葉俊華 楊啟臺(tái) 劉章雄 郭 勇 李英慧 關(guān)榮霞 邱麗娟,*

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大豆引進(jìn)種質(zhì)抗胞囊線蟲(chóng)病、抗花葉病毒病和耐鹽基因型鑒定及優(yōu)異等位基因聚合種質(zhì)篩選

葉俊華1,2楊啟臺(tái)2,3劉章雄2郭 勇2李英慧2關(guān)榮霞2邱麗娟2,*

1東北農(nóng)業(yè)大學(xué)農(nóng)學(xué)院, 黑龍江哈爾濱 150030;2國(guó)家農(nóng)作物基因資源與遺傳改良重大科學(xué)工程 / 農(nóng)業(yè)部種質(zhì)資源利用重點(diǎn)實(shí)驗(yàn)室 / 中國(guó)農(nóng)業(yè)科學(xué)院作物科學(xué)研究所, 北京 100081;3蚌埠醫(yī)學(xué)院生物科學(xué)系, 安徽蚌埠 233000

我國(guó)從美國(guó)、俄羅斯、日本等26個(gè)國(guó)家或地區(qū)共引進(jìn)大豆種質(zhì)3218份, 僅對(duì)部分種質(zhì)進(jìn)行了大豆胞囊線蟲(chóng)病(Soybean cyst nematode, SCN)、大豆花葉病毒病(Soybean mosaic virus, SMV)和鹽敏感性的抗性鑒定, 但基因型的系統(tǒng)分析尚未見(jiàn)報(bào)道。本研究針對(duì)大豆抗胞囊線蟲(chóng)病3個(gè)基因(、、)和耐鹽基因()開(kāi)發(fā)KASP標(biāo)記5個(gè), 結(jié)合與大豆花葉病毒抗性相關(guān)的1個(gè)SCAR標(biāo)記(SCN11), 對(duì)1489份大豆引進(jìn)種質(zhì)進(jìn)行基因型鑒定。結(jié)果表明, 具有優(yōu)異等位基因的種質(zhì)共1084份; 攜帶3個(gè)位點(diǎn)優(yōu)異等位基因的種質(zhì)19份, 包括抗胞囊線蟲(chóng)病3個(gè)位點(diǎn)(、、)疊加(Peking型)種質(zhì)3份, 聚合抗胞囊線蟲(chóng)病基因和抗花葉病毒病標(biāo)記7份, 聚合抗胞囊線蟲(chóng)病和耐鹽基因2份, 聚合抗胞囊線蟲(chóng)病、抗花葉病毒病和耐鹽基因7份; 攜帶4個(gè)位點(diǎn)優(yōu)異等位基因的種質(zhì)9份, 包括聚合抗胞囊線蟲(chóng)病基因和抗花葉病毒病標(biāo)記6份, 聚合抗胞囊線蟲(chóng)病和耐鹽基因2份, 聚合抗胞囊線蟲(chóng)病、抗花葉病毒病和耐鹽7份; 攜帶5個(gè)位點(diǎn)優(yōu)異等位基因8份, 聚合了抗胞囊線蟲(chóng)病、抗花葉病毒病和耐鹽優(yōu)異等位變異。在這些攜帶優(yōu)異等位變異的種質(zhì)中, 44份已由前人證明具有相應(yīng)的抗性。攜帶3個(gè)或3個(gè)以上優(yōu)異等位基因的36份種質(zhì)中, 有52.78%種質(zhì)的一種或兩種特性已被報(bào)道。在不攜帶抗性優(yōu)異等位變異的種質(zhì)中, 93份已證明有耐鹽性或?qū)MV3號(hào)株系抗性, 這些種質(zhì)可能存在新的抗性(等位)基因。本研究利用高通量分子標(biāo)記篩選出的攜帶抗病、抗逆優(yōu)異等位基因的種質(zhì)為我國(guó)大豆資源表型鑒定、抗源的快速篩選及利用提供理論依據(jù)和新思路。

大豆種質(zhì); 分子標(biāo)記; KASP技術(shù); 基因型鑒定

引進(jìn)種質(zhì)在我國(guó)大豆品種改良中發(fā)揮了重要作用, 通過(guò)對(duì)1923?1995年間育成和推廣的651份大豆品種統(tǒng)計(jì), 有224份品種可追溯到46個(gè)國(guó)外種質(zhì)[1]。其中包括綏農(nóng)14、合豐25等優(yōu)良品種。綏農(nóng)14擁有國(guó)外品種十勝長(zhǎng)葉、Amsoy的血緣, 具有良好的遺傳基礎(chǔ)和農(nóng)藝特征, 曾獲得國(guó)家科技進(jìn)步二等獎(jiǎng); 合豐25具有早熟、高產(chǎn)穩(wěn)產(chǎn)、廣適、抗逆性強(qiáng)等特點(diǎn), 曾連續(xù)13年保持全國(guó)大豆品種播種面積第一的記錄。分子標(biāo)記遺傳多樣性分析結(jié)果表明, 美國(guó)和日本的品種與中國(guó)品種間存在明顯遺傳差異, 是重要的優(yōu)異基因來(lái)源, 有利于改善我國(guó)大豆育種遺傳基礎(chǔ)狹窄的問(wèn)題[2-5]。系統(tǒng)比較分析發(fā)現(xiàn), 引進(jìn)種質(zhì)株型好、抗倒伏、抗病性較好, 對(duì)提高大豆產(chǎn)量、增強(qiáng)抗病、抗逆性、改善品質(zhì)具有重要意義[6]。因此, 對(duì)引進(jìn)種質(zhì)的研究將促進(jìn)其有效利用[7]。

大豆種質(zhì)的鑒定評(píng)價(jià)是合理利用的前提。傳統(tǒng)的種質(zhì)鑒定是表型鑒定, 其鑒定不僅耗時(shí)費(fèi)力, 鑒定結(jié)果準(zhǔn)確性易受到環(huán)境條件的影響。如大豆胞囊線蟲(chóng)(soybean cyst nematode, SCN)鑒定至少在30 d以上[8]且鑒定數(shù)量有限。由于抗病、抗逆性鑒定條件及人力物力的限制, 難以對(duì)龐大數(shù)目的種質(zhì)進(jìn)行系統(tǒng)鑒定。因此, 自“八五”以來(lái), 我國(guó)對(duì)引進(jìn)國(guó)外大豆種質(zhì)抗SCN、抗SMV (soybean mosaic virus)和耐鹽性的鑒定數(shù)目不到總數(shù)的10%。

大豆基因組測(cè)序的完成促進(jìn)了重要性狀基因定位和克隆, 促進(jìn)了基于抗病、耐逆性狀的分子標(biāo)記鑒定?？勾蠖拱揖€蟲(chóng)方面, 已克隆SCN抗性的主效基因和, 發(fā)現(xiàn)了微效基因[9]。Kadam等[10]利用KASPar (Kompetitive Allele-Specific PCR)技術(shù)明確了95份大豆種質(zhì)的、及其他QTL基因型。史學(xué)暉等[11]對(duì)105份種質(zhì)鑒定發(fā)現(xiàn), Rhg4-389鑒定的抗性等位變異對(duì)抗病種質(zhì)的選擇效率為97.1%。在抗大豆花葉病毒方面, Zheng等[12]定位了東北最強(qiáng)株系的抗性基因, 開(kāi)發(fā)并驗(yàn)證了與SMV緊密連鎖的SCAR標(biāo)記——SCN11。利用SCN11對(duì)中品95-5117的12份系譜材料進(jìn)行檢測(cè), 抗性條帶的選擇效率約為63.6%[13]。在耐鹽方面, Guan等[14]圖位克隆了大豆耐鹽基因, 從53份大豆種質(zhì)中鑒定出9種單倍型, 包括2種耐鹽單倍型和7種鹽敏感單倍型, 通過(guò)對(duì)172份微核心種質(zhì)及12份美國(guó)大豆種質(zhì)進(jìn)行鑒定, 單倍型H1對(duì)耐鹽性的選擇效率為91.9%[14]。

為了加快大豆優(yōu)異種質(zhì)鑒定效率, 本研究開(kāi)發(fā)了大豆抗胞囊線蟲(chóng)病3個(gè)基因(、、)和耐鹽基因()的KASP標(biāo)記, 并利用與大豆花葉病毒病抗性相關(guān)的SCAR標(biāo)記(SCN11), 對(duì)1489份引進(jìn)種質(zhì)進(jìn)行了分析, 鑒定出攜帶優(yōu)異等位基因且已具有抗性的種質(zhì), 同時(shí), 篩選出少量攜帶優(yōu)異等位基因的種質(zhì), 為表型高效鑒定提供了依據(jù), 發(fā)掘出具有抗性但不攜帶優(yōu)異等位基因的種質(zhì), 是新抗性(等位)基因挖掘的重要材料。本研究結(jié)果為大豆新基因發(fā)掘和新品種培育提供了材料和技術(shù)支撐。

1 材料與方法

1.1 試驗(yàn)材料

本研究利用引進(jìn)大豆種質(zhì)共1489份, 分別來(lái)自亞洲、歐洲和美洲。其中以來(lái)自美洲(包括美國(guó)、加拿大和巴西3個(gè)國(guó)家)的種質(zhì)數(shù)目最多, 達(dá)935份, 來(lái)自歐洲(包括俄羅斯、瑞典、德國(guó)等13個(gè)國(guó)家)的有371份, 來(lái)自亞洲(包括日本、泰國(guó)、韓國(guó)等6個(gè)國(guó)家或地區(qū))的種質(zhì)共91份, 解放前留下的大豆種質(zhì)共27份, 另有65份無(wú)來(lái)源記載。

1.2 大豆基因組DNA的提取與質(zhì)量檢測(cè)

將參試種質(zhì)每份播種5粒, 收集新鮮葉片。在TECAN液體自動(dòng)化工作站平臺(tái)進(jìn)行大豆基因組DNA提取, 以96孔板為單位, 用改良的CTAB法[15]從大豆葉片中提取基因組DNA。提取后的DNA, 每96孔板抽檢12個(gè)樣品, 進(jìn)行瓊脂糖電泳檢測(cè), 并取2 μL DNA在BioTeK Synergy HIM上測(cè)定OD260、OD230值以及DNA濃度質(zhì)檢。

1.3 標(biāo)記的開(kāi)發(fā)與鑒定

大豆SCN抗性及耐鹽相關(guān)的5個(gè)SNP位點(diǎn)采用KASP標(biāo)記進(jìn)行檢測(cè)。根據(jù)選取的5個(gè)SNP位點(diǎn)及其側(cè)翼序列(表1), 用Primer軟件設(shè)計(jì)3￠末端PCR擴(kuò)增引物,m值在55~65°C之間。每個(gè)SNP位點(diǎn)設(shè)計(jì)兩條SNP特異性引物和一條通用引物[16]。標(biāo)記的驗(yàn)證與檢測(cè)在DouglasArray Tape平臺(tái)上進(jìn)行, 在SOELLEX高通量PCR水浴中完成PCR反應(yīng)。PCR反應(yīng)體系5 μL, 包括2 μL模板DNA (濃度約20 ng L–1)、2.5 μL 2×Master Mix (LGC Genomics, Hoddeston, UK)、0.07 μL Primer Mix、0.43 μL ddH2O。反應(yīng)程序?yàn)?4°C熱激15 min; 94°C變性20 s, 65°C退火和延伸60 s, 10個(gè)循環(huán), 每循環(huán)降低0.8°C; 94°C變性20 s, 57°C退火和延伸60 s, 26個(gè)循環(huán)。使用ARAYA熒光閱讀儀讀取熒光信號(hào), 由Krake軟件根據(jù)讀取結(jié)果按照分型明確、NTC (無(wú)樣品陰性對(duì)照)無(wú)特異性擴(kuò)增的原則進(jìn)行樣品SNP分型。

與大豆花葉病毒抗性相關(guān)的位點(diǎn)用SCAR標(biāo)記[12]進(jìn)行檢測(cè)。以基因組DNA為模板, 反應(yīng)體系10 μL, 包括50 ng基因組DNA 2 μL、10×PCR緩沖液1 μL、2 mmol L–1的dNTPs 1 μL、2 μmol L–1上下游引物各0.2 μL、聚合酶0.2 μL (全式金生物技術(shù)有限公司)、ddH2O 5.4 μL。PCR在ABI (Applied Biosystems, 美國(guó))公司的PCR擴(kuò)增熱循環(huán)儀上進(jìn)行, 反應(yīng)程序?yàn)?5°C預(yù)變性5 min; 95°C變性30 s, 59°C退火30 s, 72°C延伸70 s, 34個(gè)循環(huán); 最后72°C延伸8 min, 于4°C保存。采用濃度為1.5%的瓊脂糖凝膠電泳, 130V電壓下電泳25 min, 經(jīng)EB染色后在紫外燈下觀察結(jié)果, 記錄結(jié)果。

表1 抗SCN、抗SMV和耐鹽相關(guān)標(biāo)記的類型及引物序列

FAM和HEX分別代表引物為FAM標(biāo)簽序列和HEX標(biāo)簽序列; COM代表通用引物。

FAM and HEX represent FAM labeled sequences and HEX labeled sequence, respectively; COM represent common primer.

1.4 數(shù)據(jù)分析

使用Microsoft Excel 2007進(jìn)行等位基因分布頻率計(jì)算。

2 結(jié)果與分析

2.1 5個(gè)位點(diǎn)的優(yōu)異等位基因頻率分析

利用抗大豆胞囊線蟲(chóng)病基因(、、)和耐鹽基因的KASP標(biāo)記對(duì)引進(jìn)種質(zhì)的分型結(jié)果顯示, 兩種純合基因型和雜合基因型呈明顯的簇狀分離(圖1-A~E), 證明了開(kāi)發(fā)KASP標(biāo)記的有效性。通過(guò)PCR擴(kuò)增檢測(cè)結(jié)果可以看出, 大豆花葉病毒病抗性相關(guān)標(biāo)記SCN將參試種質(zhì)分為擴(kuò)增片段980 bp的抗病優(yōu)異等位基因和擴(kuò)增片段1070 bp的感病等位基因(圖1-F)。標(biāo)記和源自同一個(gè)位點(diǎn)(), 共同決定了耐鹽單倍型H1, 記作。用6個(gè)分子標(biāo)記對(duì)1489份引進(jìn)大豆種質(zhì)鑒定, 除去分型結(jié)果不明確的種質(zhì), 對(duì)有效鑒定的種質(zhì)分析結(jié)果顯示, 功能標(biāo)記rhg1、Rhg4-389、SCN3-11、SALT3優(yōu)異等位基因的頻率較低(分別為0.022、0.025、0.049和0.170), 而連鎖標(biāo)記SCN11的優(yōu)異等位基因頻率較高(0.645) (表2)。

2.2 5個(gè)位點(diǎn)抗性種質(zhì)的來(lái)源分布規(guī)律

各位點(diǎn)的抗性種質(zhì)來(lái)源分布顯示, SCN主效位點(diǎn)、的抗性種質(zhì)主要來(lái)自美洲, 分別為24份和30份; 少量來(lái)自亞洲, 分別為4份和2份。微效基因的抗性種質(zhì)多數(shù)(37份)來(lái)自美洲, 但部分種質(zhì)(16份)來(lái)自歐洲。耐鹽單倍型的大豆種質(zhì)中來(lái)自美洲的最多(53.19%), 其次是歐洲(31.06%), 亞洲最少(4.68%)。SCAR標(biāo)記SCN11鑒定出的抗SMV種質(zhì)960份, 占總數(shù)的64.47%, 且美洲(700份)多于歐洲(147份)和亞洲(57份) (圖2)。

圖1 KASP和SCAR標(biāo)記對(duì)部分種質(zhì)基因型檢測(cè)結(jié)果

A~E: 每個(gè)圓點(diǎn)各對(duì)應(yīng)一份檢測(cè)種質(zhì)。紅色或藍(lán)色表示純合基因型; 綠色表示雜合基因型; 粉色表示檢測(cè)無(wú)信號(hào)或信號(hào)較弱; 紫色表示有信號(hào)但無(wú)明確分型; 黑色代表NTC, 即無(wú)模板對(duì)照。F: M是DL2000; 1、3為感SMV基因型; 2、4為抗SMV基因型。

A–E: each dot corresponds to an accession tested. Red or blue dots represent homozygous genotypes; green dots represent heterozygotes; pink dots represent no signal detected or weak signals; purple dots represent signals which cannot be classified; black dots represent NTC, no template control. F: M is DL2000; 1, 3 represent sensitivity to soybean mosaic virus; 2, 4 represent resistance to soybean mosaic virus.

表2 6個(gè)抗性標(biāo)記對(duì)引進(jìn)種質(zhì)的分型結(jié)果

1)R/T表示抗病或耐鹽, S表示感病或鹽敏感。

1)R/T indicates resistance or salt tolerance; S indicates susceptibility or salt sensitivity.

圖2 5個(gè)位點(diǎn)抗性等位基因或單倍型分布及來(lái)源

2.3 基于優(yōu)異等位基因的優(yōu)異種質(zhì)篩選

按表型特性進(jìn)行分析, 篩選出僅具有單一性狀抗性(單抗)的種質(zhì)共907份, 包括抗SCN種質(zhì)21份、抗SMV種質(zhì)796份、耐鹽種質(zhì)90份。篩選出具有2種抗性（雙抗）種質(zhì)160份, 兼抗SCN和SMV種質(zhì)32份; 抗SCN且耐鹽的種質(zhì)13份; 抗SMV且耐鹽的種質(zhì)115份。篩選出同時(shí)具有3種抗性(三抗)種質(zhì)17份。

按基因型進(jìn)行分析, 鑒定出攜帶優(yōu)異等位基因的種質(zhì)1084份, 包括具有單位點(diǎn)優(yōu)異等位基因種質(zhì)901份, 以SCN11標(biāo)記篩選出的種質(zhì)數(shù)目最多; 攜帶2個(gè)位點(diǎn)優(yōu)異等位基因種質(zhì)147份, 抗SCN雙位點(diǎn)疊加種質(zhì)最少, 而耐鹽和抗SMV的基因聚合種質(zhì)數(shù)量最多(表3); 攜帶3個(gè)或3個(gè)以上優(yōu)異等位基因的種質(zhì)36份, 其中有聚合3個(gè)位點(diǎn)的種質(zhì)19份、聚合4個(gè)位點(diǎn)的種質(zhì)9份、聚合5個(gè)位點(diǎn)的種質(zhì)8份(表4)。

3 討論

3.1 基于分子標(biāo)記的基因型檢測(cè)為提高種質(zhì)鑒定和利用效率創(chuàng)造了條件

20世紀(jì)40年代以來(lái), 我國(guó)多次引入國(guó)外大豆種質(zhì)[5], 目前引進(jìn)大豆種質(zhì)已超過(guò)3000余份, 并保存在國(guó)家種質(zhì)庫(kù)[19]。20世紀(jì)80年代, 我國(guó)開(kāi)始對(duì)大豆種質(zhì)的農(nóng)藝性狀、抗病性及抗逆性進(jìn)行評(píng)價(jià), 而美國(guó)已從種質(zhì)中篩選出了抗病、抗逆及抗蟲(chóng)種質(zhì)[6]。由于基于表型鑒定篩選種質(zhì)受到人力、財(cái)力、物力的影響, 鑒定結(jié)果受環(huán)境影響很大, 鑒定種質(zhì)的數(shù)量非常有限。迄今為止, 僅對(duì)不超過(guò)500份引入種質(zhì)的抗病性、抗逆性等性狀進(jìn)行了不完全鑒定。

表3 5個(gè)位點(diǎn)對(duì)引進(jìn)種質(zhì)的篩選

表4 攜帶3個(gè)或3個(gè)以上抗性等位基因的36份種質(zhì)

(續(xù)表4)

+表示該種質(zhì)來(lái)自泰國(guó);#表示該種質(zhì)來(lái)源不詳; 其余種質(zhì)均來(lái)自美國(guó);*表示該種質(zhì)此表型特性已有報(bào)道

+represents the accession is from Thailand;#represents the source of this accession is unknown; other accessions are from the United States.* represents the resistance or tolerance of the accession has been reported.

目前基于大豆重要性狀相關(guān)的分子標(biāo)記對(duì)種質(zhì)進(jìn)行基因型鑒定的報(bào)道較少。例如，利用大豆灰斑病抗性基因連鎖的3個(gè)SSR標(biāo)記, 對(duì)45份地方種質(zhì)進(jìn)行檢測(cè), 鑒定效率分別為72.7%、81.8%、83.3%[20]；用大豆白粉病基因連鎖標(biāo)記Sat_366和Sat_393檢測(cè)2個(gè)雜交分離F2群體, 鑒定效率分別為92.7%和60.3%[21]。用大豆黃種皮基因緊密連鎖的標(biāo)記ls1-22對(duì)355分析大豆核心種質(zhì)等材料，鑒定選擇效率為76.23%[21]。利用色素合成基因的4個(gè)功能標(biāo)記分析272份種質(zhì), 對(duì)茸毛色的檢測(cè)效率在80%以上[22]。利用與生育期相關(guān)的SSR標(biāo)記Satt431、Satt215和Satt557對(duì)47份黃淮育成品種進(jìn)行鑒定, 對(duì)熟期組MGIII和MGV的選擇效率分別為83.33%和90.00%[23]。本研究選用的功能標(biāo)記Rhg4-389及單倍型的鑒定效率在90%以上, 連鎖標(biāo)記SCN11的鑒定效率在60%以上, 揭示了在分子水平上直接對(duì)目的性狀進(jìn)行選擇的可能性。通過(guò)高通量基因型鑒定明確了1489份引入種質(zhì)的3個(gè)性狀基因型, 鑒定種質(zhì)數(shù)量占引進(jìn)種質(zhì)總數(shù)近50%, 選擇出具有優(yōu)異等位基因的種質(zhì), 其中已證明的有耐鹽種質(zhì)20份, 抗SCN種質(zhì)21份, 抗SMV3號(hào)小種的種質(zhì)12份, 可直接利用[25]。攜帶3個(gè)或3個(gè)以上抗性等位基因的優(yōu)異種質(zhì)36份, 雖然有33份表型尚需進(jìn)一步驗(yàn)證, 但提高了篩選優(yōu)異種質(zhì)的目標(biāo)性。本研究除了抗大豆花葉病毒SCAR標(biāo)記SCN11外, 抗SCN和耐鹽基因開(kāi)發(fā)了可進(jìn)行高通量檢測(cè)的KASP標(biāo)記, 降低了檢測(cè)成本, 為上萬(wàn)份種質(zhì)資源的高效發(fā)掘與利用提供了新思路。

3.2 KASP技術(shù)在大豆基因型鑒定中的應(yīng)用

KASP技術(shù)是由LGC公司(http://www.lgcgroup. com/)設(shè)計(jì)和創(chuàng)制, 與芯片技術(shù)同屬于高通量的SNP測(cè)序技術(shù)。與下一代測(cè)序(NGS)技術(shù)相比, 高通量SNP檢測(cè)具有快速高效、敏感度高、使用方便、結(jié)果可靠、價(jià)格低廉等優(yōu)勢(shì)。芯片技術(shù)適用于對(duì)100到1 000 000個(gè)以上的SNP進(jìn)行檢測(cè), 對(duì)少量SNP進(jìn)行檢測(cè)時(shí), KASP技術(shù)具有經(jīng)濟(jì)高效的優(yōu)勢(shì)[26]。與基于芯片的Illumina GoldenGate相比, KASP技術(shù)分型的錯(cuò)誤率(0.7%~1.6%)低于芯片GoldeGate平臺(tái)(2.0%~2.4%), 且用于分子標(biāo)記輔助回交選擇時(shí), 使用KASP技術(shù)分型將比使用其他高通量平臺(tái)節(jié)省7.9%~46.1%的費(fèi)用[27]。KASP技術(shù)以價(jià)格低廉、高效靈活性[28], 已在水稻[29]、玉米[30-31]、小麥[32-36]、蠶豆[37]等作物的研究中應(yīng)用。在大豆研究中，Patil等[38]開(kāi)發(fā)了基于種子組成性狀相關(guān)等位基因KASP標(biāo)記, 并應(yīng)用于明確突變來(lái)源和基因定位。Patil等[39]還對(duì)耐鹽基因多個(gè)與結(jié)構(gòu)變異相關(guān)的SNP進(jìn)行KASP檢測(cè), 驗(yàn)證了這些SNP與表型的高度相關(guān)。Pham等[40]定位來(lái)自PI594891和PI594774中抗灰斑病的2個(gè)候選基因, 開(kāi)發(fā)KASP標(biāo)記用于檢測(cè)群體基因型與表型間的關(guān)聯(lián)。本研究利用抗胞囊線蟲(chóng)病基因和耐鹽基因開(kāi)發(fā)的5個(gè)KASP標(biāo)記, 系統(tǒng)檢測(cè)了1489份引進(jìn)種質(zhì)基因型，占引進(jìn)種質(zhì)的46.2%，開(kāi)創(chuàng)了我國(guó)大豆種質(zhì)重要性狀基因型快速鑒定的新局面。

3.3 優(yōu)異等位基因種質(zhì)的發(fā)掘?yàn)檫z傳育種提供了材料支撐

利用目的基因或與目的基因緊密連鎖的分子標(biāo)記可聚合多個(gè)優(yōu)異等位基因。Maroof等[41]利用SSR標(biāo)記對(duì)SMV感病大豆Essex的近等基因系中含有位點(diǎn)進(jìn)行檢測(cè), 發(fā)現(xiàn)、和可通過(guò)兩基因和三基因等位基因聚合可產(chǎn)生抗性, 但則表現(xiàn)出晚期易感。Wang等[42]用SMV抗性基因、、連鎖的10個(gè)SSR標(biāo)記對(duì)雜交后代群體檢測(cè), 并對(duì)標(biāo)記聚合株系進(jìn)行SMV抗性評(píng)價(jià), F7代選出抗21個(gè)SMV菌株的純合株系5個(gè)。分子標(biāo)記輔助選擇不僅應(yīng)用于聚合同一性狀的多個(gè)基因, 還可以聚合多個(gè)性狀的多個(gè)基因。Kumar等[43]通過(guò)分子輔助回交育種將抗細(xì)菌性枯萎病基因和抗稻癭蚊的基因、聚合到水稻恢復(fù)系RPHR-1005; Hur等[44]利用標(biāo)記鑒定了BC4F6群體, 篩選出聚合水稻細(xì)菌枯萎病基因和及與耐冷QTL的株系A(chǔ)BL132-36。姚姝等[45]通過(guò)對(duì)雜交分離后代進(jìn)行分子標(biāo)記輔助選擇, 創(chuàng)制出聚合水稻抗稻瘟病基因、和低直鏈淀粉含量基因的新品系“南粳0051”。

分子標(biāo)記輔助選擇在多基因聚合育種的成功應(yīng)用, 提高了育種效率, 但在種質(zhì)資源基因型鑒定方面鮮見(jiàn)報(bào)道。本研究檢測(cè)的5個(gè)位點(diǎn)涉及抗SCN、抗SMV和耐鹽3個(gè)性狀, 篩選出攜帶抗SCN的3個(gè)位點(diǎn)優(yōu)異等位基因(Peking型)種質(zhì)19份, 其中15份具有抗SCN特性[46-50], 包括Centennial、Franklin、Forrest等; 用耐鹽基因的2個(gè)標(biāo)記篩選出235份種質(zhì), 其中20份已證明有耐鹽性[51], 包括Altona、Mansoy、Baekun Kong、Lee68等; 用抗SMV標(biāo)記篩選出960份大豆種質(zhì), 其中12份已證明表現(xiàn)為抗SMV3號(hào)株系[52-53], 包括L88-8440、L82-951、L84-2112、Columbia和L93-3327。除此之外, 仍有81份種質(zhì)具有耐鹽性, 但經(jīng)標(biāo)記檢測(cè)不屬于的耐鹽單倍型H1, 推斷少數(shù)種質(zhì)[14]可能為耐鹽單倍型H2, 還可能存在影響耐鹽性的新位點(diǎn); 有5份引進(jìn)種質(zhì)經(jīng)SCN11檢測(cè)為SMV感病型, 卻表現(xiàn)為抗SMV3號(hào)株系, 包括L88-8431[54]、L78-379[55]、L83-4744 (內(nèi)部資源)、L83-4483 (內(nèi)部資源)和新八達(dá)2號(hào)[56], 一個(gè)原因可能是標(biāo)記與基因間發(fā)生了交換, 這也是功能標(biāo)記比連鎖標(biāo)記檢測(cè)效率高的原因, 另一個(gè)原因可能是存在控制SMV抗性的新位點(diǎn)。有6份抗SCN種質(zhì)(Bedford、Yale、Fayette、PI 209332、Cartter、Bell)在SCN三個(gè)位點(diǎn)鑒定結(jié)果顯示均為感病基因型, 與它們的實(shí)際抗性表型不符[57-62], 其原因是這些種質(zhì)為PI88788型, 在的另一處發(fā)生變異所致[63]。與現(xiàn)有資源相比, 新增可能抗SCN的種質(zhì)4份, 可能具有耐鹽特性的種質(zhì)215份, 可能抗SMV的種質(zhì)948份。攜帶3個(gè)或3個(gè)以上抗性等位基因的36份種質(zhì)中, 其中16份種質(zhì)的一種或兩種特性已被報(bào)道, 且可能存在新特性; 17份種質(zhì)尚未有3種特性的鑒定報(bào)道, 可能存在抗SCN、SMV或耐鹽性。

本研究基于主要性狀已知基因(、、、)開(kāi)發(fā)的功能標(biāo)記鑒定出的優(yōu)異種質(zhì)數(shù)目較少, 分別占鑒定種質(zhì)總量的2.24%、2.50%、4.87%和17.03%; 而基于主要性狀緊密連鎖的標(biāo)記(SCN11)鑒定出的種質(zhì)數(shù)目較多, 占鑒定種質(zhì)總量的64.47%。這是由于基因的連鎖標(biāo)記的遺傳效應(yīng)值依賴于該標(biāo)記與基因的連鎖緊密程度[64], 而功能標(biāo)記的遺傳效應(yīng)具有可靠性和普適性[65], 能夠準(zhǔn)確地檢測(cè)目的基因, 因此, 基于功能基因篩選的優(yōu)異種質(zhì)效率高于與基因連鎖的標(biāo)記。然而, 無(wú)論是用功能基因標(biāo)記還是連鎖標(biāo)記, 一旦選擇出優(yōu)異種質(zhì), 其分子標(biāo)記的選擇效率都會(huì)提高, 可應(yīng)用于分子標(biāo)記輔助選擇育種。

4 結(jié)論

抗病耐逆性是與大豆產(chǎn)量、品質(zhì)相關(guān)的重要性狀, 而通過(guò)分子標(biāo)記進(jìn)行基因型鑒定是進(jìn)行優(yōu)異種質(zhì)篩選的有效手段。本研究鑒定出攜帶至少1種優(yōu)異等位基因的種質(zhì)1084份, 44份已由前人證明相應(yīng)的抗性; 攜帶3個(gè)或3個(gè)以上優(yōu)異等位基因的種質(zhì)有36份, 其中52.78%種質(zhì)的一種或兩種優(yōu)異特性已被報(bào)道。在不攜帶抗性優(yōu)異等位變異的種質(zhì)中, 93份具有耐鹽性或SMV3號(hào)株系抗性報(bào)道, 這些種質(zhì)可能存在新的抗性(等位)基因。

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Genotyping of SCN, SMV Resistance, Salinity Tolerance and Screening of Pyramiding Favorable Alleles in Introduced Soybean Accessions

YE Jun-Hua1,2, YANG Qi-Tai2,3, LIU Zhang-Xiong2, GUO Yong2, LI Ying-Hui2, GUAN Rong-Xia2, and QIU Li-Juan2,*

1College of Agriculture, Northeast Agricultural University, Harbin 150030, Heilongjiang, China;2National Key Facility for Gene Resources and Genetic Improvement / Key Laboratory of Crop Germplasm Utilization, Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China;3Department of Biotechnology, Bengbu Medical College, Bengbu 233000, Anhui, China

China has introduced 3218 soybean accessions from 26 countries such as the United States, Russia and Japan, and some of them have been carried out soybean cyst nematode (SCN), soybean mosaic virus (SMV) and salinity tolerance resistance evaluation. However, the systematic genotyping of these accessions has not been reported yet. In this study, five robust functional markers have been developed for KASP assays, three SCN loci (,,) and salinity tolerance gene () included. A total of 1489 introduced soybean accessions were genotyped by these markers with high-throughput assay as well as a SCAR marker (SCN11) which is related to soybean mosaic virus resistance. The results showed that there were 1084 accessions detected with favorable alleles; where accessions detected with resistant alleles at three loci were as much as 19, including three pyramiding SCN genes (,,) which were Peking type and seven pyramiding SCN and SMV , two pyramiding SCN and salinity favorable alleles, as well as seven pyramiding SCN, SMV and salinity favorable alleles; and accessions detected with four favorable alleles were as much as nine accessions, including six pyramiding SCN and SMV resistance alleles, one accession detected with SCN and salinity tolerance and two detected with SCN, SMV and salinity favorable alleles, eight detected with all the favorable alleles in this study. Among the elite accessions mentioned above, it has been proved that 44 accessions resistant to SCN, SMV-3 or tolerant to salinity. Among the 36 accessions with three or more favorable alleles, 52.78% had been reported of one or two characteristics. Among the accessions without resistance or tolerance alleles, it has been reported that 93 accessions were tolerant to salinity or resistant to SMV-3, where new resistance or tolerance genes could be found. Screening out the accessions with high-throughput SNP detection assays for resistance and tolerance alleles in soybean provides information for their further phenotyping, screening and breeding.

soybean germplasm; molecular markers; KASP; genotyping

2018-03-04;

2018-06-12;

2018-07-02.

10.3724/SP.J.1006.2018.01263

邱麗娟, E-mail: qiulijuan@caas.cn

E-mail: yejunhua1994@qq.com

本研究由國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2016YFD0100304, 2016YFD0100201)和中國(guó)農(nóng)業(yè)科學(xué)院科技創(chuàng)新工程項(xiàng)目資助。

This study was supported by the National Key R&D Program (2016 YFD0100304, 2016YFD0100201) and the Agricultural Science and Technology Innovation Program of CAAS.

URL: http://kns.cnki.net/kcms/detail/11.1809.S.20180702.0852.004.html