羅江陶，鄭建敏，鄧清燕，劉培勛，蒲宗君

重要育種親本川麥44對衍生品種的遺傳貢獻

羅江陶，鄭建敏，鄧清燕，劉培勛，蒲宗君*

四川省農(nóng)業(yè)科學(xué)院作物研究所/農(nóng)業(yè)農(nóng)村部西南地區(qū)小麥生物學(xué)與遺傳育種重點實驗室，成都 610066

【目的】小麥品種川麥44不僅本身具有高產(chǎn)、穩(wěn)產(chǎn)、廣適等特性，而且以其為親本已選育審定新品種11個，是小麥育種的一個重要親本。明確川麥44的遺傳特性，鑒定其含有的重要基因或QTL位點，為更好地利用川麥44選育新品種提供理論支撐?！痉椒ā坷脽晒庠浑s交明確小麥-外源易位對川麥44及其衍生品種的影響以及川麥44及其衍生品種在染色體層面的遺傳規(guī)律。利用660K SNP芯片數(shù)據(jù)分析川麥44對其衍生品種的遺傳貢獻，明確衍生品種中來源于川麥44的高傳遞率區(qū)段。利用已知的小麥基因功能標(biāo)記及QTL連鎖標(biāo)記，對川麥44中有利于育種的重要基因位點進行鑒定。【結(jié)果】細胞學(xué)鑒定表明川麥44不含四川小麥品種中常見的2條易位染色體6VS/6AL和1RS/1BL。其衍生品種中，僅昌麥32和昌麥34含1對1RS/1BL易位染色體，其余品種不含有小麥-外源易位染色體。系譜分析表明，昌麥32和昌麥34的易位染色體遺傳自另外一個雜交親本——昌麥19。1RS/1BL易位的導(dǎo)入可能是昌麥32和昌麥34表現(xiàn)為弱筋的原因之一。除了小麥-外源易位染色體，多個染色體的核型在川麥44及其10個衍生品種中表現(xiàn)出多態(tài)性。其中，4A染色體有2種類型，80%的衍生品種與川麥44相同核型相同；5A染色體有4種類型，與川麥44相同的頻率為40%；6B染色體有2種類型，與川麥44相同的頻率為40%，7B染色體有2種類型，與川麥44相同的頻率為40%。660K SNP芯片分析共鑒定到1 106個分布于川麥44所有染色體上的高遺傳率區(qū)段，平均長度為1.57 Mb。從基因組層面來看，B基因組的區(qū)段總長度和總數(shù)均最大。從不同染色體來看，區(qū)段最長的3條為別為4A、2B和5B，區(qū)段數(shù)最多的3條染色體分別為4A、2B和3B。利用61個已知的小麥基因功能標(biāo)記及13個產(chǎn)量相關(guān)QTL連鎖SNP標(biāo)記分析川麥44及其衍生品種，再與之前獲得的川麥44高傳遞率區(qū)段對比，發(fā)現(xiàn)有9個基因的標(biāo)記和3個QTL位點標(biāo)記錨定在川麥44高傳遞率區(qū)段內(nèi)，這些基因被認為是潛在的川麥44高被選擇基因。依據(jù)功能標(biāo)記或連鎖標(biāo)記的等位類型推斷，其中2個功能基因、和3個QTL位點、、可能是川麥44攜帶的重要優(yōu)勢等位基因或位點，在培育衍生品種過程中被優(yōu)先選擇保留。5個基因或QTL位點分別對穗發(fā)芽、有效分蘗數(shù)、千粒重和穗長4個性狀具有正向效應(yīng)?！窘Y(jié)論】重要育種親本川麥44基因組片段在衍生品種中的長度短，具有較高的遺傳配合力，易于與不同的同源染色體重組，不易導(dǎo)致連鎖累贅問題。、、、和是利用川麥44育種的5個重要靶基因位點，可加強對其在分子標(biāo)記輔助育種中應(yīng)用。

川麥44；遺傳貢獻；有益基因；QTL；高傳遞率

0 引言

【研究意義】小麥?zhǔn)侵袊钪匾募Z食作物之一，其生產(chǎn)發(fā)展離不開優(yōu)異種質(zhì)資源的創(chuàng)新與利用。在新品種選育過程中，一些品種（系）表現(xiàn)出優(yōu)異的豐產(chǎn)性、抗病性和高配合力等優(yōu)點，從而被育種家廣泛應(yīng)用，進而培育出較多的衍生品種，這類材料被育種家稱為骨干親本[1]。骨干親本的利用對提高品種產(chǎn)量、促進品種更新?lián)Q代具有重要意義。【前人研究進展】前人對于小麥骨干親本在衍生品種中遺傳貢獻的研究，主要是運用農(nóng)藝性狀分析、品質(zhì)分析[2]、抗性鑒定、SSR分子標(biāo)記檢測[3]及通量更高的SNP全基因組掃描[4-6]等方法估算骨干親本的遺傳貢獻。這些研究僅從表型或基因組水平上進行，而未從基因水平上估算骨干親本對衍生品種的遺傳貢獻。對育種家而言，充分解析骨干親本中具體哪些優(yōu)異性狀基因直接傳遞到現(xiàn)有的衍生品種，對更好地利用骨干親本進行新品種選育，提高育種效率，培育高產(chǎn)、抗病新品種具有重要意義。隨著SNP檢測技術(shù)的發(fā)展，已開發(fā)出基于KASP技術(shù)的小麥功能基因標(biāo)記，涉及矮稈基因[7]、產(chǎn)量性狀相關(guān)基因[8-17]、春化基因[18-21]、光周期基因[22-24]、抗病基因[25-29]、抗倒伏基因[30]、抗旱基因[31]、抗逆基因[32]、抗穗發(fā)芽基因[33-36]、品質(zhì)相關(guān)基因[37-46]、開花基因[47]，這些基因功能標(biāo)記將為研究骨干親本中的基因傳遞提供技術(shù)支持?！颈狙芯壳腥朦c】川麥44是由四川省農(nóng)業(yè)科學(xué)院作物研究所選育的中強筋品種，具有矮稈、高產(chǎn)、廣適等特性。截至目前，利用川麥44作為直接親本已培育出新品種11個。鄭建敏等[48]于2018年利用系譜分析方法，初步分析了川麥44的核質(zhì)貢獻。次年，利用小麥660K SNP芯片從DNA水平上分析了川麥44對其6個衍生品種的遺傳貢獻[49]。然而，相關(guān)研究還不夠深入，并且還未從功能基因或QTL位點層面解析川麥44的育種貢獻?！緮M解決的關(guān)鍵問題】本研究利用熒光原位雜交對川麥44及其10個衍生品種進行分析，以明確是否存在外緣染色體的影響以及川麥44及其衍生品種在染色體層面的遺傳規(guī)律；利用目前已知的部分小麥基因功能標(biāo)記及Ye等[50]鑒定出的四川小麥產(chǎn)量性狀QTL連鎖分子標(biāo)記對川麥44及其10個衍生品種進行分析，并結(jié)合660K SNP數(shù)據(jù)，篩選川麥44中可能的重要基因，為進一步利用川麥44進行新品種選育提供參考。

1 材料與方法

1.1 材料

材料包括10個川麥44的衍生品種，以及川麥44和另外6個參與雜交組配的親本材料（西昌19、川麥42、川麥36、貴農(nóng)21、川麥30和川農(nóng)23）。10個衍生品種中，川麥63、川麥1131、川麥1145、川麥1826均來源于雜交組合川麥44/川農(nóng)23；川麥66和川麥68均來源于川麥42/98-266//川麥44；川麥67的雜交組合為川麥42/川麥36//川麥44；川麥601的雜交組合為貴農(nóng)21/川麥30//川麥42/川麥44；昌麥32和昌麥34的雜交組合為川麥44/昌麥19。

1.2 原位雜交分析

隨機選取川麥44及其衍生品種和親本的種子各5粒，在墊有濕潤濾紙的培養(yǎng)皿中發(fā)芽。待根尖長至3—4 cm時，剪取根尖。參考Luo等[51]方法進行根尖的處理和體細胞制片。原位雜交探針包括2個寡核苷酸序列探針Oligo-pSc119.2-1和Oligo-pTa535[51]，由成都擎科生物科技有限公司合成（中國，成都）。使用配備CCD鏡頭的奧林巴斯BX63熒光顯微鏡進行雜交信號檢測并采集圖像。

1.3 衍生品種中川麥44基因組區(qū)段分析

所用的660K SNP芯片分型數(shù)據(jù)來源于鄭建敏等[49]研究，包括川麥44及其8個衍生品種（川麥63、川麥1131、川麥1145、川麥66、川麥68、川麥67、川麥601和昌麥32）。參照Hao等[52]方法，以中國春參考基因組v1.0為參考，將所有21條染色體分割成1 Mb大小的連續(xù)區(qū)間，統(tǒng)計每1 Mb區(qū)間范圍內(nèi)，衍生品種與其親本在該區(qū)間內(nèi)相同標(biāo)記比例。如果衍生品種一個區(qū)間內(nèi)標(biāo)記的分析結(jié)果與某個親本相同的比例高于其他親本，且相同標(biāo)記的比例大于0.50，則判斷該區(qū)段來源于此親本，否則判斷為無法識別。統(tǒng)計僅能夠識別來源的區(qū)段用于計算川麥44對其衍生品種的遺傳貢獻，計算公式為：遺傳貢獻率=川麥44來源區(qū)段數(shù)/總共識別出的區(qū)段數(shù)×100%。如果一個來源于川麥44的區(qū)段在8個衍生品種中的頻率大于50%，則將其定義為高傳遞頻率區(qū)段。高傳遞頻率區(qū)段在染色體上的分布畫圖采用R包ggplot2（v.2.2.1）繪制。

1.4 基因功能標(biāo)記和四川小麥品種產(chǎn)量相關(guān)性狀QTL位點分析

61個涉及生育期、穗發(fā)芽、產(chǎn)量性狀、品質(zhì)、抗逆性、株高基因和抗病性的小麥基因功能基因標(biāo)記[7-47]由北京中玉金標(biāo)記有限公司完成。功能標(biāo)記的檢測平臺為KASP，分析的材料包括川麥44及其衍生的10個普通小麥品種。對應(yīng)基因在染色體上的位置通過BLAST中國春參考基因組v1.0確定（http://202.194.139.32/blast/viroblast.php，比對參數(shù)為默認值）。四川小麥品種與產(chǎn)量相關(guān)的QTL及其連鎖分子標(biāo)記信息從Ye等[50]中獲得。

2 結(jié)果

2.1 川麥44及其衍生品種染色體核型分析

熒光原位雜交分析表明，川麥44不含有四川小麥品種中常見的2條易位染色體6VS/6AL和1RS/1BL。其衍生的10個品種中，昌麥32和昌麥34為1RS/1BL易位系（圖1）。川麥44及其10個衍生品種的核型在部分染色體上呈現(xiàn)多態(tài)型。其中，4A染色體有2種多態(tài)型，衍生品種中與川麥44相同的頻率為80%；5A染色體有4種多態(tài)型，與川麥44相同的頻率為40%；6B染色體有2種多態(tài)型，與川麥44相同的頻率為40%，7B染色體有2種多態(tài)型，與川麥44相同的頻率為40%。

2.2 川麥44選擇優(yōu)勢基因組區(qū)段及潛在的功能基因

由于沒有98—266的660K芯片數(shù)據(jù)，僅來源于川麥44/川農(nóng)23雜交組合的川麥63、川麥1131、川麥1145；川麥42/川麥36//川麥44雜交組合的川麥67；貴農(nóng)21/川麥30//川麥42/川麥44雜交組合的川麥601和川麥44/昌麥19雜交組合昌麥32用于分析川麥44的遺傳貢獻情況。

川麥44對川麥63、川麥1131和川麥1145的遺傳貢獻為分別為67.0%（5 827/8 692）、72.8%（6 391/8 774）和71.3%（6 300/8 836），略高于理論值50%；對川麥67的遺傳貢獻為22.4%（1 994/8 684），顯著低于理論值50%；對川麥601的遺傳貢獻為16.3%（1 189/7 275），略低于理論值25%；對昌麥32的遺傳貢獻為21.8%（2 237/10 285），顯著低于理論值50%。

紅色方框表示1RS/1BL易位系；白色方框表示染色體多態(tài)性類型

以區(qū)段在6個后代中的傳遞頻率大于50%為標(biāo)準(zhǔn)，篩選到1 721個高傳遞頻率片段。將相鄰的連續(xù)片段進行整合后，得到1 106個染色體區(qū)段，片段平均長度為1.57 Mb（1—15 Mb），分布在所有21條染色體上（圖2）。從基因組層面來看，B基因組的區(qū)段總長度和總數(shù)均最大（表1）。從不同染色體來看，區(qū)段最長的3條分別為4A（165 Mb）、2B（161 Mb）和5B（125 Mb），區(qū)段數(shù)最多的3條染色體分別為4A（100個）、2B（78個）和3B（72個）。

表1 川麥44傳遞頻率大于50%的染色體區(qū)段統(tǒng)計情況

2.3 基因功能分子標(biāo)記檢測

為了尋找可能支撐川麥44作為骨干親本的重要功能基因，利用北京中玉金標(biāo)記公司開發(fā)的61個功能標(biāo)記檢測川麥44及其10個衍生品種。這些功能標(biāo)記來自于17條染色體上的52個基因，包括產(chǎn)量相關(guān)基因標(biāo)記16個、光周期及春化相關(guān)基因標(biāo)記10個、抗病相關(guān)基因標(biāo)記8個、抗穗發(fā)芽相關(guān)基因標(biāo)記6個、品質(zhì)相關(guān)基因標(biāo)記17個、抗倒伏基因標(biāo)記1個、抗逆基因標(biāo)記2個和矮稈基因標(biāo)記1個（電子附表1）。整體來看，這61個標(biāo)記在10個衍生品種中的多樣性非常低，平均一致頻率為0.91（0.50—1.00）。將61個標(biāo)記在染色體上的物理位置與前面660K芯片分析得到的高傳遞頻率區(qū)段進行比較，有9個基因（9/52=17.31%）的標(biāo)記（12個功能標(biāo)記）落在了高傳遞頻率區(qū)段內(nèi)，這些基因被認為是潛在的川麥44高被選擇基因（表2），其中包括產(chǎn)量相關(guān)基因2個、抗病相關(guān)基因1個、品質(zhì)相關(guān)基因4個、抗穗發(fā)芽相關(guān)基因2個。然而，從基因等位類型的功能注釋來看，9個基因中，僅2個在川麥44中為有利等位類型（和），分別對應(yīng)性狀為小麥品質(zhì)和抗穗發(fā)芽。結(jié)合2個SNP標(biāo)記可以判斷，川麥44的基因等位類型為d；而衍生的10個品種中，除川麥68為b等位類型外，其余9份材料與川麥44相同。

紅色箭頭表示川麥44的有益基因；綠色箭頭表示川麥44有益QTL位點

2.4 川麥44潛在的選擇優(yōu)勢QTL位點

Ye等[50]利用165份四川小麥品種（系）進行了產(chǎn)量性狀的關(guān)聯(lián)分析，得到13個與產(chǎn)量性狀相關(guān)的QTL位點，與18個SNP標(biāo)記連鎖。為進一步尋找川麥44中潛在的重要基因位點，對川麥44中這些QTL位點（連鎖SNP標(biāo)記）的等位類型進行了分析。結(jié)果表明，69%（9/13）的QTL在川麥44中均為有利等位類型，連鎖12個SNP標(biāo)記。衍生品種與川麥44相同，且位點頻率≥50%的QTL有9個（9/13=69.23%），其中3個錨定在川麥44高傳遞率區(qū)段內(nèi)，包括、和（表3）。這3個QTL可能是川麥44的優(yōu)勢位點，其中，位于2AS和4AL染色體臂的這兩個QTL位點對千粒重具有正向效應(yīng)，而位于5AL的QTL位點對穗長具有正向效應(yīng)。

3 討論

中強筋小麥川麥44來源于雜交組合96夏440/貴農(nóng)21，具有高產(chǎn)、分蘗力強、成穗率高、矮稈、早熟等優(yōu)異特點。谷蛋白分析表明其含優(yōu)質(zhì)亞基5+10[53]，可能是其表現(xiàn)為中強筋的支撐基因之一。本研究利用基因功能標(biāo)記發(fā)現(xiàn)，川麥44含有，并且該基因在其衍生品種中的頻率高達90%，川麥68是唯一與川麥44不同的品種（等位類型為）。研究表明與有效分蘗和千粒重顯著關(guān)聯(lián)，并且在的4種單倍型中（A1a—A1d），A1d對千粒重的貢獻最大[54-55]。因此，可能是川麥44強分蘗力和高產(chǎn)性狀的一個重要支撐基因。雖然，基因功能標(biāo)記分析發(fā)現(xiàn)，川麥44含有抗穗發(fā)芽基因的有利等位位點且在衍生品種中的傳遞頻率也較高（60%），但是其本身并對穗發(fā)芽的抗性并不好。穗發(fā)芽性狀受復(fù)雜的基因網(wǎng)絡(luò)控制，并且其發(fā)生受天氣環(huán)境的影響非常大[56]。因此，該位點可能在育種過程中并沒有受到顯著的選擇壓（60% vs 50%的隨機選擇頻率）。另外，本研究利用Ye等[50]發(fā)現(xiàn)的四川小麥品種產(chǎn)量性狀關(guān)聯(lián)的SNP標(biāo)記，鑒定到3個可能對川麥44作為重要育種親本非常重要的QTL位點、和。其中，影響穗長的位點可能具有重要利用價值，因為其在衍生品種中的傳遞頻率為100%，具有明顯的選擇優(yōu)勢

表2 川麥44高傳遞率基因

加粗字體表示川麥44中高傳遞率有利基因

The bold indicate the favorable genes with high transmission rate in Chuanmai 44

表3 衍生品種與川麥44位點相同且頻率≥50%產(chǎn)量相關(guān)QTL

本研究分析的10個川麥44衍生品種中，有4個為弱筋，其余都為中筋。細胞學(xué)分析表明，弱筋品種中的昌麥32和昌麥34均含有1對1RS/1BL易位染色體，易位染色體來自另外一個雜交親本-昌麥19。1RS/1BL易位染色體在給小麥帶來抗病、抗逆和豐產(chǎn)性的同時，也使得其加工品質(zhì)變差，主要表現(xiàn)在面團黏性增加，面筋強度減弱，面團形成時間變短，面包體積減小等[57]。因此，1RS/1BL易位染色體可能是昌麥32和昌麥34表現(xiàn)為弱筋的原因之一。

4 結(jié)論

在川麥44中共檢測到2個高傳遞率的有利基因：抗穗發(fā)芽基因與有效分蘗和千?；?；3個對產(chǎn)量性狀具有正向效應(yīng)且高傳遞率頻率的QTL位點，其中，2個增加千粒重（和），1個增加穗長（）。穗長有利位點在衍生品種中的傳遞頻率為100%，可能具有重要利用價值。

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The genetic contribution of the important breeding parent Chuanmai 44 to its derivatives

LUO JiangTao, ZHENG JianMin, Deng QingYan, LIU PeiXun, PU ZongJun*

Crop Research Institute of Sichuan Academic of Agricultural Sciences/Key Laboratory of Wheat Biology and Genetic Improvement on Southwestern China, Ministry of Agriculture and Rural Areas, Chengdu 610066

【Objective】Common wheat variety Chuanmai 44 has the characteristics of high yield, stable yield and wide adaptability.Ten new varieties have been selected and approved in breeding program using Chuanmai 44 as parent.It indicates Chuanmai 44 is an important breeding parent.To clarify the genetic base of Chuanmai 44 as a vital parent in breeding exercise and identify important genes or QTL within it will be helpful in breeding new elite varieties using Chuanmai 44.【Method】Fluorescencehybridization was applied to Chuanmai 44 and its ten derived varieties to identify whether there were wheat-alien translocations, and to analyze the chromosome diversity among them.The 660K SNP array data of Chuanmai 44 and its derived varieties were used to calculate the genetic contribution of Chuanmai 44 to its derived varieties and clarify the high transmission genomic segments.Functional molecular markers within cloned genes and linked molecular markers for yield-related traits were used to identify important genes or QTL in Chuanmai 44 for breeding.【Result】Chuanmai 44 did not harbor the 6VS/6AL and 1RS/1BL translocation chromosomes which both frequently existed in wheat varieties in Sichuan.Only two out of its ten derivatives, Changmai 32 and Changmai 34, contained 1RS/1BL translocation, which is inherited from another parent Changmai 19.The existence of 1RS/1BL translocation in the two varieties may explain their weak gluten phenotype.Except wheat-relative translocation, the karyotypes of Chuanmai 44 and its 10 derivative varieties also showed polymorphisms on some chromosomes.For instance, there were two types of chromosome 4A among derivatives, and 80% of them showed the same as Chuanmai 44.Chromosomes 5A, 6B and 7B had 4, 2 and 2 karyotypes, respectively.These three chromosomes in the derivative population of Chuanmai 44 showed the same karyotype with Chuanmai 44 in a frequency of 40%.660K SNP chip analysis identified 1127 genomic segments with high transmission frequency (>50%) within its derived varieties.These genomic segments located on all 21 chromosomes and their mean length was 1.57 Mb.B genome owned the most number and the largest length of the high transmission frequency segments.Chromosomes 4A, 2B and 5B were the three chromosomes with the longest high transmission frequency segments.Chromosomes 4A, 2B and 3B were the three chromosomes with the most number of high transmission frequency segments.Combing the genotype data of 61 functional markers of cloned wheat gene and 13 SNP markers linked with yield-related QTL and the distribution of Chuanmai 44 high transmission genomic regions, we discovered that there are 9 genes markers and 3 QTL markers are anchored in the high transmission rate section of Chuanmai 44.The twelve markers responding to two favorable alleles and three QTL, including,,,,, which exhibited positive effect on preharvest sprouting resistance, effective tiller number, thousand grain weight and spike length, respectively.【Conclusion】The length of genomic segments retained within its derived varieties was short.It suggested that Chuanmai 44 as a breeding parent had high genetic combining ability, and its chromosomes were easy to recombine with different homologous chromosomes in resulting hybrids, which is beneficial to reduce linkage drag.Therefore, it plays an important role as a skeleton parent in breeding excercise.,,,andwere the important loci in Chuanmai 44, which should be widely used in further breeding program under molecular marker assisted selecting.

Chuanmai 44; genetic contribution; beneficial gene; QTL; high transmission rate

2021-02-03；

2021-04-06

國家重點研發(fā)計劃（2016YFD0101600，2017YFD0100905）、四川省科技計劃（2018JY0627）、四川省財政創(chuàng)新能力提升工程項目（2016ZYPZ-015）、四川省育種攻關(guān)項目（2021YFYZ0002）

羅江陶，E-mail：jtluohao@163.com。通信作者蒲宗君，E-mail：pzjun68@163.com

（責(zé)任編輯 李莉）