菜豆普通細(xì)菌性疫病抗性基因定位

朱吉風(fēng) 武 晶 王蘭芬 朱振東 王述民

中國農(nóng)業(yè)科學(xué)院作物科學(xué)研究所, 北京100081

菜豆普通細(xì)菌性疫病抗性基因定位

朱吉風(fēng) 武 晶 王蘭芬 朱振東 王述民*

中國農(nóng)業(yè)科學(xué)院作物科學(xué)研究所, 北京100081

菜豆普通細(xì)菌性疫病是世界上危害普通菜豆生產(chǎn)的最嚴(yán)重的病害之一。龍蕓豆5號(hào)是我國黑龍江省的主栽品種, 對(duì)菜豆普通細(xì)菌性疫病表現(xiàn)出良好的抗性。為定位來源于龍蕓豆5號(hào)的抗性基因, 本研究構(gòu)建了包含785個(gè)單株的F2分離群體?；谠撊后w構(gòu)建了一張包含206個(gè)SSR標(biāo)記, 總長度1648.42 cM, 標(biāo)記間平均遺傳距離8.00 cM的遺傳圖譜。圖譜包含12個(gè)連鎖群, 各連鎖群平均長度137.37 cM, 連鎖群上標(biāo)記數(shù)量3～35個(gè)。結(jié)合溫室表型鑒定結(jié)果, 采用QTL IciMapping v4.0軟件的完備區(qū)間作圖法進(jìn)行QTL定位和效應(yīng)估計(jì)。接種14 d后在Pv06染色體上檢測(cè)到一個(gè)抗病QTL。該位點(diǎn)位于標(biāo)記p6s249與p6s183之間, 加性效應(yīng)值為0.44, 說明增效基因來源于龍蕓豆5號(hào), LOD值為5.93, 表型貢獻(xiàn)率為4.61%, 該抗病QTL的效應(yīng)值相對(duì)較低, 將在培育穩(wěn)定持久的抗菜豆普通細(xì)菌性疫病的品種中發(fā)揮作用。最后, 對(duì)抗性基因緊密連鎖的11對(duì)SSR引物與菜豆普通細(xì)菌性疫病抗性的關(guān)聯(lián)分析表明, SSR標(biāo)記p6s249與菜豆普通細(xì)菌性疫病抗性極顯著關(guān)聯(lián)(P＜0.001), 該標(biāo)記可用于抗病分子育種。

普通菜豆; 普通細(xì)菌性疫病; SSR; QTL; 關(guān)聯(lián)分析

普通菜豆(Phaseolus vulgaris L.)是人類生活中重要的食用豆類之一, 富含蛋白質(zhì)、碳水化合物、維生素等人體所必需的營養(yǎng)成分[1]。2014年全世界普通菜豆籽粒產(chǎn)量為2500萬噸, 占食用豆總產(chǎn)量的50%左右(FAOSTAT 2015)。然而, 由地毯草黃單胞桿菌菜豆致病變種(Xanthomonas axonopodis pv. phaseoli, Xap)[2]所引起的菜豆普通細(xì)菌性疫病(common bacterial blight, CBB)嚴(yán)重威脅著普通菜豆的生產(chǎn), 已成為全世界普通菜豆生產(chǎn)中的主要病害[3]。菜豆普通細(xì)菌性疫病為種傳細(xì)菌性病害, 在普通菜豆全生育期內(nèi)皆可發(fā)生, 危害寄主植物的莖、葉、嫩莢和種子, 致使產(chǎn)量及品質(zhì)下降; 由該病導(dǎo)致的普通菜豆產(chǎn)量損失一般為 20%～60%, 嚴(yán)重時(shí)高達(dá)80%, 甚至絕產(chǎn)[4]。這就迫切要求我們鑒定抗菜豆普通細(xì)菌性疫病的優(yōu)異種質(zhì), 并從中挖掘優(yōu)異基因或有效功能標(biāo)記, 將其有效應(yīng)用于普通菜豆抗病分子育種。

迄今為止, 國內(nèi)外已報(bào)道定位了24個(gè)抗菜豆普通細(xì)菌性疫病的QTL[3,5-9], 分布于普通菜豆的11條連鎖群上[3,6,9]。其中, 位于染色體Pv07上的菜豆普通細(xì)菌性疫病抗病位點(diǎn)最多, 有 5個(gè)[10-12]; 而在染色體Pv01[11]、Pv04[7]、Pv06[7]和Pv10[10-11]上各檢測(cè)到的一個(gè)抗病位點(diǎn)。在已定位的抗病位點(diǎn)中, 對(duì)SAP6[13]、BC420[7]和SU91[14]的研究較為深入。SAP6來源于普通菜豆(Montana 5)[13], 而BC420、SU91均來源于普通菜豆的近緣種寬葉菜豆(PI 319443)[15]。SAP6位于染色體Pv10[13], 可解釋35%的表型變異[13]。BC420位于染色體Pv06[7], 可解釋62%～63%的表型變異[12]。SU91位于染色體 Pv08[14], 可解釋 14%～17%的表型變異[16]。由于已報(bào)道的24個(gè)抗性基因的分子標(biāo)記多為顯性標(biāo)記如RAPD、SCAR標(biāo)記等, 不能鑒別雜合子和純合子, 限制了其在抗病分子育種中的應(yīng)用。在已定位的抗病基因中, 僅針對(duì)BC420和SU91基因開發(fā)了共顯性標(biāo)記且被廣泛應(yīng)用于普通菜豆的抗病分子育種[17]。為此, 需要定位克隆新的菜豆普通細(xì)菌性疫病抗性基因, 并開發(fā)共顯性標(biāo)記, 為抗病分子輔助選擇育種提供更多、更有效的分子標(biāo)記。

普通菜豆龍蕓豆5號(hào)在溫室和田間對(duì)我國菜豆普通細(xì)菌性疫病病菌Xap菌株XS2均表現(xiàn)出較好的抗性。目前, 關(guān)于龍蕓豆5號(hào)的抗病性遺傳分析研究尚未報(bào)道。本研究利用龍蕓豆5號(hào)與感病材料龍蕓豆4號(hào)配置的 F2雜交群體, 以 SSR分子標(biāo)記定位來源于龍蕓豆5號(hào)的抗病基因, 并為抗病分子育種提供有效的分子標(biāo)記。

1 材料與方法

1.1 試驗(yàn)材料

普通菜豆抗病品種龍蕓豆5號(hào)(龍5)和感病品種龍蕓豆4號(hào)(龍4)為黑龍江省農(nóng)業(yè)科學(xué)院的2個(gè)育成品種, 由中國農(nóng)業(yè)科學(xué)院作物科學(xué)研究所提供。以龍5為父本、龍4為母本, 通過雜交和自交獲得F2分離群體共785個(gè)單株, 用于表型鑒定、遺傳分析和抗病QTL定位。

取保存于-80℃超低溫冰箱的Xap菌株XS2于牛奶吐溫培養(yǎng)基[18]上活化 4～5 d (28℃±2℃), 然后轉(zhuǎn)入牛肉膏蛋白胨液體培養(yǎng)基中振蕩培養(yǎng)48 h (200轉(zhuǎn)min-1, 28℃±2℃), 用滅菌蒸餾水稀釋至菌液濃度為1×108cfu mL-1, 用于接種。

1.2 表型鑒定

以親本材料龍5為抗病對(duì)照、龍4為感病對(duì)照,將親本與F2群體播種于口徑為23 cm × 18 cm × 18 cm的花盆中, 每盆5粒, 每親本材料4盆, 控制溫室白天溫度為28℃±2℃、晚上溫度為20℃±2℃, 待第一片三出復(fù)葉完全展開后采用針刺葉片法接種[19]。接種 14 d后調(diào)查接種植株葉片的發(fā)病情況, 參照Zapata等[20]評(píng)級(jí)標(biāo)準(zhǔn), 調(diào)查記錄每株接種葉片的發(fā)病嚴(yán)重度, 并計(jì)算親本龍5和龍4的平均發(fā)病級(jí)別,利用軟件SAS 9.1對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。

1.3 基因組DNA提取及PCR擴(kuò)增

采用改良CTAB法[21]提取龍5、龍4及F2單株的基因組DNA。用于遺傳分析的SSR引物包括兩部分, 共計(jì)3186對(duì)。第1部分來源于已發(fā)表文獻(xiàn)[22-23],計(jì)381對(duì); 第2部分是本實(shí)驗(yàn)室基于普通菜豆全基因組序列[24]所開發(fā), 計(jì)2805對(duì)。上述SSR引物全部用于親本間的多態(tài)性分析, 選取在親本間有明顯多態(tài)性且?guī)颓宄囊锓治鯢2群體的遺傳連鎖性。

PCR擴(kuò)增體系為15 μL, 含20 ng模板DNA、0.2 μmol L-1正反引物(Invitrogen, USA)、0.25 mmol L-1dNTPs (dATP、dCTP、dGTP與dTTP)、1.5 μL的10×Taq緩沖液(含1.5 mmol L-1Mg2+)以及1 U DNA聚合酶。PCR擴(kuò)增程序?yàn)?5℃預(yù)變性5 min; 95℃變性30 s, 53℃退火45 s, 72℃延伸45 s, 循環(huán)35次; 最后72℃延伸10 min。PCR擴(kuò)增產(chǎn)物經(jīng)8%非變性聚丙烯酰胺凝膠電泳檢測(cè), 銀染法[25]染色后觀察記錄結(jié)果。

1.4 QTL定位與關(guān)聯(lián)性分析

利用QTL IciMapping v4.0[26]構(gòu)建F2群體的遺傳連鎖圖譜, 設(shè)置LOD≥3.0、最大遺傳距離30 cM, 選用Kosambi作圖函數(shù)[27]作圖。參照普通菜豆全基因組序列[24], 將連鎖群與染色體一一對(duì)應(yīng)。

結(jié)合構(gòu)建的遺傳連鎖圖譜和表型鑒定結(jié)果, 采用完備區(qū)間作圖法(inclusive composite interval mapping, ICIM)[28]檢測(cè)龍5中LOD值≥3.0的抗性基因位點(diǎn)。

利用 TASSEL 2.1軟件[29]中一般線性模型(general linear model, GLM)對(duì)目標(biāo)區(qū)段所在染色體上的SSR標(biāo)記與CBB抗感結(jié)果進(jìn)行標(biāo)記-性狀關(guān)聯(lián)分析, 結(jié)合QTL檢測(cè)結(jié)果確定與抗病性緊密關(guān)聯(lián)的位點(diǎn)。

2 結(jié)果與分析

2.1 表型鑒定結(jié)果

接種14 d后, 抗病親本龍5接菌葉片沒有出現(xiàn)感病癥狀, 表現(xiàn)出較強(qiáng)抗性(圖 1), 平均發(fā)病級(jí)別為3.88 (圖2); 感病親本龍4接菌葉片的接種區(qū)和對(duì)照區(qū)均枯萎變色(圖 1), 平均發(fā)病級(jí)別為 7.64 (圖 2),表現(xiàn)為感病。F2群體表現(xiàn)出明顯的抗感分離, 最大值超過了高值親本, 最小值低于低值親本, 表明性狀同時(shí)具有正向和負(fù)向超親優(yōu)勢(shì), 且呈連續(xù)分布(圖2), 其偏度(Skewness)值為 0.46, 峰度(Kurtosis)值為-0.30, 二者的絕對(duì)值均小于 0.5, 符合正態(tài)分布, 適合進(jìn)行QTL定位分析。

圖1 接種Xap菌株XS214 d后龍5與龍4的發(fā)病情況Fig. 1 Phenotype of Long 5 and Long 4 inoculated with Xap strain, XS2

圖2 接種Xap菌株XS214 d后F2群體的抗性等級(jí)分布Fig. 2 Frequency distribution of ratings in F2population for resistance to Xap strain, XS2

2.2 遺傳連鎖分析

利用3186對(duì)SSR引物擴(kuò)增親本龍5和龍4基因組 DNA表明, 有 228對(duì)引物在親本間具有多態(tài)性。利用這些多態(tài)性引物對(duì)785個(gè)F2單株進(jìn)行遺傳連鎖分析, 獲得含206個(gè)SSR標(biāo)記的遺傳連鎖圖譜,共分12個(gè)連鎖群, 其中最短連鎖群為Pv02, 僅含3個(gè)標(biāo)記、最長連鎖群為Pv08, 含35個(gè)標(biāo)記(圖3)。依據(jù)標(biāo)記的染色體信息, 確定每個(gè)連鎖群對(duì)應(yīng)的染色體, 染色體Pv03由于標(biāo)記密度不夠而斷開為2個(gè)連鎖群(Pv03a與Pv03b), 其余連鎖群與其染色體一一對(duì)應(yīng)(圖3)。該遺傳圖譜總長度為1648.42 cM, 平均遺傳距離為8.00 cM; 標(biāo)記間最短遺傳距離為0.60 cM, 位于Pv06上的p6s267與p6s123標(biāo)記之間; 標(biāo)記間最大遺傳距離為 36.92 cM, 位于 Pv06上的p6s126與 p6s277標(biāo)記之間(圖 3); 標(biāo)記密度最大的是 Pv04連鎖群, 其標(biāo)記間平均遺傳距離只有5.65 cM。

2.3 抗病QTL定位與關(guān)聯(lián)性分析

結(jié)合遺傳連鎖圖譜和 F2表型鑒定結(jié)果, 利用軟件QTL IciMapping v4.0進(jìn)行ICIM定位分析, 接種后14 d僅在Pv06染色體上檢測(cè)到一個(gè)抗病QTL, 該位點(diǎn)位于分子標(biāo)記 p6s249與 p6s183之間(圖 4-A),其LOD值為5.93 (圖4-B), 共解釋4.61%的表型變異。此外, 在 Pv06染色體上檢測(cè)到的抗病 QTL加性效應(yīng)值為0.44, 說明增效基因來源于龍蕓豆5號(hào),且該抗病QTL能增強(qiáng)龍蕓豆5號(hào)對(duì)菜豆普通細(xì)菌性疫病的抗性。

利用GLM法對(duì)抗病QTL所在連鎖群(Pv06)上的所有標(biāo)記關(guān)聯(lián)性分析表明, 該連鎖群上有 11對(duì)SSR引物與 CBB發(fā)病等級(jí)顯著相關(guān)(P＜0.05), 其中個(gè)SSR標(biāo)記p6s302、p6s281、p6s267、p6s249、p6s210、PVBR163與CBB抗性的相關(guān)性達(dá)到P＜0.001的顯著水平(表 1)。結(jié)合本研究所檢測(cè)到的抗性 QTL的側(cè)翼標(biāo)記(p6s249與p6s183)(圖4-A), 發(fā)現(xiàn)與菜豆普通細(xì)菌性疫病抗性位點(diǎn)緊密關(guān)聯(lián)的標(biāo)記為 p6s249, 該引物在G19833基因組中預(yù)擴(kuò)增目標(biāo)片段為216 bp (表1)。p6s249標(biāo)記在龍5基因組中擴(kuò)增出的目標(biāo)片段位于201～238 bp之間, 在龍4擴(kuò)增出的片段位于238～242 bp之間(圖5)。

3 討論

由地毯草黃單胞桿菌菜豆致病變種[2]引起的菜豆普通細(xì)菌性疫病是影響全世界菜豆生產(chǎn)的一種主要病害[3]。研究表明菜豆普通細(xì)菌性疫病的抗性是受少數(shù)主效基因控制的數(shù)量性狀[8,12]。目前, 已報(bào)道的24個(gè)與CBB抗性相關(guān)的QTL中, 位于染色體Pv07上有 5個(gè)[10-12], Pv02上 4個(gè)[7,10-11], Pv08上 3個(gè)[5,14], Pv03[7,10]、Pv05[7]、Pv09[11]和 Pv11[10]上各 2 個(gè), Pv01[11]、Pv04[7]、Pv06[7]和Pv10[10-11]上各1個(gè)。這些抗病QTL中, 位于Pv06染色體上的(BC420)因其具有較高貢獻(xiàn)率而得到深入研究[6,17]。

本研究在Pv06染色體上檢測(cè)到一個(gè)抗Xap菌株XS2的 QTL, 該抗病位點(diǎn)位于分子標(biāo)記 p6s249與p6s183之間(圖4-A), LOD值為5.93 (圖4-B), 貢獻(xiàn)率為 4.61%, 抗病效應(yīng)值較低, 可能將在培育穩(wěn)定持久的抗菜豆普通細(xì)菌性疫病的品種中發(fā)揮作用。依據(jù)SSR引物p6s249與p6s183在G19833物理圖譜上的位置可初步斷定本研究獲得的抗病QTL位于Pv06染色體28～30 Mb的物理距離處(圖4-C)。根據(jù)Shi等[17]所確定的位于 Pv06染色體上的抗性基因BC420的候選基因序列比對(duì)分析, 該抗病位點(diǎn)位于第6染色體4 Mb的物理距離處, 與本研究檢測(cè)到的抗病位點(diǎn)相距較遠(yuǎn)(圖4-C), 因此我們所發(fā)現(xiàn)的抗病位點(diǎn)可能是一個(gè)來源于普通菜豆的新抗病位點(diǎn)。

圖3 利用F2群體構(gòu)建的SSR分子標(biāo)記遺傳連鎖圖譜Fig. 3 Linkage map of F2population with SSR markers

圖4 F2群體中抗病QTL的定位Fig. 4 Mapping of QTL for CBB resistance in F2populationB: 紅線代表在Pv06上檢測(cè)到的抗病QTL的LOD值。B: The red line means the LOD score of the major QTL for CBB resistance on chromosome Pv06.

圖5 p6s249在龍5、龍4和F2基因組中的擴(kuò)增結(jié)果Fig. 5 Amplification products of p6s249 in Long 5, Long 4, and F2populationA: p6s249在F2中擴(kuò)增出的與龍5一致的帶型; B: p6s249在F2中擴(kuò)增出的與龍4一致的帶型; H: p6s249在F2中擴(kuò)增出的雜合帶型; P1: p6s249在龍5中擴(kuò)增出的帶型; P2: p6s249在龍4中擴(kuò)增出的帶型。A: products of p6s249 in F2consistent with Long 5; B: products of p6s249 in F2consistent with Long 4; H: products of the hybrid in F2by p6s249; P1: products of p6s249 in Long 5; P2:products of p6s249 in Long 4.

表1 SSR引物與CBB抗性間的關(guān)聯(lián)分析Table 1 Testing of association between SSR primers and CBB resistance

利用與目的基因緊密連鎖的 DNA標(biāo)記進(jìn)行分子標(biāo)記輔助選擇有利于加快作物育種進(jìn)程、提高育種效率[30]。目前, DNA標(biāo)記已被廣泛應(yīng)用于水稻[31]、玉米[32]、小麥[33]等作物的分子育種。在眾多的DNA標(biāo)記中, SSR標(biāo)記因其具有多態(tài)性高、共顯性、數(shù)量豐富、信息量高、重復(fù)性好、DNA需求量低、易檢測(cè)、簡單經(jīng)濟(jì)等優(yōu)點(diǎn), 成為作物分子標(biāo)記輔助選擇育種中最好用的一種標(biāo)記類型[34]。此外, 由DNA的特殊序列標(biāo)記(如RFLP、RAPD等)轉(zhuǎn)換而來的STS、SCAR和SNP等標(biāo)記也同樣被廣泛應(yīng)用于分子標(biāo)記輔助選擇育種[34]。分子標(biāo)記輔助選擇育種技術(shù)在菜豆普通細(xì)菌性疫病抗病育種中也得到應(yīng)用, 特別是3個(gè)重要的SCAR標(biāo)記BC420、SU91和SAP6已被用于菜豆普通細(xì)菌性疫病的 MAS育種[6,35]。然而,由于SCAR標(biāo)記同其他共顯性標(biāo)記一樣不能區(qū)分雜合位點(diǎn), 在一定程度上限制了它的應(yīng)用。Shi等[17]開發(fā)了共顯性候選基因標(biāo)記 BC420-CG14和 SU91-CG11來代替SU91和BC420用于CBB抗病育種。本研究獲得了一個(gè)與CBB極顯著關(guān)聯(lián)的SSR標(biāo)記p6s249, 該標(biāo)記所在位點(diǎn)遠(yuǎn)離 BC420, 為一個(gè)新抗病位點(diǎn)連鎖的標(biāo)記; 此外, 該標(biāo)記來源于普通菜豆,更易于普通菜豆育種選擇, 所以p6s249可能有助于今后的MAS抗病育種。

4 結(jié)論

定位了 1個(gè)普通菜豆抗菜豆普通細(xì)菌性疫病QTL, 該位點(diǎn)位于標(biāo)記p6s249與p6s183之間, 共解釋 4.61%的表型變異率, 效應(yīng)值較小, 將在培育穩(wěn)定持久抗菜豆普通細(xì)菌性疫病品種中發(fā)揮作用。p6s249與菜豆普通細(xì)菌性疫病抗性極顯著關(guān)聯(lián)(P＜0.001), 可被用于抗病分子育種。

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Mapping of Common Bacterial Blight Resistance Gene in Common Bean

ZHU Ji-Feng, WU Jing, WANG Lan-Fen, ZHU Zhen-Dong, and WANG Shu-Min*
Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Common bacterial blight disease is a serious disease affecting the production of common bean (Phaseolus vulgaris L.) worldwide. The common bean germplasm Longyundou 5 is the main cultivar carrying common bacterial blight resistance gene in Heilongjiang province. To study the genetic mechanisms behind this resistance, we constructed 785 F2plants from Longyundou 5. Linkage analysis was performed on this population by using SSR markers. A linkage map covering 1648.42 cM with an average marker distance of 8.00 cM among 206 SSR markers was constructed. This map contained 12 linkage groups with a mean group length of 137.37 cM and the number of loci ranging from three to thirty-five. Combined with the results of phenotypic evaluation, QTL analysis was performed by the inclusive composite interval mapping method with QTL IciMapping v4.0. A QTL was detected between p6s249 and p6s183 on chromosome Pv06, with an additive effect of 0.44, which means the favourable gene of this locus is from Longyundou 5. The LOD score of the QTL was 5.93 and the total phenotypic variation at 14 days after inoculation was 4.61%. These results showed the effect of the QTL was low, which will play an important role in breeding durable resistant varieties of common bean. Association analysis between 11 SSR primers linked with common bacterial blight resistance and the disease rating showed that SSR marker p6s249 is excellently associated (P＜0.001) with common bacterial blight resistance and can be used for marker assisted selection.

Phaseolus vulgaris L; Common bacterial blight; SSR; QTL; Association analysis

10.3724/SP.J.1006.2017.00001

本研究由國家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(xiàng)(CARS-09), 國家科技支撐計(jì)劃項(xiàng)目(2013BAD01B03-18a)和中國農(nóng)業(yè)科學(xué)院科技創(chuàng)新工程項(xiàng)目資助。

This study was supported by the China Agriculture Research System (CARS-09), the National Key Technology R&D Program of China (2013BAD01B03-18a), and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.

*通訊作者(Corresponding author): 王述民, E-mail: wangshumin@caas.cn

聯(lián)系方式: E-mail: zhujifeng0224@163.com

稿日期): 2016-05-04; Accepted(接受日期): 2016-07-11; Published online(

日期): 2016-07-28.

URL: http://www.cnki.net/kcms/detail/11.1809.S.20160728.0816.006.html