趙 蝶 胡文靜 程曉明 王書平 張春梅 李東升 高德榮

揚麥4號/偃展1號RIL群體株高QTL挖掘及其對赤霉病抗性的效應分析與驗證

趙 蝶1,2胡文靜2,3,*程曉明2王書平1張春梅2李東升2高德榮2,3,*

1長江大學農學院, 湖北荊州 434025;2江蘇里下河地區(qū)農業(yè)科學研究所 / 農業(yè)農村部長江中下游小麥生物學與遺傳育種重點實驗室, 江蘇揚州 225007;3揚州大學 / 江蘇省糧食作物現(xiàn)代產業(yè)技術協(xié)同創(chuàng)新中心 / 江蘇省植物功能基因組學重點實驗室, 江蘇揚州 225009

小麥的株高(plant height, PH)性狀與赤霉病(fusarium head blight, FHB)抗性的關系密切。本研究利用揚麥4號/偃展1號(YM4/YZ1)雜交組合衍生的重組自交系(recombinant inbred lines, RIL)群體, 利用55K單核苷酸多態(tài)性(single-nucleotide polymorphism, SNP)芯片數(shù)據(jù), 結合3年共6個環(huán)境下RIL群體及其親本的株高數(shù)據(jù), 挖掘株高性狀的遺傳位點。同時利用土壤表面赤霉病麥粒拋撒法和單花滴注法鑒定株高位點對赤霉病抗侵染(Type I)和抗擴展(Type II)兩種類型的效應。在染色體2D、4B、4D、5A和7D上檢測到7個與株高相關的數(shù)量性狀位點(quantitative trait loci, QTL), 經(jīng)過比對,可能是新的株高位點。、和的矮稈效應來源于揚麥4號, 其余4個QTL的矮稈效應來源于偃展1號。和均在6個環(huán)境下被檢測到, 表型變異貢獻率(PVE)范圍分別為19.48%～44.11%和10.48%～13.71%。研究發(fā)現(xiàn)和位點上的高稈等位變異(YM4等位變異)分別降低侵染型平均病小穗率(average percentage of infected spikelets, PIS) 34.97%和19.09%,和位點上的矮稈等位變異(YM4等位變異)分別降低擴展型平均病小穗率(average percentage of diseased spikelets, PDS) 24.73%和14.56%。的矮稈等位變異來源于阿夫。利用小麥中國春2.1版本的參考基因組信息分析區(qū)間, 發(fā)現(xiàn)一共有146個有注釋功能的高置信基因, 主要涉及合成細胞色素P450、脫水反應元件結合蛋白、乙烯響應轉錄因子、轉錄因子MYC2和細胞壁受體相關激酶等。進一步將位點緊密連鎖SNP標記轉化成育種可用分子標記KASP-5A, 并在126份小麥品種(系)中初步驗證其對株高和赤霉病抗性的效應。研究結果可為的育種應用和精細定位奠定基礎。

小麥; 株高; 赤霉病; QTL;; 候選基因

小麥(L.)是世界上最重要的糧食作物之一, 提供了我們日常飲食中20%以上的卡路里和蛋白質[1]。由于人口的不斷增加和全球耕地面積的持續(xù)減少, 高產仍然是小麥育種計劃的主要目標之一[2]。株高(plant height, PH)是一個與株型和產量潛力相關的關鍵性狀[3]。因此, 利用矮稈基因提高產量已成為產量育種的主要策略之一。例如, 在小麥綠色革命期間, 將半矮稈品種引入小麥適當?shù)亟档椭旮咧? 并對全球潛在糧食產量的提高做出了顯著貢獻[4]。眾所周知, 小麥株高通常受多基因控制,其數(shù)量性狀位點(quantitative trait loci, QTL)廣泛分布在小麥21條染色體上[5]。其中兩個矮稈基因和在小麥中分布最廣并在小麥株高矮化過程中起到了重要作用[6-7],和均編碼DELLA蛋白, 參與赤霉素(gibberellic acid, GA)信號的傳導過程, 此類基因突變之后對GA信號不敏感而導致株高變矮, 這兩個基因的特性及其功能標記對小麥矮化和高產育種起到了促進作用[8-11]。另一個在小麥育種中被廣泛應用的矮稈基因是, 它被定位在染色體2D短臂上, 對GA3反應為敏感型[12]。編碼核糖核酸酶h樣結構域蛋白, 分別被Chai等[13]和Xiong等[14]證實為的候選基因。隱性基因來源于赤小麥, 位于7B染色體短臂上,為GA3敏感型, 對小麥有較強的矮化作用[15]。是一個對GA敏感的顯性矮化基因, 位于染色體5A長臂上, 通過激活來降低植物高度, 顯著縮短干細胞長度, 減少GA生物合成[16]。小麥半矮化是由于表達增加和GA含量降低造成的[17]。位于染色體7AS上, 其短莖性狀可能與細胞數(shù)量減少有關[18]。位于6A染色體長臂上的一個GA敏感型矮稈基因,可能是其候選基因, 其過表達可顯著降低GA水平并引起植株矮化, 但對產量性狀無不利影響[19]。Borrill等[20]發(fā)現(xiàn)一個潛在替代傳統(tǒng)矮化基因的新型矮化基因, 與傳統(tǒng)矮化基因相比,的矮稈等位變異降低了植株高度, 并增加了莖稈強度, 其編碼一個自激活活性NB-LRR基因, 而不是赤霉素信號或代謝的組成部分, 自激活活性的等位基因()引起致病相關基因的上調, 導致活性氧的產生, 活性氧能促進交聯(lián)和細胞壁硬化, 導致生長減少。與小麥其他產量相關性狀相似, 株高是復雜的數(shù)量性狀, 受遺傳和環(huán)境因素的交互作用影響較大。QTL定位為在遺傳水平上分析這些復雜的數(shù)量性狀提供了一種有效的方法, 同時也為小麥株高的分子標記輔助育種奠定基礎[21-23]。赤霉病(fusarium head blight, FHB), 主要由禾谷鐮刀菌(Schwabe)引起, 嚴重危害小麥或其他谷物的生產和糧食安全。中國的長江中下游麥區(qū)和東北春麥區(qū)常遭受到赤霉病的危害[24-26], 近年來, 由于以小麥-玉米為主的耕作制度和少耕留茬, 赤霉病已成為黃淮流域小麥的主要病害之一[27-28]。小麥對赤霉病的抗性具有多種表現(xiàn)形式, 其中抗侵染(Type I)和抗擴展(Type II)是兩種最主要的類型, 抗侵染反映的是小麥抵抗赤霉菌初侵染的能力, 而抗擴展反映的是在赤霉菌侵染后宿主抵御其通過菌絲生長沿著穗軸延伸的能力[29]。小麥抗赤霉病育種瓶頸之一是同時提高小麥赤霉病抗性和產量。已知的一些赤霉病抗源例如蘇麥3號、望水白的產量潛力太低, 株高達到130～150 cm, 易倒伏, 應用起來較困難[30]。相關研究發(fā)現(xiàn)小麥的一些農藝性狀, 如芒的長短、小穗密度、株高、抽穗期、穗長等對赤霉病的侵染和擴展均有不同程度的影響, 其中, 株高等對赤霉病的侵染影響較大, 有研究表明在自然條件下植株高可使小麥減輕受病原菌感染程度[31-35]。控制株高的QTL/基因中矮稈基因和對赤霉病的侵染能力具有顯著的效應[30,36-39], 而株高位點對赤霉病的擴展嚴重度具有顯著效應的相關報道罕見[30]。

揚麥4號(Yangmai 4, YM4)是長江中下游流域20世紀80年代育成的第一個赤霉病抗性達到中抗的小麥品種, 在赤霉病大流行年份依然可以抵抗赤霉病的危害, 其親本是南大2419、勝利麥(1-3-2系)和阿夫選系[26]。偃展1號(Yanzhan 1, YZ1)是一個矮稈、早熟、高產、抗旱、抗寒但高感赤霉病的小麥品種。揚麥4號株高為90～100 cm, 偃展1號的株高為70～80 cm, 經(jīng)過前期分子標記檢測表明揚麥4號不攜有和兩個主效矮稈基因, 偃展1號攜有基因。目前對揚麥4號和偃展1號的株高遺傳基礎尚未有解析, 本研究的目的是: (1) 利用揚麥4號/偃展1號(YM4/YZ1)重組自交系(recombinant inbred lines, RIL)群體解析揚麥4號和偃展1號的株高遺傳基礎; (2) 利用RIL群體研究所定位到的株高位點對赤霉病抗侵染和抗擴展的效應; (3) 開發(fā)主效穩(wěn)定的株高QTL相應的KASP (kompetitive allele specific PCR)分子標記, 并在自然群體中驗證其對株高和赤霉病抗性的效應, 為小麥分子標記輔助育種奠定基礎。

1 材料與方法

1.1 試驗材料及田間試驗

將揚麥4號與黃淮麥區(qū)的優(yōu)良冬小麥品種偃展1號進行雜交, 利用單粒傳方法獲得F10代的RIL群體, 共151個家系。揚麥4號/偃展1號種群在小麥生長季分別種植于2018—2019年間荊州試驗點(海拔35～45 m, 北緯33.46度, 東經(jīng)118.22度, 年降水量910 mm, 簡稱19JZ)、揚州試驗點西區(qū)和揚州試驗點東區(qū)(海拔10～20 m, 北緯32.24度, 東經(jīng)119.26度, 年降水量1020 mm, 簡稱19YZW和19YZE), 2019—2020年間揚州試驗點西區(qū)和揚州試驗點東區(qū)(簡稱20YZW和20YZE), 2020—2021年間揚州試驗點東區(qū)(簡稱21YZE)。田間試驗分為3組進行, 分別是株高評價、Type I型赤霉病抗性評價和Type II型赤霉病抗性評價。田間試驗采用完全隨機設計, 2個重復。每個RIL和2個親本平均播撒30粒種子, 每行133 cm, 雙行間隔25 cm。田間試驗和疾病控制的管理遵循當?shù)貥藴首龇? 2種抗赤霉病評價苗圃中未使用任何殺菌劑。采用與RIL相同的方案, 于2019—2020年間和2020—2021年間的揚州試驗點西區(qū)種植126個小麥品種(系)。待到成熟期, 在揚麥4號/偃展1號群體和2個親本中隨機選取生長狀況相近的每個家系的10株代表性植株, 從主莖穗植株基部到主莖穗頂部(不包括芒)測量株高。田間赤霉病評價在2019—2020年間和2020—2021年間的揚州試驗站進行, 采用土壤表面赤霉病麥粒拋撒法和單花滴注法分別對揚麥4號/偃展1號群體和2個親本的抗侵染型(Type I)和抗擴展型(Type II)的赤霉病抗性進行鑒定。Type I型接種在孕穗前5 d進行第1次接種, 孕穗期再進行第2次接種, 等到開花后10 d開始調查, 記錄至少一個小穗出現(xiàn)明顯赤霉病癥狀的病小穗數(shù)和每穗總小穗數(shù), 計算出每個RIL家系每次重復(共20穗)的平均病小穗率(average percentage of infected spikelets, PIS)。Type II型接種是在花期用Hu等[40]描述的4種禾谷鐮孢()菌株的混合物接種, 在接種21 d后, 記錄每個RIL家系(共20穗)的病小穗的平均百分比(average percentage of diseased spikelets, PDS), 作為赤霉病嚴重程度的衡量標準。接種后根據(jù)胡文靜等[30]方法進行田間保濕和調查赤霉病。從每天早上07:00—18:00, 每30 min噴霧5 min, 以提供有利于赤霉病感染的高度濕潤條件。接種4周后, 觀察接種穗的病小穗數(shù)和每穗總小穗數(shù)。計算每個RIL的平均病小穗率作為赤霉病嚴重程度的度量。2019— 2021兩年間的126個小麥品種(系)采用與RIL相同的實驗方法進行株高和2種抗赤霉病類型的調查。

1.2 統(tǒng)計分析

利用Microsoft Excel 2019對數(shù)據(jù)進行基本處理和統(tǒng)計分析; 利用SPSS的Tukey進行等位變異之間PIS和PDS的T測驗。株高的廣義遺傳力(2)計算公式為22G/(2G+2G×E/E+2eER)[41-42], 其中E和R分別為環(huán)境次數(shù)和重復數(shù),2G為基因型方差,2G×E為環(huán)境對基因型的互作方差,2e為殘差。使用IciMapping v4.1計算2和最優(yōu)線性無偏估計值()[43]。

1.3 基因分型、連鎖圖譜構建、QTL分析

采用CTAB法[44]從幼苗中提取基因組DNA, 凝膠電泳檢測DNA完整性和數(shù)量。利用中國金標記(北京)生物技術有限公司的小麥55k單核苷酸多態(tài)性(single-nucleotide polymorphism, SNP)芯片對揚麥4號、偃展1號親本品種和RIL群體進行基因分型, 然后選擇、、和等與其他已知基因相關的KASP標記和SSR (simple sequence repeat)對親本及RIL群體進行基因分型, 檢測多態(tài)性[11,25,28,45]。標記數(shù)據(jù)的質控和遺傳連鎖圖譜構建參考Hu等[39]。在構建連鎖圖譜之前, 對SNP數(shù)據(jù)進行質控, 然后經(jīng)過去冗余(刪除缺失率大于30%和最小等位基因頻率小于5%的SNP)、分群(LOD=8), 最終得到1440個上圖SNP, 構建成長度為3574.10 cM的覆蓋小麥21條染色體的遺傳連鎖圖譜, 標記之間的平均遺傳距離是2.58 cM[39]。利用IciMapping v4.1 (https://www.isbreeding.net/)的完備區(qū)間作圖法(inclusive composite interval mapping, ICIM)檢測與小麥株高顯著相關的QTL, LOD閾值設為3.0[43]。圖譜繪制使用MapChart 2.3 (https://www.wur.nl/en/ show/Mapchart.htm)。我們利用QTL區(qū)間的側翼SNP的序列信息, 將本研究中鑒定的QTL與先前報道的QTL或基因進行比較(http://202.194.139. 32/blast/blast.html; http://202.194.139.32/genes/), 重疊置信區(qū)間內的QTL被視為同一個[46]。

1.4 KASP標記的開發(fā)與驗證

根據(jù)初步QTL作圖結果, 參照Li等[47]方法將目標QTL的側翼標記轉化為KASP標記, 追蹤相應性狀。隨后進行KASP測定。使用PHERAstar (BMG LABTECH, 德國)對PCR反應進行熒光檢測[45]。使用KlusterCaller軟件(LGC Genomics, Beverly, 美國)將開發(fā)成功的KASP標記在RIL群體中檢測, 與芯片數(shù)據(jù)進行比較[48]。為了進一步驗證主效QTL在不同遺傳背景下的效應, 對126個來自國內外的小麥品種(系)進行KASP標記的檢測和株高、PIS和PDS的考察。

1.5 候選基因預測

根據(jù)主效QTL目標區(qū)間側翼標記的物理位置從小麥參考基因組2.1版本(http://202.194.139.32/ jbrowse-1.12.3-release)中提取物理區(qū)間的高置信基因, 利用Triticeae-GeneTribe (http://wheat.cau.edu. cn/TGT/)發(fā)掘基因在擬南芥和水稻的同源基因并分析其參與的生物進程和分子功能[49-50]。

2 結果與分析

2.1 親本和RIL群體株高表現(xiàn)

統(tǒng)計分析顯示(表1), 兩個親本的株高具有極顯著差異(<0.01)。(最優(yōu)線性無偏估計值)下親本揚麥4號和偃展1號的株高分別為98.40 cm和75.21 cm, RIL群體株高變異范圍為65.62～119.98 cm, 平均值為97.14 cm。株高性狀在6個環(huán)境下的廣義遺傳力(2)為0.81 (表1)。

表1 不同環(huán)境下親本及揚麥4號/偃展1號的株高表型變異及遺傳力

E1、E2、E3、E4、E5和E6分別表示2019JZ、2019YZW、2019YZE、2020YZW、2020YZE和2021YZE的環(huán)境, BLUE表示最優(yōu)線性無偏估計值。**表示兩親本間的株高差異顯著(< 0.01)。

E1, E2, E3, E4, E5, and E6 indicate the environment of 2019JZ, 2019YZW, 2019YZE, 2020YZW, 2020YZE, and 2021YZE, respectively. BLUE represents the best linear unbiased prediction.**indicates significant difference in plant height between the two parents at< 0.01.

2.2 株高QTL定位分析

共檢測到7個株高相關QTL, 分別位于染色體2D、4B、4D、5A和7D上(圖1)。、和加性效應為負, 說明矮稈效應來源于揚麥4號, 其余的4個QTL加性效應均為正, 說明矮稈效應來源于偃展1號(表2)。和可同時在6個環(huán)境下被檢測到, 株高貢獻率范圍分別為19.48%～44.11% 和10.48%～13.71%, LOD值范圍分別是15.50～35.95和8.31～12.70 (表2)。的緊密連鎖標記是Rht-D1_SNP, 位于矮稈基因的物理區(qū)間。在5個環(huán)境下被檢測到, 表型貢獻率范圍為4.13%～11.09%。和在4個環(huán)境下被檢測到, 表型貢獻率范圍分別為3.16%～10.76%和3.60%～9.75% (表2)。和在2個環(huán)境下被檢測到, 株高貢獻率范圍分別為3.72%～7.06%和8.04%～8.64% (表2)。

連鎖群右邊是標記名稱, 左邊是遺傳位置(cM), 連鎖群中的黑色矩形代表QTL區(qū)域。E1、E2、E3、E4、E5和E6分別表示2019JZ、2019YZW、2019YZE、2020YZW、2020YZE和2021YZE的環(huán)境,表示最優(yōu)線性無偏估計值。

The marker name is on the right of the linkage group, the genetic position (cM) is on the left, and the black rectangle in the linkage group represents the QTL region. E1, E2, E3, E4, E5, and E6 indicate the environment of 2019JZ, 2019YZW, 2019YZE, 2020YZW, 2020YZE, and 2021YZE, respectively.represents the best linear unbiased prediction.

表2 揚麥4/偃展1號群體株高的QTL分析

E1、E2、E3、E4、E5和E6分別表示2019JZ、2019YZW、2019YZE、2020YZW、2020YZE和2021YZE的環(huán)境, BLUE表示最優(yōu)線性無偏估計值。b加性效應為正說明矮稈效應來源于偃展1號, 加性效應為負說明矮稈效應來源于揚麥4號。

E1, E2, E3, E4, E5, and E6 indicate the environment of 2019JZ, 2019YZW, 2019YZE, 2020YZW, 2020YZE, and 2021YZE, respectively. BLUE represents the best linear unbiased prediction.bPositive additive effect indicated that the dwarf effect originates from Yanzhan 1, and the negative additive effect indicates that the dwarf effect originates from Yangmai 4.

2.3 QTL位點對抗赤霉病的效應

分析本研究中定位到的株高相關QTL對赤霉病侵染和擴展嚴重度的影響, 結果表明,和對赤霉病侵染嚴重度(PIS值)和赤霉病擴展嚴重度(PDS值)均無顯著效應(圖2-A, C, F)。對PIS值無顯著效應, 對PDS值具有極顯著效應, 攜帶矮稈等位基因型(YM4等位變異)的家系比攜帶高稈等位基因型(YZ1等位變異)的家系的PDS值降低24.73% (<0.01) (圖2-B);對PIS值無顯著效應, 對PDS值具有極顯著效應, 攜帶矮稈等位基因型(YM4等位變異)的家系比攜帶高稈等位基因型(YZ1等位變異)的家系的PDS值降低14.56% (<0.01) (圖2-E)。和對PIS值均具有極顯著效應, 攜帶高稈等位基因型(YM4等位變異)的家系比攜帶矮稈等位基因型(YZ1等位變異)的家系的PIS值分別降低34.97% (<0.01)和19.09% (<0.01) (圖2-D, G)。

圖2 揚麥4號/偃展1號RIL群體株高QTL對赤霉病抗性的效應

PIS: 侵染型平均病小穗率, 土壤表面赤霉病麥粒拋撒法對赤霉病抗性的測定; PDS: 擴展型平均病小穗率, 單花滴注法對赤霉病抗性的測定。‘×’在箱形圖中為平均值標記; 箱形圖中的點表示離群點; 數(shù)據(jù)框中的水平線表示中位數(shù)。*和**分別代表與偃展1號相比<0.05和<0.01。

PIS: the average percentage of infected spikelets, the measure of FHB resistance in soil surface inoculation; PDS: the average percentage of diseased spikelets, the measure of FHB resistance in point inoculation. ‘×’ in the data box indicates the mean value; the dots in the boxplots are the outliers; the horizontal line in the data box indicates the median. * and ** represent significant difference at< 0.05 and< 0.01 compared with Yanzhan 1, respectively.

2.4 KASP標記開發(fā)及驗證

定位到的株高QTL中,和是效應值較大且穩(wěn)定的位點,位于矮稈基因區(qū)間[11]。根據(jù)另一主效位點目標區(qū)間兩側的分子標記側翼序列, 最終成功將位于峰值區(qū)間緊密連鎖的SNP標記AX109341178轉化為KASP標記并命名為KASP-5A[51]。該SNP堿基突變是C/G (揚麥4號是C, 偃展1號是G), 共用引物序列是5'- TCTGCGGGCACATCAGTTAG-3', 特異性引物1序列是5′-GAAGGTGACCAAGTTCATGCTTGGACA CCGAAGTAGTTCCC-3′, 尾部添加能夠與FAM熒光結合的特異性序列; 特異性引物2序列是5′- GAAGGTCGGAGTCAACGGATTTGGACACCGAAGTAGTTCCT-3′, 尾部添加能夠與HEX熒光結合的特異性序列。

2.5 QPh.yas-5A在自然群體中的效應分析

利用126份自然群體試驗材料驗證位點對株高和赤霉病抗性的效應(附表1)。研究發(fā)現(xiàn)位點上的矮稈等位變異(YM4等位變異)仍可顯著降低株高和赤霉病擴展嚴重度, 株高和PDS值分別降低2.38% (<0.05)和28.96% (<0.01) (圖3-A, C)。其對赤霉病侵染嚴重度無顯著影響, 與RIL群體結果一致(圖2-E和圖3-B)。

PH: 株高; PIS: 侵染型平均病小穗率, 土壤表面赤霉病麥粒拋撒法對赤霉病抗性的測定; PDS: 擴展型平均病小穗率, 單花滴注法對赤霉病抗性的測定?！痢谙湫螆D中為平均值標記; 箱形圖中的點表示離群點; 數(shù)據(jù)框中的水平線表示中位數(shù)。*和**分別代表與偃展1號相比< 0.05和<0.01。

PH: plant height; PIS: the average percentage of infected spikelets, the measure of FHB resistance in soil surface inoculation; PDS: the average percentage of diseased spikelets, the measure of FHB resistance in point inoculation. ‘×’ in the data box indicates the mean value; the dots in the boxplots are the outliers; the horizontal line in the data box indicates the median. * and ** represent significant difference at< 0.05 and< 0.01 compared with Yanzhan 1, respectively.

2.6 QPh.yas-5A區(qū)間的候選基因發(fā)掘

進一步利用小麥中國春參考基因組2.1版本的信息, 在的定位區(qū)間挖掘到191個高置信基因, 其中有146個基因有注釋功能, 主要涉及合成細胞色素P450 (水稻的同源基因是、和, 擬南芥的同源基因是)、脫水反應元件結合蛋白(水稻的同源基因是、和, 擬南芥的同源基因是)、乙烯響應轉錄因子(水稻的同源基因是和, 擬南芥的同源基因是和)、轉錄因子MYC2 (水稻的同源基因是)和細胞壁受體相關激酶等(水稻的同源基因是和) (附表2)。

3 討論

3.1 QTL的比較分析

本研究共檢測到7個株高QTL。由表2可得,的物理位置區(qū)間是18.40～20.42 Mb, 位于的置信區(qū)間附近,在染色體2D上511.67～515.21 Mb區(qū)間, 與以往研究中檢測到的株高 QTL/基因存在差異, 推測是一個新的株高位點[22,52]。位于4B染色體上589.85 Mb到590.54 Mb之間, 與之前報道的[53]、[54]、[55]和[56]具有相近的置信區(qū)間, 這一區(qū)間與(544.65 Mb)的QTL相差45.20 Mb[57]。的物理區(qū)間在62.23～68.26 Mb, 與此前在7D染色體上檢測到的株高QTL或基因的定位區(qū)間不同, 但是與春化基因在相同物理區(qū)間[57-59],是在7D染色體上檢測到的另一個與株高有關的QTL, 其物理位置區(qū)間為147.70～395.89 Mb, 與控制抽穗期和株高相關基因(237.61 Mb)在相同的置信區(qū)間[60]。的物理位置區(qū)間是19.19 Mb至19.59 Mb, 與矮稈基因重疊[61]。經(jīng)過檢測, 發(fā)現(xiàn)偃展1號和揚麥4號分別攜帶的矮稈等位變異和高稈等位變異。的物理位置區(qū)間為516.44～536.32 Mb, 距離Yan等[62]在揚麥158/偃展1號 RIL群體中挖掘到的(506.00 Mb) 10 Mb和Zhang等[63]挖掘到的矮稈效應來源于KN9204的(479.12～487.31 Mb) 29 Mb, 與Schnurbusch等[64]在‘Arina×Forno’ F5:7雜交群體中挖掘出來的(530.98 Mb)和胡文靜等[51]在人工合成小麥C615中挖掘到的株高位點(519.89 Mb)位置重疊。此外,與位于5A染色體上的矮稈基因(698.89 Mb)相距較遠[16]。

3.2 株高位點的抗赤霉病遺傳效應分析

以往研究中發(fā)現(xiàn),,對赤霉病具有顯著效應, 其中增加株高的基因型,和對赤霉病抗性具有顯著的增效作用[36-38]。本研究結果表明和的矮稈等位變異可顯著增加PIS值, 而與矮稈等位變異可顯著降低PDS值。其中對株高的效應值僅次于, 且在所有環(huán)境中均能檢測到, 在育種中可加以利用。Hu等[46]利用同一RIL群體進行了抗擴展型赤霉病QTL定位, 共挖掘到了5個抗赤霉病QTL, 其在2D和5A染色體上分別定位到了(528.39～531.66 Mb)和(547.10～548.10 Mb), 與本研究定位到的2D和5A染色體上株高QTL雖然不在同一區(qū)間, 但是相距較近:與相距13.18 Mb,與相距10.78 Mb。因此, 我們推測在2D和5A的這兩個相近區(qū)間上存在株高和抗赤霉病的基因, 并且具有連鎖關系, 還需通過精細定位和克隆進行驗證。

3.3 QPh.yas-5A 的矮稈效應來源和育種價值分析

矮稈效應來源于揚麥4號, 揚麥4號是江蘇里下河地區(qū)農業(yè)科學研究所用南大2419×勝利麥雜交后的F5代, 再與阿夫雜交育成[26], 利用KASP-5A對南大2419、勝利麥和阿夫進行檢測, 結果表明僅阿夫在該位點呈現(xiàn)與揚麥4號相同的等位變異, 說明的矮稈等位變異來源于阿夫。利用的功能標記KASP-5A在126份小麥品種(系)中進行分子檢測, 再與自然群體的株高、PIS和PDS表型數(shù)據(jù)結合進行分析, 結果表明對赤霉病抗擴展和對株高的效應與RIL群體中的結果一致。位點的矮稈等位變異不僅顯著降低小麥株高還顯著增加赤霉病抗擴展能力, 因此具有較大的育種應用潛力。中國早期的地方品種望水白、白三月黃等和早期栽培品種例如蘇麥3號、揚麥4 號等均攜有、位點的高稈等位變異, 其中蘇麥3號和揚麥4號攜有的矮稈等位變異, 望水白、白三月黃和蘇麥3號的常年株高是125～140 cm, 但是揚麥4號的株高常年是92～100 cm, 經(jīng)過檢測發(fā)現(xiàn)望水白、白三月黃和蘇麥3號均不攜有的矮稈等位變異, 說明對揚麥4號的降稈起到重要作用。一般小麥品種(系)的PDS值小于25%可以界定為赤霉病抗擴展達到抗-中抗的水平, 株高小于85 cm可以界定為矮稈, 我們以此為依據(jù)結合的分子標記輔助選擇發(fā)掘RIL群體和自然群體中矮稈且抗性好的材料, 發(fā)現(xiàn)RIL群體中家系RIL-31、RIL-32、RIL-50、RIL-136、RIL-146和RIL-147攜有矮稈等位變異, 株高小于85 cm, 且PDS值小于25%, 自然群體中鄂麥174、寧1616、寧17342、寧麥24、寧麥15318、蘇麥5號、揚輻麥3048、揚輻麥4188、揚輻麥9、揚麥20、揚麥21和鎮(zhèn)麥11這12個品種攜有矮稈等位變異, 株高小于85 cm, 且PDS值小于25%, 這些篩選出的品種(系)可以作為抗赤霉病遺傳改良的優(yōu)異抗源。

3.4 QPh.yas-5A區(qū)間的候選基因分析

的定位區(qū)間挖掘到的高置信基因主要涉及合成細胞色素P450、脫水反應元件結合蛋白、乙烯響應轉錄因子、轉錄因子MYC2和細胞壁受體相關激酶等。前人報道中證實小麥對赤霉病病原體和真菌毒素的應答與細胞色素P450相關基因的表達顯著相關[65], 靶向沉默宿主誘導的細胞色素P450的CYP51 (lanosterol C-14α-demethylase)基因是一種控制真菌病害的方法[66]。乙烯響應轉錄因子(ethylene-responsive transcription factor)作為轉錄激活劑, 與GCC-box致病相關啟動子元件結合, 在植物發(fā)育過程中參與基因表達的調控, 或受脅迫因子和脅迫信號轉導通路組分的介導[67-68]。細胞壁受體相關激酶(wall-associated receptor kinase 5)如絲氨酸/蘇氨酸蛋白激酶可能作為細胞外基質成分的信號受體,與果膠的結合可能在控制細胞擴張、形態(tài)發(fā)生和發(fā)育方面具有重要意義[69]。綜合上述分析結果表明區(qū)間不僅與植物生長發(fā)育相關, 與抗病相關的機制也具有一定的聯(lián)系, 因此精細定位和克隆該基因對小麥產量與抗病育種具有一定的價值。

4 結論

定位到7個株高的QTL, 其中和在6個環(huán)境中均能檢測到, 表型貢獻率范圍分別為19.48%～44.11%和10.48%～13.71%,與已知矮稈基因一致。和位點的矮稈等位變異可以顯著降低株高和赤霉病擴展嚴重度。分析目標區(qū)間的候選基因發(fā)現(xiàn)該區(qū)間與植物生長發(fā)育和對病害的響應有關。進一步開發(fā)位點的分子標記KASP-5A, 可促進其應用于小麥株高性狀的遺傳改良。

附表 請見網(wǎng)絡版: 1) 本刊網(wǎng)站http://zwxb. chinacrops.org/; 2) 中國知網(wǎng)http://www.cnki.net/; 3) 萬方數(shù)據(jù)http://c.wanfangdata.com.cn/Periodical- zuowxb.aspx。

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Detection and verification of QTL for plant height in Yangmai 4/Yanzhan 1 recombinant inbred lines population and their genetic effects on Fusarium head blight resistance

ZHAO Die1,2, HU Wen-Jing2,3,*, CHENG Xiao-Ming2, WANG Shu-Ping1, ZHANG Chun-Mei2, LI Dong-Sheng2, and GAO De-Rong2,3,*

1College of Agriculture, Yangtze University, Jingzhou 434025, Hubei, China;2Lixiahe Institute of Agriculture Sciences / Key Laboratory of Wheat Biology and Genetic Improvement for Low and Middle Yangtze Valley, Ministry of Agriculture and Rural Affairs, Yangzhou 225007, Jiangsu, China;3Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops / Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Yangzhou University, Yangzhou 225009, Jiangsu, China

Plant height (PH) is associated with fusarium head blight (FHB) resistance in wheat. In this study, a recombinant inbred lines (RIL) population derived from the cross of Yangmai 4/Yanzhan 1 (YM4/YZ1) was used to mine the quantitative trait loci (QTL) of PH traits using 55K SNP (single-nucleotide polymorphism) chip data, combined with the PH data of RIL population and their parents in six environments for three continuous years.Soil surface inoculation method and single spikelet inoculation were used to identify the FHB resistance to infection (Type I) and spread (Type II), respectively. Seven QTLs related to PH were detected on chromosomes 2D, 4B, 4D, 5A, and 7D, and onlymay be a new QTL of PH after comparing with previous studies. The dwarfing effects of,, andwere derived from YM4, and the dwarfing effects of the other four QTLs were derived from YZ1. Bothandcould be detected in six environments, and phenotypic variation explained (PVE) rates ranged from 19.48%–44.11% and 10.48%–13.71%, respectively. The increasing alleles at theand(YM4 allele) significantly reduced the average percentage of infected spikelets (PIS) by 34.97% and 19.09%, respectively. The dwarfing alleles atand(YM4 allele) significantly reduced the average percentage of diseased spikelets (PDS) by 24.73% and 14.56%, respectively. The dwarfing allele ofwas derived from Funo. Furthermore, we preliminary analyzedthe genes within the physical interval ofusing the reference genome information of wheat version 2.1. A total of 146 high-confidence annotated genes were detected in the target interval, which were mainly involved in the synthesis of cytochrome P450, dehydration response element-binding protein, ethylene response transcription factor, transcription factor MYC2, and cell wall receptor-associated kinases. The SNP marker closely linked towas further converted into kompetitive allele-specific PCR marker KASP-5A, and its effect on plant height and FHB resistance was then verified in 126 wheat cultivars (lines). The results of this study could provide a solid foundation for future fine mapping.

; plant height; fusarium head blight; QTL;; candidate gene

2023-05-24;

2023-06-15.

通信作者(Corresponding author): 胡文靜, E-mail: huren2008@126.com; 高德榮, E-mail: gdr@wheat.org.cn

10.3724/SP.J.1006.2023.31005

E-mail: zd2021720791@163.com

2023-01-10;

本研究由國家自然科學基金項目(31901544), 泰州市科技計劃項目(TN202117), 江蘇現(xiàn)代農業(yè)產業(yè)單項技術研發(fā)(CX(21)3063)和江蘇省重點研發(fā)項目(BE2021335)資助。

This study was supported by the National Natural Science Foundation of China (31901544), the Taizhou Science and Technology Project (TN202117), the Jiangsu Modern Agricultural Industry Single Technology Research and Development (CX(21)3063), and the National Key Research and Development Program of Jiangsu (BE2021335).

URL: https://kns.cnki.net/kcms2/detail/11.1809.S.20230613.1216.006.html

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).