張亞東,梁文化,赫磊,趙春芳,朱鎮(zhèn),陳濤,趙慶勇,趙凌,姚姝,周麗慧,路凱,王才林
水稻RIL群體高密度遺傳圖譜構(gòu)建及粒型QTL定位
張亞東,梁文化,赫磊,趙春芳,朱鎮(zhèn),陳濤,趙慶勇,趙凌,姚姝,周麗慧,路凱,王才林
江蘇省農(nóng)業(yè)科學(xué)院糧食作物研究所/江蘇省優(yōu)質(zhì)水稻工程技術(shù)研究中心/國(guó)家水稻改良中心南京分中心,南京 210014
【目的】水稻粒型是與產(chǎn)量直接相關(guān)的重要農(nóng)藝性狀,影響稻米的外觀品質(zhì)和商品價(jià)值。挖掘新的水稻粒型相關(guān)基因,對(duì)揭示水稻粒型調(diào)控的遺傳機(jī)理研究有重要意義,同時(shí)可為水稻粒型分子育種提供新的基因資源?!痉椒ā恳詷O端粒型差異的粳稻TD70和秈稻Kasalath,以及雜交構(gòu)建的186個(gè)家系的重組自交系群體為研究材料,利用高通量測(cè)序技術(shù)對(duì)親本和RIL株系進(jìn)行深度測(cè)序。統(tǒng)計(jì)186個(gè)RIL基因型數(shù)據(jù),利用滑動(dòng)窗法(SNP/InDel數(shù)目為15),將窗口內(nèi)SNP/InDel信息轉(zhuǎn)換成窗口的基因型,預(yù)測(cè)染色體上的重組斷點(diǎn)構(gòu)建RIL群體的BinMap遺傳圖譜,結(jié)合2年的粒長(zhǎng)、粒寬、粒厚和千粒重的表型數(shù)據(jù),運(yùn)用QTL IciMapping軟件,采用復(fù)合區(qū)間作圖法對(duì)RIL群體的4個(gè)性狀進(jìn)行QTL定位?!窘Y(jié)果】構(gòu)建了一張包含12 328個(gè)Bin標(biāo)記的高密度遺傳圖譜,該圖譜各染色體Bin標(biāo)記數(shù)為763—1 367個(gè),標(biāo)記間平均物理距離為30.26 kb。粒長(zhǎng)、粒寬、粒厚和千粒重4個(gè)性狀在RIL群體中呈近正態(tài)連續(xù)分布,且2年間的變化趨勢(shì)相似,符合QTL作圖要求。2018年對(duì)粒長(zhǎng)、粒寬、粒厚及千粒重進(jìn)行QTL分析,共檢測(cè)到40個(gè)粒型QTL,其中,粒長(zhǎng)12個(gè),粒寬9個(gè),粒厚8個(gè),千粒重11個(gè),2019年對(duì)粒長(zhǎng)、粒寬、粒厚及千粒重進(jìn)行QTL分析,檢測(cè)到56個(gè)籽粒相關(guān)的QTL,粒長(zhǎng)15個(gè),粒寬11個(gè),粒厚13個(gè),千粒重17個(gè)。分析定位到的96個(gè)粒型QTL位點(diǎn),連續(xù)2年都能檢測(cè)到的QTL位點(diǎn)有11個(gè),其中7個(gè)為已克隆的粒型基因位點(diǎn),4個(gè)為未知的新位點(diǎn),分別分布于第1、3、4、5染色體上,分別為粒長(zhǎng)和、粒厚、粒寬。【結(jié)論】構(gòu)建了一張包含12 328個(gè)Bin標(biāo)記的分子遺傳連鎖圖譜,解析大粒粳稻資源的粒型基因,獲得了、、、等4個(gè)新的粒型QTL,可用于后續(xù)粒型調(diào)控基因的精細(xì)定位及克隆研究。
水稻;Bin圖譜;粒型;QTL定位
【研究意義】水稻粒型是與產(chǎn)量性狀直接相關(guān)的重要農(nóng)藝性狀,影響稻米的外觀品質(zhì)和商品價(jià)值[1-3]。重要農(nóng)藝性狀(如產(chǎn)量、品質(zhì)性狀)QTL定位、克隆是高產(chǎn)優(yōu)質(zhì)水稻分子育種的基礎(chǔ)和前提;而連鎖圖譜,尤其是高密度遺傳連鎖圖譜,又是QTL定位、克隆的基礎(chǔ)。因此,構(gòu)建高密度遺傳連鎖圖譜對(duì)QTL定位和分子育種具有很大的實(shí)際意義[4]?!厩叭搜芯窟M(jìn)展】基于傳統(tǒng)方法的分子標(biāo)記(如RFLP、AFLP、SSR、CAPs、STS、ETS)構(gòu)建的遺傳圖譜存在標(biāo)記數(shù)量少、分布不均、覆蓋密度低等缺陷,對(duì)后續(xù)QTL精細(xì)定位和克隆極為不利[5-6]。隨著高通量測(cè)序技術(shù)發(fā)展出來的SNP、Indel等標(biāo)記,由于遺傳穩(wěn)定性高、分布廣泛、多樣性高、數(shù)量大等特點(diǎn),在水稻復(fù)雜數(shù)量性狀研究中得到了高度重視和廣泛應(yīng)用。隨著下一代測(cè)序(next-generation sequencing,NGS)技術(shù)的發(fā)展,快速高效地進(jìn)行SNP大規(guī)模開發(fā)及基因型分析的成本越來越低、質(zhì)量越來越高。取一定數(shù)量的連續(xù)SNP標(biāo)記作為判斷染色體重組事件的最小單位(recombination bin),判斷子代每個(gè)Bin的來源,得到每個(gè)子代的全基因組物理圖譜,從而構(gòu)建出的遺傳圖譜稱之為Bin圖譜[7]。Bin圖譜是基于SSR/RFLP標(biāo)記的傳統(tǒng)遺傳圖譜后的新一代遺傳圖譜,通過高通量測(cè)序進(jìn)行,自動(dòng)化程度高、構(gòu)建時(shí)間短、精確度高,可以直接進(jìn)行QTL定位后續(xù)的候選篩選和分子標(biāo)記的直接開發(fā)。Bin圖譜已成功應(yīng)用于谷子[8]、水稻[9]、玉米[10]等的QTL定位中。近年來,已有多個(gè)控制粒型的基因被克隆,幾乎分布于水稻所有染色體上。證實(shí)了、、、等通過MAPK信號(hào)途徑[11-15];、、、、等通過泛素蛋白酶體降解途徑[16-20];、、等通過G蛋白信號(hào)通路[21-24];、、等通過植物激素途徑[25-27];、、、、、、等通過轉(zhuǎn)錄調(diào)控途徑來調(diào)控水稻粒型[28-35]?!颈狙芯壳腥朦c(diǎn)】目前,粒型基因克隆較多,但已知水稻粒型仍不足以解釋其復(fù)雜的分子調(diào)控機(jī)制。【擬解決的關(guān)鍵問題】本研究利用重測(cè)序技術(shù),對(duì)特大粒粳稻TD70和小粒秈稻Kasalath構(gòu)建的包含186個(gè)株系RIL群體為作圖群體,采用基因分型測(cè)序(genotyping-by-sequencing,GBS)技術(shù)構(gòu)建Bin標(biāo)記的高密度遺傳圖譜,對(duì)粒型QTL進(jìn)行檢測(cè),以期鑒定到新的、可穩(wěn)定遺傳的粒型QTL,為水稻粒型基因的克隆、功能解析及分子育種提供依據(jù)。
用來源于天鵝谷///9520//(72-496/御糯)后代粒重超親本的粳型超大粒品系TD70和秈稻小粒型品種Kasalath雜交,通過單粒傳法,從F2代開始構(gòu)建TD70/Kasalath的重組自交系群體。群體包含186個(gè)株系,基因型鑒定為F8世代,表型調(diào)查為F9—F10世代。上述材料于2018—2019年種植在江蘇省農(nóng)業(yè)科學(xué)院試驗(yàn)田,每行8株,行距為26.7 cm,株距為16.7 cm(單苗種植),田間管理與大田相同。
稻谷成熟后,親本及每個(gè)RIL株系按單株收取5個(gè)植株的種子進(jìn)行粒型考察。每個(gè)單株隨機(jī)挑選10粒飽滿種子使用游標(biāo)卡尺(精度0.01 mm)測(cè)量粒長(zhǎng)、粒寬和粒厚,千粒重則利用電子天平(精度0.001 g)測(cè)定單株1 000粒風(fēng)干種子的重量。每個(gè)性狀以5株的平均值為最終的表型值。
2018年在水稻分蘗盛期采取幼嫩的葉片,進(jìn)行親本和RIL群體單株DNA的提取。用1%瓊脂糖凝膠電泳檢測(cè)基因組DNA完整性,用Nanodrop2000微量核酸蛋白檢測(cè)儀檢測(cè)DNA的濃度與純度。質(zhì)量檢測(cè)合格的DNA,采用NEB Next? UltraTMII DNA文庫(kù)制備試劑盒進(jìn)行測(cè)序文庫(kù)的構(gòu)建,委托安諾優(yōu)達(dá)基因科技(北京)有限公司負(fù)責(zé)完成測(cè)序,采用的測(cè)序平臺(tái)為NovaSeq 6000,測(cè)序模式為Paired-end 150 bp。測(cè)序得到原始數(shù)據(jù),用軟件包FastQC(Ver 0.11.9)和fastp(Ver 0.20.0)對(duì)原始測(cè)序數(shù)據(jù)進(jìn)行質(zhì)量評(píng)估和質(zhì)量過濾,主要去除接頭污染的reads,低質(zhì)量的reads以及含N比例大于5%的reads等,得到后續(xù)分析所用的高質(zhì)量Clean Reads。
基于二代高通量測(cè)序,對(duì)親本TD70和Kasalath及186個(gè)RIL株系進(jìn)行深度測(cè)序。TD70和Kasalath的Reads與Nipponbare(ssp.)參考基因組的比對(duì)率分別為98.7%和97.2%,分別得到121 491 700和131 284 874個(gè)clean reads,測(cè)序量為18.22和19.69 Gb,平均測(cè)序深度分別為40×和44×。RIL群體共獲得數(shù)據(jù)1 304 Gb的測(cè)序數(shù)據(jù),每個(gè)株系獲得的數(shù)據(jù)量為5.10—12.37 Gb,平均每個(gè)株系獲得7.01 Gb的數(shù)據(jù),測(cè)序深度為18.80×。測(cè)序質(zhì)量評(píng)估顯示,質(zhì)控后Q30最低為93.45%,平均為94.53%。從以上的數(shù)據(jù)統(tǒng)計(jì)顯示測(cè)序數(shù)據(jù)產(chǎn)量和質(zhì)量均較好可以進(jìn)行下一步分析。用軟件BWA軟件將測(cè)序reads與水稻日本晴參考基因組(IRGSP-1.0)進(jìn)行對(duì)比。保留唯一比對(duì)的雙端reads,通過GATK與Freebayes檢測(cè)雙親間的SNP位點(diǎn)。對(duì)186個(gè)RIL基因型數(shù)據(jù)進(jìn)行統(tǒng)計(jì),利用滑動(dòng)窗法(SNP/InDel數(shù)目為15),將窗口內(nèi)SNP/InDel信息轉(zhuǎn)換成窗口的基因型,通過窗口周圍的基因型信息,對(duì)錯(cuò)誤的基因型進(jìn)行修正。進(jìn)一步基于根據(jù)窗口基因型,預(yù)測(cè)染色體上的重組斷點(diǎn)構(gòu)建的RIL群體的BinMap圖譜。
分別用2018和2019年RIL群體的粒長(zhǎng)、粒寬、粒厚及千粒重表型進(jìn)行QTL定位。采用軟件QTL IciMapping(Ver 4.2.53)設(shè)定PIN為0.001,步長(zhǎng)為1 cM,采用完備區(qū)間作圖(inclusive composite interval mapping,ICIM)的方法檢測(cè)全基因組內(nèi)的粒型QTL[36];LOD閾值設(shè)定為2.5,以LOD峰值作為該QTL的LOD值,以LOD峰值位置的Bin標(biāo)記估算QTL的效應(yīng),遵循McCouch[37]的原則命名QTL。
對(duì)2018和2019年親本TD70、Kasalath及186個(gè)RIL株系進(jìn)行粒長(zhǎng)、粒寬、粒厚和千粒重的考察。結(jié)果顯示,TD70、Kasalath和RIL株系粒型存在極顯著差異(圖1)。RIL群體2018年粒長(zhǎng)平均為9.72 mm,變幅為7.77—13.00 mm;粒寬平均為3.12 mm,變幅為2.37—4.34 mm;粒厚平均為2.10 mm,變幅為1.71—2.63 mm;千粒重平均為30.60 g,變幅為17.95—55.4 g。
P1:TD70;P2:Kasalath;1—14:RIL群體部分株系
綜合2018—2019年數(shù)據(jù),發(fā)現(xiàn)粒長(zhǎng)、粒寬和粒厚性狀在RIL群體中存在超親分離現(xiàn)象,但均值都在2個(gè)親本表型值范圍內(nèi),變異系數(shù)為7.35— 22.68(表1)。用SPSS(Ver 22.0)軟件對(duì)各性狀的正態(tài)性檢驗(yàn)結(jié)果顯示,粒長(zhǎng)、粒寬、粒厚和千粒重4個(gè)性狀在RIL群體中呈近正態(tài)連續(xù)分布,且2年間的變化趨勢(shì)相似,符合QTL作圖要求(圖2)。
表1 親本與RIL群體2年間粒型的表型變異
GL:粒長(zhǎng);GW:粒寬;GT:粒厚;TGW:千粒重。下同
GL: grain length; GW: grain width; GT: grain thickness; TGW: 1000-grain weight. The same as below
GL:粒長(zhǎng);GW:粒寬;GT:粒厚;TGW:千粒重;T:TD70;K:Kasalath
基于RIL群體186個(gè)株系的重測(cè)序PE reads,利用短序列比對(duì)軟件BWA將PE reads比對(duì)到日本晴參考基因組上,過濾掉多位點(diǎn)和缺失率大于80%的SNP數(shù)據(jù),保留唯一比對(duì)的SNP作圖,以提高作圖效率和精度。參考Huang等[7]方法用過濾后的SNP構(gòu)建Bin標(biāo)記。采用滑動(dòng)窗法,將窗口內(nèi)SNP/InDel信息轉(zhuǎn)換成窗口的基因型,通過窗口周圍的基因型信息,對(duì)錯(cuò)誤的基因型進(jìn)行修正。進(jìn)一步基于根據(jù)窗口基因型,預(yù)測(cè)染色體上的重組斷點(diǎn),共得到12 328個(gè)Bin標(biāo)記,標(biāo)記為RBN0001— RBN12328,每條染色體Bin標(biāo)記數(shù)為763—1 367個(gè),平均為1 027個(gè)。
將Bin區(qū)段基因型數(shù)據(jù)導(dǎo)入軟件R/qtl進(jìn)行遺傳圖譜的構(gòu)建。該遺傳圖譜總長(zhǎng)為21 295.44 cM,包含12個(gè)連鎖群,分別對(duì)應(yīng)水稻的12條染色體,各染色體遺傳距離為1 006.01—2 400.93 cM,其中第4染色體遺傳距離最大,第10染色體的遺傳距離最短,染色體平均遺傳距離為1 774.62 cM;第7染色體Bin標(biāo)記間平均遺傳距離最大,為2.13cM,第3染色體Bin標(biāo)記間距離最小,僅為1.22 cM,整個(gè)染色體組的標(biāo)記間距離為1.71 cM。圖譜標(biāo)記間平均物理距離為30.26 kb(表2和圖3)。
2.3.1 2018年粒型QTL定位 對(duì)2018年種植材料的
粒長(zhǎng)、粒寬、粒厚及千粒重進(jìn)行QTL分析,共檢測(cè)到40個(gè)粒型QTL,其中粒長(zhǎng)12個(gè),粒寬9個(gè),粒厚8個(gè),千粒重11個(gè)(圖4)。這些QTL除第6染色體外,其他11條染色體均有分布。其中,第3染色體上的QTL最多,為9個(gè),其次是第2染色體,為7個(gè),第8染色體僅檢測(cè)到1個(gè)。這些QTL的LOD值為2.88—35.15,單個(gè)QTL的貢獻(xiàn)率為1.61%—33.33%。檢測(cè)到的12個(gè)粒長(zhǎng)QTL,LOD值為3.59—27.88,貢獻(xiàn)率最高為21.26%。共檢測(cè)到9個(gè)粒寬相關(guān)的QTL,其中有3個(gè)QTL位于第4染色體。這些QTL LOD值為3.05—35.15,其中第2染色體上的位點(diǎn)LOD值為35.15,貢獻(xiàn)率為28.82%,第5染色體上的位點(diǎn)LOD值為32.44,可解釋25.59%的表型變異。粒厚相關(guān)QTL共檢測(cè)到8個(gè),其中第3染色體上檢測(cè)到3個(gè)。LOD值為2.88—31.44,表型貢獻(xiàn)率為2.10%—33.33%。千粒重共檢測(cè)到11個(gè)QTL,位于第3染色體上的對(duì)表型貢獻(xiàn)率最大為31.19%,其次為第2染色體上的位點(diǎn)可解釋14.67%的表型變異。以上QTL的增效等位基因主要來自大粒親本TD70,說明大粒親本中的QTL位點(diǎn)對(duì)籽粒大小具有明顯的增效作用(表3)。
表2 Bin圖譜信息表
圖3 Bin標(biāo)記構(gòu)建的遺傳連鎖圖譜
黑色位點(diǎn)為2018年定位的粒型QTL,綠色位點(diǎn)為2019年定位的粒型QTL,紅色位點(diǎn)表示2018和2019年均定位的粒型QTL
2.3.2 2019年粒型QTL定位 對(duì)2019年種植材料的粒長(zhǎng)、粒寬、粒厚及千粒重進(jìn)行QTL分析,檢測(cè)到56個(gè)籽粒相關(guān)的QTL,粒長(zhǎng)15個(gè),粒寬11個(gè),粒厚13個(gè),千粒重17個(gè)(圖4)。這些QTL的LOD值為2.53—33.76,單個(gè)QTL解釋的表型變異為1.02%—30.07%。這些QTL分布在水稻的12條染色體上,第2染色體上最多,為11個(gè),其次是第3染色體,為8個(gè),第9染色體上最少,為1個(gè)。在第3和第7染色體上各檢測(cè)到3個(gè)粒長(zhǎng)QTL。這些QTL的LOD值為2.58—33.76,單個(gè)QTL的貢獻(xiàn)率為1.02%—21.17%。其中第3染色體上、和第7染色體上的位點(diǎn),分別可以解釋15.44%、12.55%和21.17%的表型變異,加性效應(yīng)分別為0.456、0.444和0.529。粒寬QTL的LOD值范圍為2.53—32.03,貢獻(xiàn)率為1.60%—30.07%。其中位點(diǎn)LOD值為32.03,可解釋30.07%的表型變異,加性效應(yīng)為0.214。13個(gè)粒厚QTL的LOD值范圍為3.21—17.82,對(duì)粒厚的貢獻(xiàn)率為2.51%—16.27%。其中第2染色體上的和位點(diǎn)LOD值分別為11.80和17.82,分別可解釋9.67%和16.27%的表型變異。千粒重共檢測(cè)到17個(gè)QTL,LOD值最大為27.16,共解釋84.96%的表型變異。其中,、和的LOD值分別為22.27、27.16和23.36,可分別解釋千粒重11.34%、14.59%和12.38%的變異(表3)。
2.3.3 2年粒型QTL定位結(jié)果的比較 2018—2019年連續(xù)2年通過連鎖作圖的方法對(duì)粒長(zhǎng)、粒寬、粒厚以及千粒重4個(gè)性狀進(jìn)行QTL定位(表3)。結(jié)果顯示,2年共檢測(cè)到96個(gè)粒型QTL,其中28個(gè)QTL涉及重復(fù)交叉,關(guān)聯(lián)在11個(gè)區(qū)間內(nèi),LOD值為2.53—35.50,貢獻(xiàn)率為1.02%—35.15%。從染色體分布看,檢測(cè)到的QTL在水稻的12條染色體上皆有分布。其中,第2、第3染色體上檢測(cè)到的QTL較多,均為15個(gè),第6和第9染色體檢測(cè)到的QTL最少,均為2個(gè)。
2018年和2019年都檢測(cè)到的QTL有11個(gè),與粒長(zhǎng)相關(guān)的QTL有6個(gè)。位于第2染色體,2年的LOD值分別為7.47和8.27,貢獻(xiàn)率為4.26%和3.69%。和位于第3染色體,2年的LOD值分別為27.88和27.05,貢獻(xiàn)率為20.94%和15.44%;2年的LOD值分別為27.5和22.49,貢獻(xiàn)率分別為21.26%和12.55%。位于第4染色體,2年的LOD值分別為10.85和8.37,貢獻(xiàn)率分別為6.66%和3.86%。位于第5染色體,2年的LOD值分別為5.81和4.31,貢獻(xiàn)率分別為5.13%和4.55%。位于第7染色體,LOD值分別為10.13和33.76,貢獻(xiàn)率分別為5.99%和21.17%。、和分別位于第2、4和5染色體上,是2年均檢測(cè)到的粒寬QTL,2年的LOD值為3.22—35.15,可以解釋粒寬3.05%—30.07%的變異。2018年檢測(cè)到的與2019年檢測(cè)到位于同一區(qū)間,LOD值各為8.56和6.32,分別解釋了粒型性狀6.07%和5.01%的變異;2018年檢測(cè)到的粒長(zhǎng)與2019年千粒重位于同一區(qū)間,LOD值各為3.60和22.34,分別解釋了粒型性狀3.15%和11.34%的變異。
續(xù)表3 Continued table 3
續(xù)表3 Continued table 3
*:同區(qū)間在不同年份定位出不同粒型性狀;**:同一區(qū)間在不同年份定位出同一粒型性狀
*: different grain traits were located in the same interval in different years; **: the same grain type trait is located in the same interval in different years
基于傳統(tǒng)的分子標(biāo)記如RAPD、RFLP、SSR所構(gòu)建的遺傳圖譜過程耗時(shí)、繁瑣,涉及引物設(shè)計(jì)、PCR擴(kuò)增、核酸電泳等步驟。這些分子標(biāo)記所構(gòu)建的遺傳圖譜因分子標(biāo)記密度較低,不能提供準(zhǔn)確和完全的控制QTL的數(shù)目和座位信息[38-39]。隨著高通量測(cè)序技術(shù)的發(fā)展,測(cè)序價(jià)格變得越來越便宜,使得高通量測(cè)序技術(shù)在植物科學(xué)研究中得到了越來越廣泛的應(yīng)用。高通量測(cè)序技術(shù)的應(yīng)用極大地促進(jìn)了高密度或超高密度遺傳圖譜的發(fā)展。與傳統(tǒng)分子標(biāo)記構(gòu)建的遺傳圖譜相比,利用高通量測(cè)序技術(shù)構(gòu)建的Bin圖譜精確度高、構(gòu)建時(shí)間短[7, 40]。YU等[41]對(duì)包含241個(gè)株系的RIL群體進(jìn)行約0.06×的重測(cè)序,構(gòu)建了一個(gè)超高密度的Bin遺傳圖譜。并與傳統(tǒng)的SSR、RFLP分子標(biāo)記構(gòu)建的圖譜進(jìn)行了比較發(fā)現(xiàn),利用Bin遺傳圖譜能夠檢測(cè)到更多的QTL,而且檢測(cè)到的QTL更加精細(xì)。YANG等[40]利用簡(jiǎn)化基因組測(cè)序,構(gòu)建了一個(gè)包含2 498個(gè)Bin標(biāo)記的遺傳圖譜,定位到與種子萌發(fā)、早期生長(zhǎng)相關(guān)的42個(gè)QTL,為培育適宜直播的水稻新品奠定了基礎(chǔ)。YANG等[42]利用2 711個(gè)Bin標(biāo)記的遺傳連鎖圖對(duì)水稻幼苗活力性狀進(jìn)行了QTL定位,并結(jié)合RNA-Seq分析獲得了37個(gè)候選差異表達(dá)基因。DU等[43]利用1 910個(gè)Bin標(biāo)記的遺傳連鎖圖,對(duì)水稻粒型性狀進(jìn)行了QTL定位,并通過CRISPR/Cas9基因敲除的方式進(jìn)一步驗(yàn)證了候選基因的功能。本研究對(duì)RIL群體的186個(gè)株系進(jìn)行重測(cè)序,獲得平均覆蓋深度約為18×的高質(zhì)量數(shù)據(jù),通過基因組重測(cè)序技術(shù)構(gòu)建了包含12 328個(gè)Bin標(biāo)記,標(biāo)記間平均遺傳距離為1.73 cM,物理距離為30.26 kb的Bin圖譜。本研究通過高密度Bin圖譜檢測(cè)到的QTL數(shù)目與前人相比明顯增多,該圖譜2年共定位到96個(gè)粒型QTL位點(diǎn),11個(gè)QTL連續(xù)2年都檢測(cè)到,說明這些QTL遺傳穩(wěn)定,可以進(jìn)行分子標(biāo)記的開發(fā)和基因利用。
從染色體分布看,粒型QTL在水稻12條染色體上分布不一,本研究中第2和3染色體共發(fā)現(xiàn)30個(gè)粒型QTL,占總數(shù)近1/3,其次第4、5、7、11和12染色體檢測(cè)的粒型QTL數(shù)量范圍為7—9個(gè)。同時(shí),粒型不同性狀QTL在染色體上往往呈現(xiàn)集中分布,粒長(zhǎng)、粒厚QTL在第3染色體上較多;粒寬QTL主要在第2和4染色體;千粒重的QTL主要集中在第2和3染色體。研究表明粒型基因往往存在一因多效現(xiàn)象,本研究中有11個(gè)QTL區(qū)間具有一因多效現(xiàn)象。、和為位點(diǎn),對(duì)粒長(zhǎng)、粒厚和粒重具有重要貢獻(xiàn);、和對(duì)粒長(zhǎng)、粒厚和粒重具有作用;和為同一位點(diǎn)的QTL,該位點(diǎn)對(duì)粒長(zhǎng)和粒寬均有貢獻(xiàn);和為位點(diǎn)對(duì)粒長(zhǎng)和粒重具有明顯的增效作用,增效位點(diǎn)來源于大粒水稻TD70。
開展水稻粒型基因的定位、克隆及效應(yīng)研究,對(duì)產(chǎn)量的提高、加工品質(zhì)的改良、外觀品質(zhì)的改善具有重要意義。據(jù)Gramene網(wǎng)站(http://archive.gramene.org/qtl/)統(tǒng)計(jì),通過遺傳作圖、關(guān)聯(lián)分析等方法,目前鑒定水稻粒型相關(guān)的基因/QTL已經(jīng)超過400個(gè),這些粒型基因/QTL幾乎分布在水稻的12條染色體上[44-45],其中位于第2、3和5染色體上QTL較多。已報(bào)道的影響粒長(zhǎng)的基因主要有[46]、[47-48]、/[49-50]、[51]、/[52-53]、[54]、[32]、[33]等;粒寬相關(guān)的基因有[16]、[26]、[55]、[25, 56]、[31]等;/[17, 57]是粒寬和粒厚調(diào)控基因;[58]和[59]是千粒重的主效QTL。此外,還有一些其他的生長(zhǎng)調(diào)節(jié)因子對(duì)粒型有調(diào)控作用,如[60]、/[28, 30]等。
本研究通過高密度Bin圖譜檢測(cè)到的QTL,由于標(biāo)記本身特定的物理位置,經(jīng)比對(duì)發(fā)現(xiàn)多數(shù)位點(diǎn)與之前檢測(cè)或者已克隆的主效粒型基因具有很好的區(qū)間一致性。定位的粒長(zhǎng)QTL、和在2年均被檢測(cè)到,與前期用該群體通過傳統(tǒng)的方法檢測(cè)到的位點(diǎn)一致,進(jìn)一步分析發(fā)現(xiàn)這3個(gè)位點(diǎn)分別為已經(jīng)克隆的粒型基因、及/位點(diǎn)[60-61]。、位點(diǎn)與前期定位區(qū)間一致,進(jìn)一步分析證明這兩個(gè)位點(diǎn)為已經(jīng)克隆的粒寬主效基因和[16, 25, 62]。本研究通過高密度Bin圖譜定位到大量的粒型QTL位點(diǎn),既包含前人已經(jīng)定位或克隆的、、、、、、等粒型基因[26, 28, 31, 48, 63-65],也有新發(fā)現(xiàn)的4個(gè)2年在同一區(qū)間控制粒型的、、新位點(diǎn),這說明本研究結(jié)果是真實(shí)可靠的。
構(gòu)建了一張包含12 328個(gè)Bin標(biāo)記的分子遺傳連鎖圖譜,利用該圖譜對(duì)大粒資源的粒型性狀進(jìn)行了QTL定位,共得到96個(gè)粒型QTL。驗(yàn)證了大部分定位或克隆的粒型基因,同時(shí)新鑒定出等4個(gè)同區(qū)間粒型QTL,證實(shí)了多個(gè)粒型基因的組合可行性。
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Construction of high-Density genetic map and QTL analysis of grain shape in rice RIL population
ZHANG YaDong, LIANG WenHua, HE Lei, ZHAO ChunFang, ZHU Zhen, CHEN Tao, ZHAO QingYong, ZHAO Ling, YAO Shu, ZHOU LiHui, LU Kai, WANG CaiLin
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Jiangsu High Quality Rice R&D Center/Nanjing Branch of China National Center for Rice Improvement, Nanjing 210014
【Objective】Rice grain shape is an important agronomic trait directly related to yield , which affects the appearance quality and commercial value of rice. Research on new rice grain shape genes is of great value for revealing the genetic mechanism of rice grain shape, and it can provide some new genetic resources for molecular breeding.【Method】In the present study, a RIL population which constructed by an extra-large grainrice variety TD70 and a small-grainrice variety Kasalath was used as the research material. The phenotypic data of grain shape, such as grain length, grain width, grain thickness and thousand grain weight were investigated. Using the Genotyping-By-Sequencing approach to re-sequence the parents and RILs to obtain SNP information. The sliding window method (the number of SNP/InDel is 15) was used for genotype calling and recombination breakpoint determination. Based on these results, a high-density Bin map was constructed. Meanwhile, the compound interval mapping method of QTL IciMapping software was used to map the QTLs related to grain shape.【Result】A high-density genetic map containing 12 328 Bin markers was constructed. The number of Bin markers on each chromosome is 763 to 1367, and the average physical distance between markers was 30.26 kb. The frequency distribution of each trait for RIL population was continuous, which were consistent with the characteristics of quantitative characters, so it was suitable for the detection of QTL. QTL analysis of RIL population in 2018 showed that 40 grain-shape QTL were detected, including 12 grain length QTL, 9 grain width QTL, 8 grain thickness QTL, and 11 thousand-grain weight QTL. QTL analysis was performed of RIL population in 2019, and 56 grain-related QTL were detected, including 15 grain length QTL, 11 grain width QTL, 13 grain thickness QTL, and 17 thousand-grain weight QTL. Based on the two-year mapping results, we have mapped a total of 96 grain shape QTL. We found that 11 QTL could be detected for two consecutive years; among them, 7 QTL have been cloned and 4 new QTL were distributed on 1, 3, 4 and 5 chromosomes. Among the 4 new QTL,andwas related to grain length,related grain thickness andrelated to grain width.【Conclusion】We constructed a molecular genetic linkage map containing 12 328 Bin markers and used the map to analyze the grain shape loci of extra-large grain rice resources. Four new QTLs related to grain shape were obtained, which can be used for subsequent fine mapping, cloning and functional studies.
rice (L.); Bin genetic map; grain shape; QTL mapping
2021-06-07;
2021-08-03
國(guó)家自然科學(xué)基金(31771761)、江蘇省現(xiàn)代農(nóng)業(yè)重點(diǎn)項(xiàng)目(BE2019339)、現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(xiàng)資金(CARS-01-67)
張亞東,E-mail:zhangyd@jaas.ac.cn。通信作者王才林,E-mail:clwang@jaas.ac.cn
(責(zé)任編輯 李莉)
中國(guó)農(nóng)業(yè)科學(xué)2021年24期