10±0.51610.31±0.495
471-145HR圆柱型Cylindrical0.61±0.1070.75±0.053白色White1.50±0.101软Soft5.32±0.18513.82±0.570
BC3F3HR橄榄型Oval1.33±0.1901.06±0.071浅橙色Light orange3.10±0.124脆Fragile10.91±0.46012.49±0.513
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3 讨论

研究表明,利用与抗病基因紧密连锁的分子标记辅助聚合多个抗性基因,可以提高选择的准确性,是培育具有持久抗性和综合抗性品种(系)的有效策略[15, 16, 17, 18]。目前,分子标记辅助育种研究在水稻、玉米和番茄等作物上已有较多的报道,但在甜瓜抗蔓枯病育种方面还鲜有报道,主要是因为与抗性基因紧密连锁的分子标记还比较少。

利用分子标记辅助选择抗病基因的可靠性主要取决于连锁程度,标记与基因连锁的越紧密,辅助选择的准确性就越高[19, 20, 21]。本研究所利用的分子标记CMCT505和SGSB1800是由本课题组创建的,与抗病基因Gsb-1和Gsb-6的遗传连锁距离分别为5.2和2.0 cM,理论上可以用来准确检测甜瓜所含有的抗蔓枯病基因。另外,从抗性鉴定结果分析表明,改良‘白皮脆’材料均因携带抗病基因Gsb-1和Gsb-6而提高了对蔓枯病的抗性水平,说明分子标记CMCT505和SGSB1800可以准确检测Gsb-1和Gsb-6两个抗性基因。为提高分子标记辅助聚合育种的效率和准确度,理想的方法是选用目标基因内标记,但要求对基因已经精细定位甚至克隆[22, 23]。本课题组对甜瓜不同抗蔓枯病基因正在进一步的研究中,希望可以在抗性基因两侧找到更加紧密连锁的分子标记,完善检测体系,提高分子标记辅助甜瓜抗蔓枯病聚合育种的效率。

传统的甜瓜抗蔓枯病苗期接种鉴定孢子液浓度为5×105 mL-1,可以有效区分出抗、感材料的差异,但不能准确区分抗性材料之间的差异[9, 24]。因此,本试验采用不同梯度浓度接种法,以准确鉴定单基因抗源和聚合基因材料抗性能力的差异。结果显示:单基因抗源PI140471和PI420145对不同蔓枯病菌表现出一定程度的选择性抗性,而聚合基因材料却一直表现为抗病,说明抗病基因Gsb-1和Gsb-6的聚合可以提高甜瓜对蔓枯病的抗性和抗谱,这与Matsumoto等[12]在黄瓜上和朱明涛等[13]在番茄上的研究结果相一致。甜瓜蔓枯病抗性是由多基因控制的数量性状,并受其他微效基因修饰增加作用贡献[7, 8],果实形状及果肉颜色受主要基因控制,表现出较高的遗传力,受环境影响较小[25, 26, 27, 28]。因此,对甜瓜品种进行蔓枯病抗性改良时,在早代对蔓枯病抗性及果实性状可进行定向选择,对表现高抗且果实性状与轮回亲本一致或相近的单株继续回交或自交留种。本研究在BC3F3世代即获得蔓枯病抗性显著提高且果实性状良好的改良‘白皮脆’材料,对甜瓜抗蔓枯病品种的选育具有重要的意义。

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