劉　靜,喬麟軼,,張曉軍,李　欣,詹海仙,郭慧娟,張小輝,馮建寧,暢志堅,

(1.山西大學(xué) 研究生院,山西 太原　030006;2.山西省農(nóng)業(yè)科學(xué)院 作物科學(xué)研究所,作物遺傳與分子改良山西省重點實驗室,山西 太原　030031;3.山西農(nóng)業(yè)大學(xué) 研究生院,山西 太谷　030801)

?

小麥抗白粉病基因Pm43定位區(qū)段內(nèi)RGA分析

劉靜1,喬麟軼1,2,張曉軍2,李欣2,詹海仙2,郭慧娟2,張小輝1,馮建寧3,暢志堅1,2

(1.山西大學(xué) 研究生院,山西 太原030006;2.山西省農(nóng)業(yè)科學(xué)院 作物科學(xué)研究所,作物遺傳與分子改良山西省重點實驗室,山西 太原030031;3.山西農(nóng)業(yè)大學(xué) 研究生院,山西 太谷030801)

利用普通小麥測序草圖可從基因組范圍內(nèi)對小麥單條染色體上的某個區(qū)段進行分析。Pm43是作物遺傳與分子改良山西省重點實驗室在小麥2D染色體長臂上定位的一個抗白粉病基因。利用信息學(xué)方法分析Pm43所在物理圖譜、遺傳圖譜和基因組圖譜上的位置,可為其精細(xì)定位乃至候選基因的確定提供參考。試驗采用Pm43兩側(cè)標(biāo)記序列進行比對,將Pm43定位于染色體C-2DL3-0.49區(qū)間的79～99 cM內(nèi),所在基因組區(qū)段為2DL_9835990～2DL_9823315。利用目前已克隆小麥抗病基因的保守基序作為探針,從目標(biāo)區(qū)段內(nèi)檢索出89條包含抗病基因類似物(Resistance gene analogues,RGA)序列的scaffold,其中,36條scaffold被診斷出含有SSR位點,之后針對SSR位點開發(fā)分子標(biāo)記。利用攜帶有Pm43的普通小麥材料CH5025、感白粉病材料臺長29以及CH5025×臺長29 的F2作圖群體的抗感池DNA,對開發(fā)的SSR標(biāo)記進行連鎖性檢測,共篩選出4個多態(tài)性標(biāo)記,從而將目標(biāo)區(qū)段進一步確定在標(biāo)記PK_9908430和NBS_9908778之間。最后經(jīng)聚類分析,篩選出與已克隆Pm基因同源性較高的1個PK序列和1個NBS序列,且在粗山羊草2D染色體和水稻第4染色體中均存在與這2個序列同源的RGA表達(dá)序列。

Pm43;小麥基因組;2D染色體;PK;NBS

隨著小麥種植密度的加大和水肥條件的改善,小麥白粉病已成為我國各大麥區(qū)普遍發(fā)生的主要病害。據(jù)統(tǒng)計,2015年全國小麥白粉病發(fā)病面積約為570萬hm2,造成小麥產(chǎn)量的嚴(yán)重?fù)p失[1]。而研發(fā)抗白粉病基因、選育抗病品種是減少其病害損失最為經(jīng)濟有效的方法[2]。

目前國際上正式命名的54個抗白粉病基因中,有5個基因已經(jīng)被克隆,其分別是來源于簇毛麥的Pm21[3]和普通小麥的Pm3b[4]、Pm3c[5]、Pm3f[6]、Pm3g[5]。其中,Pm21編碼蛋白激酶(Protein kinase,PK),Pm3b-g編碼一個核苷酸結(jié)合位點(Nucleotide binding site,NBS)結(jié)構(gòu)域。PK和NBS是廣泛存在于植物界的2種保守抗病結(jié)構(gòu),在植株抵御病原菌侵染過程中起著關(guān)鍵作用[7]。

Pm43是作物遺傳與分子改良山西省重點實驗室于2009年在小麥2D染色體長臂上定位的一個抗白粉病基因[8-9],距其兩側(cè)分子標(biāo)記Xwmc41和Xbarc11的遺傳距離分別為2.3,4.2 cM。2014年普通小麥測序草圖[10]的公布,使得從基因組范圍內(nèi)分析單條染色體上的某個區(qū)段成為可能。本試驗利用小麥基因組測序數(shù)據(jù),對Pm43初定位區(qū)段內(nèi)具有完整序列的抗病基因類似物(Resistance gene analogues,RGA)進行分析,以期為候選基因的確定提供參考依據(jù)。

1　材料和方法

1.1試驗材料

普通小麥抗白粉病材料CH5025(攜帶Pm43)和感白粉病材料臺長29的DNA模板以及CH5025×臺長29 F2作圖群體的抗感池均由作物遺傳與分子改良山西省重點實驗室提供。

1.2試驗方法

1.2.1目標(biāo)區(qū)段分析從URGI數(shù)據(jù)庫中下載小麥2DL染色體序列數(shù)據(jù)建立本地數(shù)據(jù)庫。利用Pm43的連鎖標(biāo)記序列檢索本地庫,同時在GrainGene數(shù)據(jù)庫中比對小麥2D染色體遺傳圖譜[11]和物理圖譜,以獲得Pm43在2DL染色體的區(qū)段位置信息。

1.2.2區(qū)段內(nèi)RGA序列的分離和檢測從本地庫中分離出目標(biāo)區(qū)段序列數(shù)據(jù),利用PK家族(Pfam登錄號:PF00069)和NBS家族(PF00931)的隱馬模型文件,分別檢索區(qū)段數(shù)據(jù)獲得氨基酸序列,設(shè)E≤1e-5。利用SMART服務(wù)器對檢索結(jié)果進行保守結(jié)構(gòu)域的檢查,參數(shù)為默認(rèn)值。

1.2.3RGA-SSR位點診斷、引物開發(fā)和PCR檢測利用SSRhurnter軟件查找RGA所在scaffold序列的SSR位點。利用Primer5軟件在SSR位點兩側(cè)設(shè)計引物(由生工公司合成)(表1)。

表1　與Pm43連鎖的4對RGA-SSR標(biāo)記

PCR總體系為15 μL:含1.5 μL 10× Buffer (TaKaRa),0.2 mmol/L dNTP(0.3 μL),1 U(0.15 μL)Taq酶(TaKaRa)、0.25 μmol/L(2 μL)引物和100 ng模板DNA(1.5 μL)、H2O(9.55 μL)。反應(yīng)擴增程序為:94 ℃預(yù)變性5 min;94 ℃變性45 s,58 ℃復(fù)性45 s,72 ℃延伸 1 min,共36個循環(huán);72 ℃延伸10 min。對擴增產(chǎn)物用8%非變性聚丙烯酰胺凝膠(Acr與Bis質(zhì)量比為29∶1)電泳,經(jīng)硝酸銀染色后觀察照相。

1.2.4序列聚類分析和共線性分析用ClustalX軟件進行蛋白序列多重比對,采用鄰接法(Neighbor-joining)構(gòu)建系統(tǒng)發(fā)生樹,用MEGA軟件輸出圖形,設(shè)Bootstrap值為1 000。利用RGA序列分別檢索粗山羊草GIGA數(shù)據(jù)庫和水稻RGAP數(shù)據(jù)庫,以獲得共線性RGA序列相關(guān)信息。

2　結(jié)果與分析

2.1Pm43所在區(qū)段確定

區(qū)段分析結(jié)果表明,Pm43(圖1-A)位于2D染色體物理圖譜的C-2DL3-0.49區(qū)間(圖1-B)及遺傳圖譜[11]的79～99 cM內(nèi)(圖1-C)。Pm43兩側(cè)最近的分子標(biāo)記Xwmc41和Xbarc11[9]分別位于2DL基因組[10]序列2DL_9835990和2DL_9823315上,由此確定了Pm43所在基因組區(qū)段(圖1-D)。

圖1　Pm43(A)所在2DL物理圖譜(B)、遺傳圖譜(C)和基因組圖譜(D)的區(qū)段分析

2.2區(qū)段內(nèi)RGA-SSR標(biāo)記開發(fā)與連鎖性檢測

通過信息學(xué)檢索,從Pm43所在基因組區(qū)段共獲得89條包含RGA的基因組序列(scaffold),其中,64條含PK序列,25條含NBS序列。SSR診斷結(jié)果表明,上述RGA-scaffold中有36條含有簡單重復(fù)序列位點,從中隨機取1/2序列,根據(jù)序列上SSR位點設(shè)計引物,共開發(fā)出18對RGA-SSR標(biāo)記,以“RGA名稱+scaffold編號”暫時命名,這些標(biāo)記線性排列于Pm43基因組區(qū)段內(nèi)(圖2-A)。

經(jīng)過連鎖性檢測,PK_9908430、NBS_9862754、NBS_9906982和NBS_9908778在抗感親本間和抗感池間均表現(xiàn)出多態(tài)性(圖2-B、圖3),初步推斷,這4對RGA-SSR標(biāo)記可能與Pm43連鎖。

*.序列能在植株中表達(dá);Pr.抗病親本CH5025;Ps.感病親本臺長29;Br.抗病池;Bs.感病池。

M.500 bp DNA ladder;Pr.抗病親本CH5025;Ps.感病親本臺長 29;Br.抗病池;Bs.感病池。

2.3小區(qū)段內(nèi)RGA聚類和共線性分析

進一步檢索發(fā)現(xiàn),在多態(tài)性標(biāo)記PK_9908430和NBS_9908778之間的基因組范圍內(nèi)共存在22個PK序列和7個NBS序列。聚類分析結(jié)果表明,PK序列中Ta2dlLoc010614與已克隆的Pm21相似性最高(圖4-A),NBS序列中Ta2dlLoc017419與Pm3b、Pm3c、Pm3f、Pm3g的相似性最高(圖4-B)。

由這2個RGA序列檢索粗山羊草和水稻數(shù)據(jù)庫可知,PK序列Ta2dlLoc010614與粗山羊草2D染色體上的PK序列AEGTA01197、水稻第4染色體上的PK序列Os04g39180同源,NBS序列Ta2dlLoc017419也分別在粗山羊草2D和水稻第4染色體上找到了同源序列(圖2-D、E),表明Pm43區(qū)段序列與粗山羊草2D染色體和水稻第4染色體對應(yīng)區(qū)段具有較好的共線性。更重要的是,對應(yīng)的AetRGA和OsRGA均為在植株中表達(dá)的序列,由此推斷Ta2dlLoc010614和Ta2dlLoc017419很可能也在小麥抗病生理過程中發(fā)揮作用。

圖4　Pm43小區(qū)段內(nèi)PK序列(A)和NBS序列(B)的聚類分析

3　結(jié)論與討論

3.1利用小麥基因組數(shù)據(jù)分析Pm43所在區(qū)段的可行性

普通小麥基因組草圖的完成,對于抗病基因的精細(xì)定位乃至克隆有著巨大的推動作用。Pm43是源于八倍體小偃麥的一個抗病基因,與其他近緣屬相比,偃麥草染色體(尤其是J/Js染色體)與小麥染色體存在很高的同源性[12-13],容易發(fā)生交換,且交換后的染色體在其結(jié)構(gòu)上更接近于小麥。因此,最初與小麥發(fā)生交換的外源片段本身與小麥基因組存在一定程度的相似性,加之與普通小麥品種歷經(jīng)10余年的雜交、回交過程,目前在攜帶Pm43的材料中用基因組原位雜交(GISH)技術(shù)已經(jīng)檢測不到任何外源信號,表明Pm43及其兩側(cè)現(xiàn)存的外源序列已經(jīng)與小麥基因組高度相似。此外,RGA相關(guān)結(jié)構(gòu)在植物界中十分保守,因此,對Pm43所在小麥基因組區(qū)段內(nèi)具有抗病結(jié)構(gòu)的序列進行分析具有可行性。

3.2目標(biāo)區(qū)段內(nèi)RGA分析

PK和NBS是植物界中最重要的兩類抗病結(jié)構(gòu),典型的PK類抗病基因有大麥Rpg1[14]、番茄Pti1[15]、水稻Xa3/21/26[16]等,而NBS類抗病基因更是數(shù)量龐大,僅在小麥中目前已經(jīng)克隆了Pm3b[4]、Pm3c[5]、Pm3f[6]、Pm3g[5]、Yr10[17]、Lr1[18]、Lr10[19]、Lr21[20]、Sr33[21]、Sr35[22]、Cre1和Cre3[23]12個抗病基因,此外,Lr24[24]和Lr35[25]也被證實可能為NBS類基因。本試驗利用信息學(xué)方法對Pm43所在基因組區(qū)段內(nèi)包含PK或NBS結(jié)構(gòu)的scaffold進行檢索,隨后開發(fā)RGA-SSR標(biāo)記進行連鎖性檢測,將目標(biāo)區(qū)段進一步確定在標(biāo)記PK_9908430和NBS_9908778之間,從中選出了與已克隆Pm基因同源性較高的1個PK序列和1個NBS序列,這2個序列均能在粗山羊草和水稻基因組中找到可表達(dá)的同源RGA序列,因此,今后有必要對這些序列的功能進行深入研究,為Pm43候選基因的確定提供參考依據(jù)。

[1]全國農(nóng)業(yè)技術(shù)推廣服務(wù)中心.2015年全國小麥重大病蟲害發(fā)生趨勢預(yù)報[EB/OL].[2015-01-20].http://cb.natesc.gov.cn/sites/cb/List_28092_151760.html.

[2]Line R F,Chen X M.Success in breeding for and managing durable resistance to wheat rusts[J].Plant Disease,1995,79(12):1254-1255.

[3]Cao A,Xing L,Wang X,et al.Serine/threonine kinase geneStpk-V,a key member of powdery mildew resistance genePm21,confers powdery mildew resistance in wheat[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(19):7727-7732.

[4]Yahiaoui N,Srichumpa P,Dudler R,et al.Genome analysis at different ploidy levels allows cloning of the powdery mildew resistance genePm3bfrom hexaploid wheat[J].The Plant Journal:for Cell and Molecular Biology,2004,37(4):528-538.

[5]Tommasini L,Yahiaoui N,Srichumpa P,et al.Development of functional markers specific for seven Pm3 resistance alleles and their validation in the bread wheat gene pool[J].Theoretical and Applied Genetics,2006,114(1):165-175.

[6]Srichumpa P,Brunner S,Keller B,et al.Allelic series of four powdery mildew resistance genes at the Pm3 locus in hexaploid bread wheat[J].Plant Physiology,2005,139(2):885-895.

[7]Meyers B C,Kozik A,Griego A,et al.Genome-wide analysis of NBS-LRR-encoding genes inArabidopsis[J].The Plant Cell,2003,15(4):809-834.

[8]劉建霞,賀潤麗,暢志堅,等.源于中間偃麥草的小麥新品系CH5026白粉病抗性的遺傳[J].華北農(nóng)學(xué)報,2008,23(1):194-198.

[9]He R,Chang Z,Yang Z,et al.Inheritance and mapping of powdery mildew resistance genePm43 introgressed fromThinopyrumintermediuminto wheat[J].Theoretical and Applied Genetics,2009,118(6):1173-1180.

[10]International Wheat Genome Sequencing Consortium (IWGSC).A chromosome-based draft sequence of the hexaploid bread wheat(Triticumaestivum)genome[J].Science,2014,345:1251-1288.

[11]Somers D J,Isaac P,Edwards K.A high-density microsatellite consensus map for bread wheat (TriticumaestivumL.)[J].Theoretical and Applied Genetics,2004,109(6):1105-1114.

[12]Chen Q,Conner R L,Laroche A,et al.Molecular cytogenetic evidence for a high level of chromosome pairing among different genomes inTriticumaestivum-Thinopyrumintermediumhybrids[J].Theoretical and Applied Genetics,2001,102(6/7):847-852.

[13]閆金龍,暢志堅,孫美榮,等.中間偃麥草抗小麥白粉病基因?qū)爰捌淇剐栽u價[J].華北農(nóng)學(xué)報,2010,25(3):225-230.

[14]Brueggeman R,Rostoks N,Kudrna D,et al.The barley stem rust-resistance geneRpg1 is a novel disease-resistance gene with homology to receptor kinases[J].Proceedings of the National Academy of Sciences of the United States of America,2002,99(14):9328-9333.

[15]Zhou J,Loh Y T,Bressan R A,et al.The tomato genePti1 encodes a serine/threonine kinase that is phosphorylated by Pto and is involved in the hypersensitive response[J].Cell,1995,83(6):925-935.

[16]Sun X,Cao Y,Wang S.Point mutations with positive selection were a major force during the evolution of a receptor-kinase resistance gene family of rice[J].Plant Physiology,2006,140(3):998-1008.

[17]Liu W,Frick M,Huel R,et al.The stripe rust resistance geneYr10 encodes an evolutionary-conserved and unique CC-NBS-LRR sequence in wheat[J].Molecular Plant,2014,7(12):1740-1755.

[18]Cloutier S,Mccallum B D,Loutre C,et al.Leaf rust resistance geneLr1,isolated from bread wheat (TriticumaestivumL.) is a member of the large psr567 gene family[J].Plant Molecular Biology,2007,65(1/2):93-106.

[19]Sela H,Spiridon L N,Petrescu A J,et al.Ancient diversity of splicing motifs and protein surfaces in the wild emmer wheat (Triticumdicoccoides) LR10 coiled coil (CC) and leucine-rich repeat (LRR) domains[J].Molecular Plant Pathology,2012,13(3):276-287.

[20]Huang L,Brooks S A,Li W,et al.Map-based cloning of leaf rust resistance geneLr21 from the large and polyploid genome of bread wheat[J].Genetics,2003,164(2):655-664.

[21]Periyannan S,Moore J,Ayliffe M,et al.The geneSr33,an ortholog of barleyMlagenes,encodes resistance to wheat stem rust race Ug99[J].Science,2013,341(6147):786-788.

[22]Saintenac C,Zhang W,Salcedo A,et al.Identification of wheat geneSr35 that confers resistance to Ug99 stem rust race group[J].Science,2013,341(6147):783-786.

[23]De Majnik J,Ogbonnaya F C,Moullet O,et al.The cre1 and cre3 nematode resistance genes are located at homeologous loci in the wheat genome[J].Molecular Plant-microbe Interactions,2003,16(12):1129-1134.

[24]張立榮,楊文香,劉大群.小麥NBS類抗病基因類似序列的多樣性和進化關(guān)系研究[J].華北農(nóng)學(xué)報,2011,26(4):23-26.

[25]高倩,王海燕,劉大群,等.小麥抗葉銹病基因Lr35的RGA分析[J].華北農(nóng)學(xué)報,2008,23(6):50-53.

Analysis of Resistance Gene Analogues from the Section of Mildew Powdery Resistance GenePm43 in Wheat

LIU Jing1,QIAO Linyi1,2,ZHANG Xiaojun2,LI Xin2,ZHAN Haixian2,GUO Huijuan2,ZHANG Xiaohui1,FENG Jianning3,CHANG Zhijian1,2

(1.Graduate School of Shanxi University,Taiyuan030006,China;2.Institute of Crop Sciences,Shanxi Academy of Agricultural Sciences,Shanxi Key Laboratory of Crop Genetics and Molecular Improvement,Taiyuan030031,China;3.Graduate School of Shanxi Agricultural University,Taigu030801,China)

Draft sequencing data of common wheat can be used to analysis a target section of single chromosome from genome-wide in wheat.A powdery mildew resistance gene,Pm43,was assigned on the long arm of wheat 2D chromosome by Shanxi Key Laboratory of Crop Genetics and Molecular Improvement.Bioinformatics methods were used to determine the position ofPm43 in the physical map,genetic map and genome map in wheat.The results provided a reference to the fine mapping or the candidate genes determining ofPm43.By aligning the sequences of flanking markers,thePm43 was mapped on the 79-99 cM of C-2DL3-0.49 section,which was between 2DL_9835990 and 2DL_9823315 in genomic region.Using the conserved motifs of cloned resistance genes in wheat as a probe,89 RGA (Resistance gene analogues)-scaffolds were retrieved from the target region,and 36 scaffolds were containing SSR loci.Then SSR markers were developed.Applying the resistant (R) parent CH5025 carriedPm43,the susceptible (S) parent Taichang 29 and two R/S bulks of F2population crossed by CH5025 and Taichang 29,the linkage of developed SSR markers andPm43 was detected.Four polymorphic markers were screened and further defined the target region between PK_9908430 and NBS_9908778.Then by cluster analysis,one PK sequence and one NBS sequence which had the highest homology with clonedPmwere screened,and their homologous RGA expressed sequences were found fromAegilopstauschiiand rice genome.

Pm43;Wheat genome;Chromosome 2D;PK;NBS

2016-01-08

國家自然科學(xué)基金項目(31171839);山西省青年基金項目(2015021145);山西省科技攻關(guān)項目(20150311001-5);山西省國際合作項目(201603D421003);山西省農(nóng)科院攻關(guān)項目(15YGG01)

劉靜(1991-),女,山西柳林人,在讀碩士,主要從事小麥抗病基因研究。

暢志堅(1959-),男,山西萬榮人,研究員,碩士生導(dǎo)師,主要從事小麥抗病育種與種質(zhì)創(chuàng)新研究。

S435.121.4+6;Q78

A

1000-7091(2016)04-0026-05

10.7668/hbnxb.2016.04.005