玉米株高和穗位高性狀全基因組關(guān)聯(lián)分析

馬雅杰 鮑建喜 高悅欣 李雅楠 秦文萱 王彥博 龍 艷 李金萍 董振營(yíng)* 萬(wàn)向元*

玉米株高和穗位高性狀全基因組關(guān)聯(lián)分析

馬雅杰1,**鮑建喜1,**高悅欣1李雅楠1秦文萱1王彥博1龍 艷1李金萍2董振營(yíng)1,2,*萬(wàn)向元1,2,*

1北京科技大學(xué)生物與農(nóng)業(yè)研究中心 / 化學(xué)與生物工程學(xué)院 / 順德研究生院 / 北京中智生物農(nóng)業(yè)國(guó)際研究院, 北京 100083;2北京首佳利華科技有限公司 / 主要作物生物育種北京市工程實(shí)驗(yàn)室 / 生物育種北京市國(guó)際科技合作基地, 北京 100192

適宜的株高和穗位高可提高植株的養(yǎng)分利用效率及抗倒伏性, 對(duì)玉米增產(chǎn)和穩(wěn)產(chǎn)具有重要意義。為揭示玉米株高和穗位高遺傳機(jī)制, 本研究以854份玉米自交系為關(guān)聯(lián)群體, 利用均勻分布于玉米10條染色體的2795個(gè)SNP標(biāo)記對(duì)4個(gè)環(huán)境下玉米株高、穗位高以及穗位系數(shù)進(jìn)行全基因組關(guān)聯(lián)分析(genome-wide association study, GWAS)。共定位到81個(gè)顯著關(guān)聯(lián)SNP位點(diǎn)(<0.0001), 其中與株高顯著關(guān)聯(lián)的SNP為35個(gè), 單個(gè)位點(diǎn)表型解釋率為0.02%～6.23%; 與穗位高顯著關(guān)聯(lián)SNP為31個(gè), 單個(gè)位點(diǎn)表型變異解釋率為0.03%～3.06%; 與穗位系數(shù)顯著關(guān)聯(lián)的SNP位點(diǎn)為24個(gè), 單個(gè)位點(diǎn)表型變異解釋率為0.03%～6.64%。進(jìn)一步鑒定出15個(gè)可在2個(gè)及以上環(huán)境共定位的穩(wěn)定SNP, 其中6個(gè)為本研究首次發(fā)現(xiàn), 9個(gè)位于前人定位QTL區(qū)間或/和關(guān)聯(lián)SNP位點(diǎn)2 Mb范圍內(nèi)。在15個(gè)穩(wěn)定SNP位點(diǎn)上下游各200 kb的置信區(qū)間共發(fā)現(xiàn)83個(gè)功能注釋基因, 結(jié)合文獻(xiàn)分析篩選出了每個(gè)位點(diǎn)最有可能的候選基因, 這些候選基因主要參與激素合成與信號(hào)轉(zhuǎn)導(dǎo)、糖類(lèi)代謝、細(xì)胞分裂調(diào)控等途徑。鑒定出6個(gè)主效SNP位點(diǎn), 并發(fā)現(xiàn)1個(gè)可同時(shí)調(diào)控株高、穗位高和穗位系數(shù)的一因多效位點(diǎn)。本研究可為分子標(biāo)記輔助選擇育種提供有效遺傳位點(diǎn), 為精細(xì)定位和克隆株高與穗位高相關(guān)性狀基因提供參考。

玉米; 株高; 穗位高; 全基因組關(guān)聯(lián)分析; 候選基因

玉米是世界重要糧食作物, 同時(shí)大量用作動(dòng)物飼料和工業(yè)原料, 在社會(huì)生產(chǎn)和生活中具有重要地位[1-3]。據(jù)國(guó)家統(tǒng)計(jì)局(http://www.stats.gov.cn/)數(shù)據(jù), 我國(guó)玉米播種面積在2020年達(dá)到4126萬(wàn)公頃, 為我國(guó)種植面積最大的農(nóng)作物。因此, 提高玉米單產(chǎn)對(duì)保障我國(guó)糧食安全具有重要意義[4-5]。適宜的株高(plant height, PH)和穗位高(ear height, EH)可提高植株的養(yǎng)分利用效率、改善株型結(jié)構(gòu), 進(jìn)而有利于提高單產(chǎn)[6-9]。同時(shí), 相對(duì)較低的株高和穗位高有利于降低玉米植株重心, 提高抗倒伏性, 從而有利于玉米穩(wěn)產(chǎn)[10-13]。

玉米株高、穗位高均屬于典型數(shù)量性狀, 目前國(guó)內(nèi)外已有通過(guò)QTL (quantitative trait locus)定位以及全基因組關(guān)聯(lián)分析(genome-wide association analysis, GWAS)等方法挖掘株高及穗位高等性狀遺傳位點(diǎn)的研究報(bào)道。對(duì)于株高性狀, Beavis等[14]較早通過(guò)構(gòu)建多個(gè)F2群體, 利用RFLP標(biāo)記定位到14個(gè)株高QTL, 分析結(jié)果顯示其中大多數(shù)QTL與已克隆株高調(diào)控基因位點(diǎn)吻合。Yan等[15]通過(guò)創(chuàng)制F2:3群體, 利用RFLP及SSR標(biāo)記構(gòu)建遺傳連鎖圖譜, 在玉米發(fā)育的5個(gè)不同時(shí)期共定位8個(gè)株高QTL, 其中3個(gè)QTL可在不同發(fā)育時(shí)期均被檢測(cè)到, 表明這些QTL在玉米不同發(fā)育時(shí)期均對(duì)株高具有調(diào)控作用。Weng等[16]利用包含44,235個(gè)有效SNP位點(diǎn)的MaizeSNP50芯片, 基于混合線性模型(mixed linear model, MLM)對(duì)284個(gè)玉米自交系進(jìn)行株高性狀GWAS分析, 共檢測(cè)到204個(gè)顯著關(guān)聯(lián)SNP位點(diǎn), 定位區(qū)間發(fā)現(xiàn)赤霉素、生長(zhǎng)素和表觀遺傳途徑相關(guān)基因, 暗示這些基因可能參與了優(yōu)良自交系矮化表型形成。對(duì)于穗位高性狀, Bai等[17]利用以Zong3為背景的近等基因?qū)胂岛突亟蝗后w以及120個(gè)SSR標(biāo)記進(jìn)行QTL定位, 共檢測(cè)到15個(gè)穗位高QTL, 其單個(gè)QTL表型變異解釋率為5.6%～31.2%, 同時(shí)檢測(cè)到3個(gè)在不同年份間較為穩(wěn)定的位點(diǎn)。Vanous等[18]利用包含252個(gè)雙單倍體株系的關(guān)聯(lián)群體和采用GBS (genotyping-by-sequencing)方法所獲得的62,077個(gè)SNP標(biāo)記進(jìn)行GWAS分析, 共鑒定出30個(gè)顯著SNP位點(diǎn)以及20個(gè)與生長(zhǎng)素和赤霉素信號(hào)等途徑相關(guān)的候選基因。針對(duì)穗位系數(shù)(EH/PH), Wang等[19]通過(guò)構(gòu)建1021個(gè)重組自交系和采用GBS方法所獲得的16,769個(gè)SNP標(biāo)記, 共定位到8個(gè)QTL, 其中一些QTL區(qū)間包含已知可調(diào)控莖稈伸長(zhǎng)的基因如、、和。劉坤等[20]以284份自交系為關(guān)聯(lián)群體, 利用56萬(wàn)個(gè)SNP標(biāo)記進(jìn)行GWAS分析, 共關(guān)聯(lián)到5個(gè)與穗位系數(shù)性狀顯著關(guān)聯(lián)的位點(diǎn), 其中位于8號(hào)染色體的位點(diǎn)可解釋10.12%的表型變異。

1 材料與方法

1.1 試驗(yàn)材料

以854份玉米自交系為供試群體材料, 將該群體分別在2020年和2021年種植于北京平谷(PG)和山東諸城(ZC) 4個(gè)環(huán)境, 即20PG、20ZC、21PG和21ZC。每材料雙行區(qū)種植, 行長(zhǎng)6.0 m, 行距0.6 m, 株距0.2 m, 其他同常規(guī)大田管理。

1.2 表型測(cè)定與分析

在玉米乳熟期, 每個(gè)家系隨機(jī)選取5個(gè)單株, 測(cè)量株高(PH)和穗位高(EH)。株高指地面至雄穗頂端的距離, 穗位高指地面至第1果穗著生節(jié)的距離, 單位均為cm, 穗位系數(shù)(EH/PH)為穗位高和株高的比值。采用IBM SPSS Statistics 23.0軟件對(duì)3類(lèi)性狀均值、標(biāo)準(zhǔn)差、變異系數(shù)、變異范圍、偏度、峰度進(jìn)行描述性統(tǒng)計(jì)分析。參照Knapp等[21]方法計(jì)算廣義遺傳力2,2g2g2gl2gy2e2。式中,2表示廣義遺傳力,g2為遺傳方差,gl2為基因型與地點(diǎn)互作方差,gy2為基因型與年份互作方差,e2為殘差方差,為地點(diǎn)數(shù),為年份數(shù)。利用Origin 2018軟件計(jì)算不同性狀各環(huán)境間的皮爾森相關(guān)系數(shù)(Pearson correlation coefficient), 利用R包“l(fā)me4”計(jì)算株高、穗位高和穗位系數(shù)的最佳線性無(wú)偏預(yù)測(cè)值(best linear unbiased prediction, BLUP)。

1.3 DNA提取與基因型檢測(cè)

在玉米五葉期, 取關(guān)聯(lián)群體植株的葉片, CTAB法[22]提取DNA, 采用由北京市農(nóng)林科學(xué)院玉米研究中心研發(fā)的包含3072個(gè)SNP位點(diǎn)的MaizeSNP3072芯片[23]進(jìn)行基因分型。去除雜合率大于10%、最小等位基因頻率小于5%和缺失大于20%的SNP, 最終篩選出2795個(gè)高質(zhì)量的SNP標(biāo)記, 利用Structure 2.3.4軟件進(jìn)行群體結(jié)構(gòu)分析[24], 利用FastTree軟件鄰接法(Neighbor-Joining, NJ)構(gòu)建關(guān)聯(lián)分析群體系統(tǒng)進(jìn)化樹(shù)。

1.4 全基因組關(guān)聯(lián)分析

利用R包“FarmCPU”進(jìn)行全基因組關(guān)聯(lián)分析[25], 以群體結(jié)構(gòu)-主成分(principal components analysis, PCA)為協(xié)變量。Bonferroni校正作為最嚴(yán)格的多重假設(shè)檢驗(yàn)校正方法, 可能導(dǎo)致較高的假陰性[26-27], 根據(jù)Bonferroni校正,= 0.05/2795 = 1.79E-05 (即–log10() = 4.75)。為減少假陰性, 本研究取–log10() = 4 (即< 1E-04)為顯著性閾值[16,28-30]。利用線性回歸方法[31]計(jì)算表型變異解釋率(phenotypic variation explained, PVE)。利用R包“CMplot”繪制曼哈頓圖及QQ-PLOT圖。

1.5 候選基因功能注釋

利用Tassel 5.0軟件計(jì)算群體LD (linkage disequilibrium)衰減距離[32], 結(jié)合Maize GDB (https:// www.maizegdb.org/)數(shù)據(jù)庫(kù)B73 (RefGen_v4)參考基因組確定區(qū)間內(nèi)候選基因, 基于UniProt數(shù)據(jù)庫(kù)(http://www.uniprot.org/)和NCBI數(shù)據(jù)庫(kù)(NCBI, http://www.ncbi.nlm.nih.gov/)基因功能注釋和對(duì)比文獻(xiàn)分析篩選出最可能的候選基因[33]。

2 結(jié)果與分析

2.1 表型統(tǒng)計(jì)分析

對(duì)于株高性狀, 4個(gè)環(huán)境下均值在179.73～209.99 cm之間, 表型變異范圍在79.00～291.00 cm之間, 變異系數(shù)在13.05%～13.40%; 對(duì)于穗位高性狀, 4個(gè)環(huán)境下均值在58.91～79.53 cm之間, 表型變異范圍在19.50～139.40 cm之間, 變異系數(shù)在19.90%～22.24%; 對(duì)于穗位系數(shù)性狀, 4個(gè)環(huán)境下均值在0.33～0.39之間, 表型變異范圍在0.18～0.59之間, 變異系數(shù)在12.82%～15.79% (表1)。3個(gè)表型性狀在各個(gè)環(huán)境變異系數(shù)均大于10%, 表明該群體中所研究農(nóng)藝性狀存在較為豐富的表型變異。3個(gè)性狀偏度和峰度絕對(duì)值均小于1, 數(shù)據(jù)分布曲線符合正態(tài)分布(圖1), 說(shuō)明株高、穗位高以及穗位系數(shù)符合數(shù)量性狀特征。

(圖1)

處理縮寫(xiě)同表1。Abbreviations are the same as those given in Table 1.

表1 株高、穗位高和穗位系數(shù)表型數(shù)據(jù)統(tǒng)計(jì)分析

a: 株高和穗位高單位為cm。PH、EH和EH/PH分別表示株高、穗位高和穗位系數(shù); PG、ZC分別表示平谷和諸城。

a: the units of plant height and ear height are in cm. PH, EH, and EH/PH represent plant height, ear height, and the ratio of EH to PH, respectively; PG and ZCrepresent Pinggu and Zhucheng, respectively.

表2 不同環(huán)境間PH、EH和EH/PH性狀相關(guān)性分析

*: 在< 0.05水平顯著相關(guān);**在< 0.01水平顯著相關(guān)。處理縮寫(xiě)同表1。

*:< 0.05;**:< 0.01. Abbreviations are the same as those given in Table 1.

相關(guān)性分析表明不同環(huán)境間同一性狀數(shù)據(jù)均呈極顯著相關(guān)(< 0.01), 株高、穗位高和穗位系數(shù)性狀相應(yīng)相關(guān)系數(shù)分別為0.716～0.847、0.699～0.799和0.711～0.788 (表2)。不同性狀之間相關(guān)性分析表明株高與穗位高性狀之間呈極顯著正相關(guān)(< 0.01),相關(guān)系數(shù)在0.424～0.706之間; 穗位高與穗位系數(shù)之間同樣呈極顯著正相關(guān)(< 0.01), 相關(guān)系數(shù)在0.522～0.809之間; 而株高與穗位系數(shù)之間只在少數(shù)環(huán)境下呈微弱正相關(guān)(表2)。株高廣義遺傳力為0.81, 穗位高廣義遺傳力為0.78, 穗位系數(shù)廣義遺傳力為0.74, 表明株高、穗位高以及穗位系數(shù)主要受遺傳因素影響(表1), 可以進(jìn)行后續(xù)分析。

2.2 全基因組關(guān)聯(lián)分析

利用2795個(gè)高質(zhì)量SNP分析854份玉米自交系遺傳多樣性, 發(fā)現(xiàn)本群體材料大致分為10個(gè)類(lèi)群, 表明具有較高遺傳多樣性(附圖1)。在考慮群體結(jié)構(gòu)情況下利用“FarmCPU”模型對(duì)4個(gè)環(huán)境株高、穗位高以及穗位系數(shù)表型值及其BLUP育種值分別進(jìn)行全基因組關(guān)聯(lián)分析。共檢測(cè)到81個(gè)顯著關(guān)聯(lián)SNP位點(diǎn), 其中與株高性狀顯著關(guān)聯(lián)的SNP位點(diǎn)有35個(gè), 單個(gè)SNP位點(diǎn)表型變異解釋率范圍為0.02%～ 6.23%; 與穗位高顯著關(guān)聯(lián)的SNP位點(diǎn)有31個(gè), 單個(gè)SNP位點(diǎn)表型變異解釋率范圍為0.03%～3.06%; 與穗位系數(shù)顯著關(guān)聯(lián)的SNP位點(diǎn)有24個(gè), 單個(gè)SNP位點(diǎn)表型變異解釋率范圍為0.03%～6.64% (附表1)。進(jìn)一步發(fā)現(xiàn)株高與穗位高性狀共定位SNP有5個(gè), 穗位高與穗位系數(shù)性狀共定位SNP有4個(gè), 而株高與穗位系數(shù)性狀未發(fā)現(xiàn)共定位SNP位點(diǎn), 這與表型相關(guān)性分析中株高與穗位系數(shù)基本無(wú)顯著相關(guān)的結(jié)果相吻合(表2和附表1)。將每個(gè)性狀中被檢測(cè)到2次及以上的SNP視為穩(wěn)定SNP位點(diǎn), 鑒定出8個(gè)與株高性狀顯著相關(guān)的穩(wěn)定SNP位點(diǎn), 分別位于1號(hào)、2號(hào)、4號(hào)、5號(hào)染色體, 其中值最低的SNP位點(diǎn)為PZE-104102768 (5.59E-06), PVE最高的SNP位點(diǎn)為PZE-101256370 (5.19%), 被檢測(cè)到次數(shù)最多位點(diǎn)為PZE-105102182, 分別在BLUP和20ZC、21PG、21ZC環(huán)境中被檢測(cè)到(表3)。與穗位高性狀顯著關(guān)聯(lián)的穩(wěn)定SNP位點(diǎn)為4個(gè), 分別位于1號(hào)、5號(hào)、8號(hào)染色體, P 值最低位點(diǎn)為PZE-108069615 (3.04E-07), PVE 最高位點(diǎn)為PZE-105098019 (3.00%) (表3)。穗位系數(shù)性狀關(guān)聯(lián)分析中檢測(cè)到4個(gè)穩(wěn)定SNP 位點(diǎn), 分別位于3號(hào)、8號(hào)染色體, P 值最低SNP 位點(diǎn)為PZE-103083718(2.27E-06), PVE 最高SNP 位點(diǎn)為PZE-108069615(6.64%) (表3)。鑒定出1個(gè)位于8號(hào)染色體穗位高與穗位系數(shù)共定位穩(wěn)定SNP 位點(diǎn)PZE-108069615, 該位點(diǎn)可解釋2.36%穗位高性狀表型變異和6.64%穗位系數(shù)性狀表型變異(表3)。

(圖2)

A、B、C分別為株高、穗位高、穗位系數(shù)性狀關(guān)聯(lián)分析的曼哈頓圖; D、E、F分別為株高、穗位高、穗位系數(shù)性狀關(guān)聯(lián)分析的QQ圖。BLUP表示最佳線性無(wú)偏預(yù)測(cè)值。其他處理縮寫(xiě)同表1。

A, B, and C were Manhattan plots for GWAS of PH, EH, and EH/PH, respectively. D, E, and F were QQ plots for GWAS of PH, EH, and EH/PH, respectively. BLUP represents best linear unbiased prediction. Other abbreviations are the same as those given in Table 1.

2.3 等位變異效應(yīng)分析

分別選取株高、穗位高和穗位系數(shù)顯著相關(guān)穩(wěn)定SNP位點(diǎn)中PVE最大的2個(gè)SNP作為主效SNP, 并分析其等位變異效應(yīng)。對(duì)于株高性狀, 發(fā)現(xiàn)2個(gè)主效SNP不同等位變異在20PG、20ZC、21PG和21ZC 4個(gè)環(huán)境均可導(dǎo)致極顯著表型差異, 如PZE-101109358位點(diǎn)的G/G等位變異在4個(gè)環(huán)境下的平均株高比A/A等位變異降低8.81～10.81 cm, PZE-101256370位點(diǎn)的A/A等位變異在4個(gè)環(huán)境下的平均株高比G/G等位變異降低8.74～11.51 cm (圖3-A)。對(duì)于穗位高性狀, 攜帶2個(gè)主效SNP不同等位變異的自交系材料在4個(gè)環(huán)境下表型同樣呈顯著差異。其中PZE-105098019位點(diǎn)的G/G等位變異在4個(gè)環(huán)境下平均穗位高比A/A等位變異降低4.24～7.81 cm; PZE-101024808位點(diǎn)的C/C等位變異在4個(gè)環(huán)境下的平均穗位高比A/A等位變異降低2.13～6.06 cm (圖3-A)。對(duì)于穗位系數(shù)性狀, PZE-103075978位點(diǎn)的G/G等位變異在4個(gè)環(huán)境下導(dǎo)致平均穗位系數(shù)比A/A等位變異降低0.020～0.031, PZE-108069615位點(diǎn)的G/G等位變異則比A/A等位變異平均穗位系數(shù)降低0.017～0.032 (圖3-A)。

21世紀(jì)已經(jīng)成為名副其實(shí)的互聯(lián)網(wǎng)時(shí)代，互聯(lián)網(wǎng)成為創(chuàng)新的典型代表，也反過(guò)來(lái)極大促進(jìn)了創(chuàng)新創(chuàng)業(yè)工作。近年來(lái)，互聯(lián)網(wǎng)逐漸從“網(wǎng)絡(luò)化”發(fā)展為“智能化”，這使得網(wǎng)絡(luò)思想政治教育一直處于追蹤和把握新技術(shù)的過(guò)程。從圖4可以看出，以報(bào)紙、雜志、電視、廣播為代表的傳統(tǒng)大眾傳媒已日趨勢(shì)微，而以門(mén)戶(hù)網(wǎng)站和“兩微一端”為代表的互聯(lián)網(wǎng)和新媒體平臺(tái)成為了青年學(xué)生了解時(shí)事熱點(diǎn)的最重要渠道。

在穩(wěn)定SNP位點(diǎn)中, 株高性狀關(guān)聯(lián)位點(diǎn)PZE-105102182被重復(fù)檢測(cè)到的次數(shù)最多, 推測(cè)該位點(diǎn)攜帶可在不同環(huán)境穩(wěn)定調(diào)控株高性狀的重要基因, 因此對(duì)該位點(diǎn)進(jìn)一步分析。發(fā)現(xiàn)A/A等位變異導(dǎo)致株高性狀在20PG、20ZC、21PG和21ZC環(huán)境平均比G/G等位變異分別降低4.51、7.04、5.48和4.64 cm (圖3-B)。進(jìn)一步分析穗位高和穗位系數(shù)性狀, 發(fā)現(xiàn)A/A等位變異導(dǎo)致20PG、20ZC、21PG和21ZC環(huán)境的平均穗位高比G/G等位變異分別降低5.42、7.01、4.91和5.02 cm, 穗位系數(shù)則分別降低0.018、0.022、0.016、0.019, 差異均達(dá)到極顯著水平(圖3-B), 表明PZE-105102182位點(diǎn)具有一因多效性, A/A為優(yōu)異等位變異, 可同時(shí)負(fù)調(diào)控株高、穗位高和穗位系數(shù)性狀。

2.4 候選基因分析

以2降至0.2所對(duì)應(yīng)物理距離為該群體LD衰減距離[34-35], 計(jì)算得到該群體的衰減距離約為200 kb, 與前人結(jié)果較為一致[36-38]。在3個(gè)性狀檢測(cè)到的15個(gè)穩(wěn)定SNP位點(diǎn)上下游200 kb置信區(qū)間共發(fā)現(xiàn)173個(gè)候選基因, 其中具有功能注釋基因?yàn)?3個(gè)。通過(guò)前期總結(jié), 發(fā)現(xiàn)玉米分生組織調(diào)控、微管活動(dòng)、蔗糖運(yùn)輸、根毛生長(zhǎng)、光敏色素合成相關(guān)基因以及激素信號(hào)、AP2類(lèi)轉(zhuǎn)錄因子等的相關(guān)基因可調(diào)控玉米株高、穗位高性狀[33]。因此, 若置信區(qū)間存在以上生物途徑相關(guān)基因, 則篩選顯著SNP標(biāo)記最近的該類(lèi)基因作為候選基因; 若置信區(qū)間無(wú)以上生物途徑相關(guān)基因, 則以距離顯著SNP標(biāo)記最近、且具有功能注釋的基因?yàn)楹蜻x基因。共篩選到15個(gè)候選基因, 進(jìn)一步分析發(fā)現(xiàn)6個(gè)候選基因與激素的合成與信號(hào)轉(zhuǎn)導(dǎo)途徑相關(guān), 分別為。5個(gè)候選基因與糖類(lèi)代謝相關(guān), 分別為; 3個(gè)候選基因參與細(xì)胞分裂調(diào)控途徑, 分別為。針對(duì)特定性狀, 2個(gè)株高性狀主效位點(diǎn)候選基因分別為編碼脫落酸(ABA)信號(hào)轉(zhuǎn)導(dǎo)途徑核質(zhì)轉(zhuǎn)運(yùn)蛋白的Zm00001d034914和編碼甘露糖苷酶的。2個(gè)穗位高性狀主效位點(diǎn)候選基因分別為編碼RING/U-box泛素連接酶的和編碼巖藻糖基轉(zhuǎn)移酶蛋白的。2個(gè)穗位系數(shù)性狀主效位點(diǎn)候選基因分別為編碼CPP轉(zhuǎn)錄因子的和編碼Dof鋅指蛋白的。

圖3 株高、穗位高和穗位系數(shù)相關(guān)重要SNP位點(diǎn)等位變異效應(yīng)分析

Fig. 3 Allelic effects of the SNPs associated with plant height (PH), ear height (EH), and the ratio of EH/PH (EH/PH) traits

A: 株高、穗位高、穗位系數(shù)主效SNP位點(diǎn)等位變異效應(yīng)分析; B: PZE-105102182位點(diǎn)對(duì)不同性狀的等位效應(yīng)分析。*: 在< 0.05水平上顯著相關(guān);**: 在< 0.01水平上顯著相關(guān)。處理縮寫(xiě)同表1。

A: the allelic effect of dominant SNP locus in plant height, ear height, and the ratio of EH to PH; B: the allelic effect of PZE-105102182 locus on different traits.*: significance correlation at< 0.05;**: significant correlation at< 0.01. Abbreviations are the same as those given in Table 1.

3 討論

3.1 株高、穗位高相關(guān)性狀定位結(jié)果分析

株高、穗位高及穗位系數(shù), 為多基因控制的數(shù)量性狀, 其直接影響植株的養(yǎng)分利用率及抗倒伏性[14-20]。全基因組關(guān)聯(lián)分析方法具有高分辨率和高通量的優(yōu)勢(shì), 可以高效鑒定調(diào)控重要性狀關(guān)鍵位點(diǎn)和其優(yōu)異等位變異[39]。本研究利用854份自交系群體進(jìn)行株高、穗位高及穗位系數(shù)性狀GWAS, 共檢測(cè)到81個(gè)顯著關(guān)聯(lián)SNP位點(diǎn), 株高與穗位高、穗位高與穗位系數(shù)均鑒定出共定位SNP, 未發(fā)現(xiàn)株高與穗位系數(shù)性狀共定位位點(diǎn)?？梢?jiàn)穗位高和株高性狀可能存在相似調(diào)控機(jī)制, 但是穗位系數(shù)與株高調(diào)控機(jī)制差異較大。

與前人研究對(duì)比分析, 發(fā)現(xiàn)本研究所鑒定15個(gè)穩(wěn)定SNP位點(diǎn)中有8個(gè)位于前人定位的QTL區(qū)間, 4個(gè)SNP位點(diǎn)與前人所鑒定相關(guān)SNP物理距離小于2 Mb。如對(duì)于株高性狀, 1號(hào)染色體穩(wěn)定SNP位點(diǎn)PZE-101058322和PZE-101109358分別位于Zhou等[40]定位QTL區(qū)間和楊曉軍等[41]定位區(qū)間。4號(hào)染色體SNP標(biāo)記PZE-104088728和PZE-104102768分別位于Zhang等[42]定位QTL區(qū)間和Zhang等[42]定位QTL區(qū)間, 同時(shí)該位點(diǎn)與Hu等[43]定位SNP位點(diǎn)PZE-104103747僅相距1.15 Mb。5號(hào)染色體SNP標(biāo)記PZE-105102182位于Tang等[44]定位區(qū)間, 同時(shí)該位點(diǎn)與Liu等[45]定位SNP位點(diǎn)chr5.s_156946316僅相距0.78 Mb。對(duì)于穗位高性狀, 1號(hào)染色體SNP位點(diǎn)PZE-101024808與Dell’Acqua等[46]定位SYN12547位點(diǎn)相距1.90 Mb, 而另一個(gè)1號(hào)染色體SNP位點(diǎn)PZE-101105515同時(shí)位于Li等[28]、Tang等[44]和Park等[47]定位QTL區(qū)間和Pan等[48]所關(guān)聯(lián)SNP位點(diǎn)1.92 Mb距離位置。5號(hào)染色體PZE-105098019同時(shí)位于楊曉軍等[41]、Li等[28]以及Li等[49]定位的QTL區(qū)間。8號(hào)染色體PZE-108069615位點(diǎn)位于Park等[47]定位的QTL區(qū)間。由于目前對(duì)穗位系數(shù)性狀研究較少, 僅發(fā)現(xiàn)8號(hào)染色體SNP位點(diǎn)PZE-108069615位于Wang等[19]定位的QTL區(qū)段。

綜上, 本研究鑒定的15個(gè)穩(wěn)定SNP位點(diǎn)中, 共有9個(gè)位點(diǎn)與前人定位研究結(jié)果相吻合, 表明本研究關(guān)聯(lián)結(jié)果具有較高可信度, 所挖掘SNP可為分子標(biāo)記輔助選擇育種提供依據(jù)。同時(shí)發(fā)現(xiàn)一些SNP位點(diǎn)與前人多個(gè)獨(dú)立研究定位結(jié)果相吻合, 暗示該位點(diǎn)附近可能存在可在不同環(huán)境穩(wěn)定調(diào)控株高或穗位高性狀的重要基因, 是后續(xù)克隆穗位高調(diào)控基因的重要目標(biāo)區(qū)域。

6個(gè)穩(wěn)定SNP位點(diǎn)為本研究所首次發(fā)現(xiàn), 包括3個(gè)株高性狀SNP位點(diǎn), 其中在BLUP和20PG、21ZC環(huán)境中均檢測(cè)到PZE-101256370, 且該位點(diǎn)PVE最高, 為主效SNP (表3)。另外3個(gè)SNP新位點(diǎn)分別為穗位系數(shù)顯著關(guān)聯(lián)PZE-103075978、PZE- 103083718和PZE-108039693, 其中PZE-103075978最高可解釋5.58%表型變異, 屬于主效SNP。這些株高和穗位系數(shù)相關(guān)新位點(diǎn)可同時(shí)在不同環(huán)境下, 具有較高可信度, 有助于進(jìn)一步解析相關(guān)性狀的遺傳結(jié)構(gòu)。

鑒定重要遺傳位點(diǎn)優(yōu)異等位變異有助于利用分子標(biāo)記輔助選擇將多個(gè)優(yōu)異等位基因進(jìn)行聚合, 從而培育優(yōu)異種質(zhì)。如李等[50]通過(guò)分子標(biāo)記輔助選擇與傳統(tǒng)育種手段相結(jié)合, 實(shí)現(xiàn)了玉米抗南方銹病、抗莖腐病以及抗矮花葉病等多個(gè)性狀的聚合育種。本研究對(duì)株高、穗位高和穗位系數(shù)性狀的主效SNP位點(diǎn)進(jìn)行分析, 發(fā)現(xiàn)該6個(gè)位點(diǎn)不同等位變異在4個(gè)環(huán)境間表型均具有顯著差異, 優(yōu)異等位變異可分別顯著降低株高、穗位高及穗位系數(shù), 說(shuō)明以上主效SNP位點(diǎn)可用于分子標(biāo)記輔助選擇育種。此外, 本研究檢測(cè)到一個(gè)可協(xié)同調(diào)控玉米株高、穗位高及穗位系數(shù)性狀的SNP位點(diǎn)PZE-105102182, 該位點(diǎn)的優(yōu)異等位變異可分別使株高、穗位高、穗位系數(shù)在4個(gè)環(huán)境下都得到顯著降低, 因此該位點(diǎn)具有一因多效性, 可作為分子標(biāo)記協(xié)同改良上述性狀。

3.2 候選基因分析

目前已克隆到多個(gè)株高、穗位高相關(guān)性狀調(diào)控基因, 主要包括激素相關(guān)基因, 如生長(zhǎng)素(auxin)、赤霉素(gibberellin, GA)、油菜素內(nèi)酯(brassinolide, BR)、獨(dú)腳金內(nèi)酯(strigolactone, SL)等, 同時(shí)微管發(fā)育、糖代謝、纖維素合成、根毛生長(zhǎng)、轉(zhuǎn)錄因子調(diào)控等過(guò)程均可影響植株莖稈發(fā)育[33]。由此, 根據(jù)已克隆基因文獻(xiàn)報(bào)道預(yù)測(cè)了穩(wěn)定SNP位點(diǎn)置信區(qū)間的最可能候選基因。結(jié)果顯示株高性狀候選區(qū)間內(nèi)存在有較多可能與莖稈發(fā)育相關(guān)的候選基因, 如PZE-101256370位點(diǎn)候選基因編碼核質(zhì)轉(zhuǎn)運(yùn)蛋白。Xu等[51]發(fā)現(xiàn)擬南芥Exportin1A (XPO1A)作為一種核質(zhì)轉(zhuǎn)運(yùn)蛋白, 參與脫落酸(ABA)信號(hào)轉(zhuǎn)導(dǎo), 戚義東等[52]發(fā)現(xiàn)脫落酸可通過(guò)拮抗赤霉素降低水稻株高, 因此推測(cè)候選基因可能通過(guò)ABA途徑參與株高性狀調(diào)控。糖類(lèi)在植物株高發(fā)育中扮演著重要角色, 為細(xì)胞分裂、伸長(zhǎng)等生命活動(dòng)提供了必要的物質(zhì)基礎(chǔ), 如已克隆株高性狀調(diào)控基因, 其突變體葉中因不能正常輸出蔗糖, 導(dǎo)致植株表現(xiàn)矮小[53]。候選基因編碼甘露糖苷酶, 其參與甘露糖苷鍵的水解過(guò)程, 甘露糖是一種單糖, 為多種多糖的組成成分, 由此推測(cè)該候選基因可能通過(guò)影響糖類(lèi)物質(zhì)合成進(jìn)而影響株高性狀。

穗位高性狀關(guān)聯(lián)位點(diǎn)PZE-105098019候選區(qū)間內(nèi)基因編碼泛素連接酶, 泛素-蛋白酶體途徑在生長(zhǎng)素、赤霉素、油菜素內(nèi)酯等影響莖稈發(fā)育的激素信號(hào)轉(zhuǎn)導(dǎo)中具有重要作用[54], 由此推測(cè)可能通過(guò)參與激素信號(hào)傳導(dǎo)。PZE-101024808候選區(qū)間基因編碼巖藻糖基轉(zhuǎn)移酶蛋白, 馮玥[55]研究發(fā)現(xiàn), 棉花巖藻糖基轉(zhuǎn)移酶家族基因?qū)τ诩?xì)胞伸長(zhǎng)具有重要作用, 推測(cè)可能同樣通過(guò)調(diào)控細(xì)胞伸長(zhǎng)進(jìn)而影響穗位高性狀。

針對(duì)穗位系數(shù)性狀, 候選基因中鑒定出較多轉(zhuǎn)錄因子編碼基因(表3)。如PZE-103075978位點(diǎn)候選基因編碼Dof鋅指蛋白, Skirycz等[56]研究發(fā)現(xiàn), 過(guò)表達(dá)擬南芥Dof轉(zhuǎn)錄因子基因, 會(huì)影響植物的細(xì)胞大小和數(shù)量, 造成植株矮化, 推測(cè)可能通過(guò)影響細(xì)胞大小和數(shù)量, 進(jìn)而調(diào)控穗位系數(shù)性狀。PZE-108069615是穗位高和穗位系數(shù)性狀共定位的SNP位點(diǎn), 該位點(diǎn)候選基因編碼CPP轉(zhuǎn)錄因子, 而CPP轉(zhuǎn)錄因子參與花器官的發(fā)育, 在控制生殖組織發(fā)育和細(xì)胞分裂過(guò)程中起著極其重要的作用[57], 推測(cè)可能通過(guò)影響生殖組織發(fā)育來(lái)影響果穗的形成, 從而影響穗位高和穗位系數(shù)性狀。此外, 本研究定位到一個(gè)一因多效位點(diǎn)PZE- 105102182, 該位點(diǎn)的優(yōu)異等位變異可使株高、穗位高、穗位系數(shù)均得到顯著降低, 因此挖掘該位點(diǎn)的候選基因?qū)f(xié)同改良植物株型具有重要意義。該位點(diǎn)候選基因編碼絲氨酸/蘇氨酸磷酸酶, 是參與催化蛋白質(zhì)去磷酸化的主要酶類(lèi)之一, 可參與激素信號(hào)轉(zhuǎn)導(dǎo)途徑[58-59], 如已克隆玉米株高調(diào)控基因編碼的絲氨酸/蘇氨酸激酶參與油菜素內(nèi)酯的信號(hào)傳導(dǎo), 其功能缺失突變體植株由于節(jié)間縮短而表現(xiàn)為矮小[60], 因此推測(cè)可能通過(guò)調(diào)控與油菜素內(nèi)酯的信號(hào)傳導(dǎo)調(diào)控玉米株型性狀, 其調(diào)控功能仍有待進(jìn)一步驗(yàn)證。

4 結(jié)論

本研究以854份自交系作為關(guān)聯(lián)群體, 利用2795個(gè)SNP標(biāo)記, 采用FarmCPU模型針對(duì)株高、穗位高、穗位系數(shù)性狀進(jìn)行全基因組關(guān)聯(lián)分析, 共檢測(cè)到81個(gè)顯著關(guān)聯(lián)SNP。在2個(gè)及以上環(huán)境中被穩(wěn)定檢測(cè)到的穩(wěn)定SNP位點(diǎn)為15個(gè), 其中6個(gè)SNP為本研究首次發(fā)現(xiàn)。在穩(wěn)定SNP位點(diǎn)上下游各200 kb置信區(qū)間內(nèi), 根據(jù)基因功能注釋及文獻(xiàn)資料, 篩選出各位點(diǎn)的最可能候選基因, 這些候選基因主要參與激素合成與信號(hào)轉(zhuǎn)導(dǎo)、糖類(lèi)代謝、細(xì)胞分裂調(diào)控等途徑。針對(duì)3個(gè)性狀分別鑒定出2個(gè)主效SNP位點(diǎn), 并鑒定出1個(gè)可同時(shí)調(diào)控株高、穗位高、穗位系數(shù)的一因多效位點(diǎn)。本研究可為分子標(biāo)記輔助選擇育種提供重要遺傳位點(diǎn), 為株高相關(guān)性狀基因精細(xì)定位和克隆提供參考和依據(jù)。

附表1 不同環(huán)境下株高、穗位高、穗位系數(shù)顯著關(guān)聯(lián)SNP位點(diǎn)匯總

(續(xù)附表1)

PH、EH、EH/PH和BLUP分別表示株高、穗位高、穗位系數(shù)和最佳線性無(wú)偏預(yù)測(cè)值; PG、ZC分別表示平谷和諸城。

PH, EH, EH/PH, and BLUP represent plant height, ear height, the ratio of EH to PH and best linear unbiased prediction, respectively; PG and ZCrepresent Pinggu and Zhucheng, respectively.

附圖1 關(guān)聯(lián)分析群體系統(tǒng)進(jìn)化樹(shù)

Fig. S1 Phylogenetic tree of the association analysis population

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Genome-wide association analysis of plant height and ear height related traits in maize

MA Ya-Jie1,**, BAO Jian-Xi1,**, GAO Yue-Xin1, LI Ya-Nan1, QIN Wen-Xuan1, WANG Yan-Bo1, LONG Yan1, LI Jin-Ping2, DONG Zhen-Ying1,2,*, and WAN Xiang-Yuan1,2,*

1Zhongzhi International Institute of Agricultural Biosciences, Shunde Graduate School, School of Chemistry and Biological Engineering, Research Center of Biology and Agriculture, University of Science and Technology Beijing (USTB), Beijing 100083, China;2Beijing Engineering Laboratory of Main Crop Bio-Tech Breeding, Beijing International Science and Technology Cooperation Base of Bio-Tech Breeding, Beijing Solidwill Sci-Tech Co. Ltd., Beijing 100192, China

Suitable plant height (PH) and ear height (EH) can improve the efficiency of nutrient utilization and lodging resistance, which is of great significance for stable and high yield in maize. In this study, an association panel including 854 maize inbred lines used to analyze the PH, EH, and the ratio of EH to PH (EH/PH) in four environments, and genome-wide association study (GWAS) was then conducted using 2795 single nucleotide polymorphism (SNP) markers distributed uniformly throughout maize genome. A total of 81 SNP loci (< 0.0001) were identified by using FarmCPU model, among which 35 SNPs were significantly associated with PH, with phenotypic variation explained (PVE) ranging from 0.020% to 6.225%; 31 SNPs were significantly associated with ear height, and PVE was from 0.026% to 3.060%; 24 SNPs were significantly associated with EH/PH, and the PVE ranged from 0.032% to 6.636%. 15 stable SNPs that were repeatedly detected in multiple environments for specific trait were further identified, among which six loci were reported for the first time in this study, and the remaining nine loci located in the previously identified quantitative trait loci (QTLs) or/and no more than 2 Mb with the known SNPs related with PH and EH traits. A total of 83 genes were annotated in the confidence intervals of the 15 stable SNPs, and the most likely candidate genes were further predicted according to the gene functional annotations and comparison with previous reports. The candidate genes were mainly involved in hormone synthesis and signal transduction, carbohydrate metabolism, cell division regulation and so on. Finally, six major SNP loci and one locus that affected PH, EH, and EH/PH simultaneously were identified. This study can provide genetic loci for molecular marker-assisted selection in maize breeding and provide valuable information for fine mapping and cloning of PH and EH related genes.

maize; plant height; ear height; genome-wide association analysis; candidate gene

10.3724/SP.J.1006.2023.23023

本研究由國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目“農(nóng)業(yè)生物種質(zhì)資源挖掘與創(chuàng)新利用”重點(diǎn)專(zhuān)項(xiàng)(2021YFD1200700)資助。

This study was supported by the National Key Research and Development Program of China (2021YFD1200700).

通信作者(Corresponding authors):董振營(yíng), E-mail: zydong@ustb.edu.cn; 萬(wàn)向元, E-mail: wanxiangyuan@ustb.edu.cn

同等貢獻(xiàn)(Contributed equally to this work)

馬雅杰, E-mail: 13292097686@163.com; 鮑建喜, E-mail: bjx1232003@126.com

2022-03-03;

2022-05-05;

2022-05-19.

URL: https://kns.cnki.net/kcms/detail/11.1809.S.20220518.1923.002.html

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