104份甘肅小麥品種主要品質(zhì)基因的分子標記檢測

柳　娜，曹　東，王世紅，楊文雄

(1.甘肅省農(nóng)業(yè)科學院 小麥研究所，蘭州　730070；2.甘肅農(nóng)業(yè)大學 農(nóng)學院，蘭州　730070)

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104份甘肅小麥品種主要品質(zhì)基因的分子標記檢測

柳娜1，曹東2，王世紅1，楊文雄1

(1.甘肅省農(nóng)業(yè)科學院 小麥研究所，蘭州730070；2.甘肅農(nóng)業(yè)大學 農(nóng)學院，蘭州730070)

摘要高分子量麥谷蛋白亞基、黃色素質(zhì)量分數(shù)和1B/1R易位系均影響小麥的加工品質(zhì)。為明確相關(guān)品質(zhì)性狀基因在104份甘肅育成小麥品種中的分布，并為小麥品質(zhì)育種提供親本材料，利用高分子質(zhì)量谷蛋白亞基 Dx5、 Bx7、 By8、 By9和黃色素質(zhì)量分數(shù)基因及1B/1R易位系的特異性標記對104份小麥品種進行檢測。結(jié)果表明，含基因 Dx5、 Bx7、 By8、 By9和1B/1R易位系的品種分別占總品種數(shù)的63.46%、76.92%、52.88%、45.19%和45.19%。含有等位基因 Psy-A1a和 Psy-A1b品種分別占總品種數(shù)的60.58%和21.15%，其黃色素質(zhì)量分數(shù)均值差異達到顯著水平(P<0.05)。總體來看，甘肅育成小麥品種中含適合加工饅頭和面粉等傳統(tǒng)食品的單個品質(zhì)優(yōu)質(zhì)基因頻率較高，而優(yōu)質(zhì)基因組合的比例較低，具有黃色素質(zhì)量分數(shù)較高的品種比例高。

關(guān)鍵詞甘肅小麥；高分子量谷蛋白亞基；品質(zhì)基因；分子標記

小麥面筋強度及其面制品色澤等主要受高分子量谷蛋白亞基(HMW-GS)、1B/1R易位系、黃色素質(zhì)量分數(shù)等因素的影響。已有研究表明，HMW-GS是影響小麥面團特性和面筋強度的關(guān)鍵因素，而且其不同類別的亞基對小麥品質(zhì)改良的貢獻各異[1-2]。Payne等[3]和Branlard等[4]研究表明， Glu-D1位點上小麥烘烤等品質(zhì)表現(xiàn)最優(yōu)的亞基為Dx5+Dy10，并可利用 Dx5標記準確快速的檢測和判斷小麥品種中是否含有Dx5+Dy10優(yōu)質(zhì)亞基。D’Ovideo等[5-6]和Bustos等[7]設(shè)計了一系列可以區(qū)分基因 1Dx5、 1Dx2、 1Dx3、 1Dx4、 1Dy10、 1Dy12和Null等的HMW-GS的特異引物。Ma等[8]和張曉科等[9]分別建立了基因 Ax2、 Bx17和 Dx5的多重PCR反應體系。Bx7亞基在小麥中分布較廣，通常與亞基By8或By9連鎖，但亞基組合Bx7+By8對面筋強度的貢獻大于其Bx7+By9[5]。1B/1R易位系即小麥的1B染色體短臂被黑麥的1R染色體短臂取代而形成[10]。在中國小麥育種進程中1B/1R易位系得到廣泛應用，同時也推進小麥育種進程[11]。Francis等[12]已開發(fā)檢測1B/1R易位系的基因標記，可準確快速方便的鑒定其是否攜帶1B/1R易位系。但與此同時也產(chǎn)生一些問題，如1B/1R易位品種的面筋質(zhì)量下降，使小麥的加工品質(zhì)變劣[10,13-15]。因此，對于品質(zhì)育種而言，判斷其親本及后代中是否含有1B/1R易位是至關(guān)重要的。中國傳統(tǒng)面條和饅頭等面制品需亮度好和低黃色素質(zhì)量分數(shù)的小麥面粉來加工。因此，研究和選育低黃色素質(zhì)量分數(shù)的新品種是中國小麥品質(zhì)育種的主要目標。而小麥籽粒中黃色素質(zhì)量分數(shù)是多基因調(diào)控的，研究表明，染色體7A和7B上的基因?qū)ζ湄暙I較大[16-17]。He等[18]開發(fā)了位于7A染色體的黃色素基因標記 YP7A，能有效區(qū)分小麥7A染色體控制高、低黃色素質(zhì)量分數(shù)的等位基因 Psy-A1a和 Psy-A1b。Parker等[19]研究得出位于小麥染色體3A和7A上控制黃色素質(zhì)量分數(shù)的主效QTL可分別解釋其表型變異的13%和60%。以上相關(guān)品質(zhì)基因分子標記的開發(fā)為小麥品質(zhì)改良提供可靠的理論依據(jù)。

高鳳梅等[20]、蘆靜等[21]、王憲國等[22]和胡鳳靈等[23]分別對黑龍江、新疆、寧夏及全國冬、春小麥的相關(guān)品質(zhì)基因進行檢測，掌握其相關(guān)品質(zhì)優(yōu)質(zhì)基因的分布情況。但有關(guān)甘肅省小麥品質(zhì)相關(guān)基因檢測的研究較缺乏。本研究選用甘肅省育成104份小麥品種資源，利用 Dx5、 YP7A、 Bx7、 By8 、 By9和 1B/1R特異性標記進行分子檢測，掌握甘肅育成品種中所含相關(guān)品質(zhì)基因的種類及分布情況，為甘肅省小麥品質(zhì)育種提供有價值的親本材料，加快品質(zhì)改良進程。

1材料與方法

1.1材 料

供試品種為104份甘肅省育成審定的小麥品種，由甘肅省農(nóng)業(yè)科學院小麥課題組保存。并在2012-2013年種植于甘肅省武威市黃羊鎮(zhèn)甘肅農(nóng)科院小麥育種基地，常規(guī)管理，成熟時收獲。這些冬小麥(72份)和春小麥(32份)基本反映甘肅省近10 a小麥育種現(xiàn)狀。

1.2試驗方法

1.2.1DNA提取選取每個品種新鮮幼嫩的小麥黃化苗，用CTAB法[24-25]提取小麥基因組DNA。為保證檢測結(jié)果的準確性，每個品種提取3批DNA，并分別檢測。

1.2.2PCR擴增與電泳檢測以D’Ovidio等[5]、He等[18]、Ma等[26]、Lei等[27]和Francis等[12]開發(fā)的功能標記 Dx5、 YP7A、 Bx7、 By9、 By8和 1B/1R進行分子檢測。引物由上海生工生物工程技術(shù)服務有限公司合成(表1)。擴增體系10 μL，含Tris-HCl 20 mmol/L(pH 8.4)，KCl 20 mmol/L，dNTPs 200 μmol/L，MgCl21.5 mmol/L，引物10 pmol/L，1.5 UTaqDNA 聚合酶，模板DNA為50 ng。

最后擴增產(chǎn)物用15 g/L的瓊脂糖凝膠電泳檢測，緩沖液為 1×TAE 溶液，160 V電壓電泳45 min，染色、掃描成像。

表1　引物序列、擴增片段及對應的基因型

1.2.3黃色素質(zhì)量分數(shù)測定采用已改進AACC14-50 法[28]，取各個小麥品種的面粉樣品3 g，加入提取液水飽和正丁醇15 mL，于振蕩器上振蕩浸提1 h后，靜置和離心。用分光光度計(波長為440 nm)測定吸光度，重復3次，取其平均值。

2結(jié)果與分析

2.1甘肅小麥中部分HMW-GS的分布

利用部分HWM-GS特異性引物檢測可以得出，在 Glu-D1位點，標記 Dx5在含亞基Dx5的品種中可擴增出450 bp的條帶(圖1)。在 Glu-B1位點，標記 Bx7在含亞基Bx7的品種中可擴增出630 bp和766 bp的2條帶型(圖2)；標記 By8在含亞基By8的品種中可擴增出527 bp的條帶(圖3)；標記 By9在含亞基By9的品種中可擴增出662 bp的帶型(圖4)。

在104份甘肅省育成的小麥品種中，含亞基 Dx5、 Bx7、 By8和 By9的品種分別有66、80、55和88份，分別占總品種數(shù)的63.46%、76.92%、52.88%和45.19%。其中，所有冬小麥品種中含亞基Dx5、Bx7、By8和By9分別占總品種數(shù)的63.89%、77.78%、55.56%和41.67%；而在所有春小麥品種中的頻率依次為62.5%、75%、46.88%和53.13%(表2)。

M.DNA ladder 2000；1.隴春23號Longchun 23；2.隴春28號Longchun 28；3.隴春22號Longchun 22；4.武春5號Wuchun 5；5.武春6號Wuchun 6；6.武春7號Wuchun 7；7.武春8號Wuchun 8；8.甘育1號Ganyu 1；9.平?jīng)?3號Pingliang 43；10.平?jīng)?4號Pingliang 44；11.涇麥1號Jingmai 1；12.天選43Tianxuan 43；13.隴春26號Longchun 26

圖1 Dx5標記擴增部分品種瓊脂糖凝膠電泳

Fig.1Agarose gel electrophoresis of some tested materials amplified by Dx5

M.DNA ladder 2000；1.隴春23號Longchun 23；2.隴春28號Longchun 28；3.隴春30號Longchun 30；4.臨農(nóng)826Linnong 826；5.隴鑒103Longjian 103；6.環(huán)冬3號Huandong 3；7.隴育1號Longyu 1；8.武春8號Wuchun 8；9.臨麥33Linmai 33；10.天選50Tianxuan 50；11.隴鑒9343Longjian 9343；12.張冬30號Zhangdong 30；13.靜麥10號Jingmai 10

圖2 Bx7標記擴增部分品種瓊脂糖凝膠電泳

Fig.2Agarose gel electrophoresis of some tested materials amplified by Bx7

M.DNA ladder 2000；1.隴鑒9811Longjian 9811；2.武春3號Wuchun 3；3.張冬30號Zhangdong 30；4.隴鑒301Longjian 301；5.銀春8號Yinchun 8；6.靈臺3號Lingtai 3；7.西峰28號Xifeng 28；8.定豐16號Dingfeng 16；9.中梁23號Zhongliang 23；10.張春21號Zhangchun 21；11.隴育2號Longyu 2；12.隴輻2號Longfu 2

圖3 By8標記擴增部分品種瓊脂糖凝膠電泳

Fig.3 Agarose gel electrophoresis of some tested materials amplified by By8

2.2甘肅小麥黃色素質(zhì)量分數(shù)等位基因的檢測

2.2.1 Psy-A1位點變異檢測利用標記 YP7A對104份甘肅小麥品種中 Psy-A1位點的等位變異檢測，在含 Psy-A1a和 Psy-A1b基因的品種中可分別擴增出194 bp和231 bp的2條帶型(圖5)。結(jié)果顯示，104份甘肅小麥中有63份擴增出194 bp片段，占總品種數(shù)的60.58%，含 Psy-A1a基因；22份品種中擴增出231 bp片段，占總品種數(shù)的21.15%，含 Psy-A1b基因，與低黃色素質(zhì)量分數(shù)相關(guān)[29]。在冬小麥品種中，44份(61.11%)品種中含有 Psy-A1a基因，17份(23.61%)品種含有 Psy-A1b基因；在春小麥品種中，19份(59.38%)品種中含有 Psy-A1a基因，5份(15.63%)品種含有 Psy-A1b基因(表3)。

M.DNA ladder 2000；1.武春3號Wuchun 3；2.武春6號Wuchun 6；3.武春7號Wuchun 7；4.定豐12號Dingfeng 12；5.甘育1號Ganyu 1；6.定豐10號Dingfeng 10；7.中天1號Zhongtian 1；8.隴育4號Longyu 4；9.臨麥32號Linmai 32；10.武都17號Wudu 17；11.靜麥2號Jingmai 2；12.航選01Hangxuan 01

圖4　 By9標記擴增部分供試材料瓊脂糖凝膠電泳

M.DNA ladder 2000；2.武春3號Wuchun 3；3.武春7號Wuchun 7；4.武春5號Wuchun 5；5.靈臺2號Lingtai 2；6.環(huán)冬4號Huandong 4；7.隴育2號Longyu 2；8.西峰28Xifeng 28；9.環(huán)冬3號Huandong 3；10.中梁23Zhongliang 23；11.中梁24Zhongliang 24；12.天選45Tianxuan 45

圖5　 YP7A標記擴增部分品種瓊脂糖凝膠電泳

2.2.2 Psy-A1基因與黃色素質(zhì)量分數(shù)的關(guān)系從表4可以看出，大多含有 Psy-A1a 基因的小麥品種黃色素質(zhì)量分數(shù)較高，均值為3.5 mg/kg，而含有 Psy-A1b基因的品種所對應的黃色素質(zhì)量分數(shù)較低，均值為2.1 mg/kg。依據(jù)t測驗結(jié)果可以看出，分別含 Psy-A1a和 Psy-A1b 2種基因的品種間黃色素質(zhì)量分數(shù)均值差異顯著。

2.3甘肅小麥1B/1R易位系的分布

利用1B/1R易位系的特異性標記對104份甘肅小麥品種進行檢測，可擴增出1 500 bp的條帶(圖6)。結(jié)果顯示，在104份小麥品種中有47份材料含有1B/1R易位系，占總品種數(shù)的45.19%。其中，冬小麥和春小麥分別有32份和15份，頻率分別為44.44%和46.88%。

含有Dx5亞基的小麥品種面筋強度高；同時具有低黃色素質(zhì)量分數(shù)的品種有利于面條和饅頭等面制品色澤的改善。在甘肅省育成的104份冬、春小麥中，有‘隴春22號’、‘靜麥10號’、‘隴鑒9343’、‘蘭天17’等37份(35.58%)小麥含有優(yōu)質(zhì)亞基Dx5，且不是1B/1R易位系。同時含亞基Dx5和 Psy-A1b基因，且不是1B/1R易位系的有‘隴春22號’和‘定西38號’等11份(10.58%)小麥品種。由此可以得出，具有多種優(yōu)質(zhì)基因的小麥品種較少。

表4　104份甘肅小麥品種黃色素質(zhì)量分數(shù)t測驗

注：不同字母表示差異達顯著水平(P<0.05)。

Note:Different letters indicate significant differences (P<0.05).

M.DNA ladder 2000；2.武春3號Wuchun 3；3.武春7號Wuchun 7；4.武春5號Wuchun 5；5.靈臺2號Lingtai 2；6.環(huán)冬4號Huandong 4；7.隴育2號Longyu 2；8.西峰28Xifeng 28；9.環(huán)冬3號Huandong 3；10.中梁23Zhongliang 23；11.中梁24Zhongliang 24；12.天選45Tianxuan 45

圖61B/1R標記擴增部分供試材料瓊脂糖凝膠電泳

Fig.6Agarose gel electrophoresis of some tested materials amplified by 1B/1R

3討 論

近年來品質(zhì)育種越來越受到許多小麥育種家的重視。不同面制品對其品質(zhì)的要求各異，傳統(tǒng)的面食品如面條和饅頭等要求色白[30]。研究表明，部分HMW-GS和LMW-GS對改良小麥面筋強度具有重要的作用，特別是優(yōu)質(zhì)亞基Dx5+Dy10等的引入，改善了面筋質(zhì)量[31]。本研究用功能性標記 Dx5、 YP7A、 Bx7、 By9、 By8和 1B/1R 易位系的特異性標記對甘肅省近年來審定的104份小麥品種進行檢測，含Dx5、Bx7、By8和By9亞基的頻率依次為63.46%、76.92%、52.88%和45.19%。與其他地區(qū)相比[20,32-33]，含亞基Dx5、By8、 By9和1B/1R易位系的分布頻率高于其他麥區(qū)，而含Bx7亞基的低于其他麥區(qū)。由此可以看出，含單個品質(zhì)優(yōu)質(zhì)亞基的分布頻率較高，這些基因在甘肅冬春麥中的分布不同，冬小麥所含的頻率高于春小麥。但為今后更好地改善小麥品種的面筋強度，更應繼續(xù)引進含優(yōu)質(zhì)基因品種，并結(jié)合相關(guān)品質(zhì)基因標記輔助選擇。在生產(chǎn)中，由于大多數(shù)冬小麥品種的品質(zhì)優(yōu)于春小麥品種，冬小麥品質(zhì)改良重視程度高于春小麥，在小麥生產(chǎn)和加工中傾向于使用冬小麥品種。

小麥品種中黃色素質(zhì)量分數(shù)的高低與面粉及面制品的色澤緊密相關(guān)[32]。甘肅省育成小麥品種表現(xiàn)出 Psy-A1a頻率(60.58%)偏高，需加強對小麥色澤的選擇，提高面粉白度來滿足人們的需求。小麥品質(zhì)育種應不宜采用含1B/1R易位系的品種作親本培育優(yōu)質(zhì)新品種[34]。而在104份甘肅小麥中，1B/1R易位系品種(45.19%)所占的比例較高。

綜上可以得出，在甘肅育成品種中含Dx5、Bx7、By8和By9亞基的頻率較高；同時含優(yōu)質(zhì)亞基Dx5和 Psy-A1b基因的小麥品種較少。說明近年來甘肅小麥品質(zhì)育種比較單一，今后應緊密結(jié)合分子標記輔助選擇，選擇攜帶優(yōu)質(zhì)基因的品種充當親本，聚合多個品質(zhì)優(yōu)質(zhì)基因，加快品質(zhì)育種進程，改善小麥品質(zhì)。

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Received 2015-01-05Returned2015-04-30

Foundation itemThe National Natural Science Foundation of China (No.31560390); Special Fund for Agro-scientific Research in the Public Interest(No.201503125-1);the Wheat Engineering Research Center of Gansu Province (No.144JTGA230); the National Agriculture Science Technology Achievement Transformation Fund (No.2014GB2G100140).

First authorLIU Na,female,assistant researcher.Research area:molecular and conventional breeding of wheat.E-mail:592905658@qq.com

(責任編輯：成敏Responsible editor:CHENG Min)

Molecular Marker Characterization of Main Quality Genes in 104 Gansu Wheat Varieties

LIU Na1, CAO Dong2, WANG Shihong1and YANG Wenxiong1

(1.Wheat Institute, Gansu Academy of Agricultural Sciences, Lanzhou730070, China;2.College of Agronomy, Gansu Agricultural University, Lanzhou730070, China)

AbstractSubunits of high molecular weight glutenin, yellow pigment mass fraction and 1B/1R translocation have main impacts on wheat processing quality.In this study, locus specific molecular markers for high molecular mass glutenin subunits of Dx5, Bx7, By8 and By9, PSY, 1B/1R translocation genes were used to characterize 104 wheat varieties to know the distribution of these genes in wheat varieties of Gansu province, and provide parent materials in molecular marker assisted breeding program for wheat with good quality.The results showed that, the frequency of cultivars with genes of Dx5, Bx7, By8, By9 and 1B/1R translocation were 63.46%, 76.92%, 52.88%, 84.62%,45.19% and 45.19%, respectively.The proportion of the allelic variations of Psy-A1a and Psy-A1b in these varieties were 60.58% and 21.15% respectively.Significant differences in yellow pigment mass fraction were detected between the genotypes with Psy-A1a and those with Psy-A1b (P<0.05).Generally, the frequency of single good quality gene for processing traditional food, such as steamed bread and flour, was high, the combination of good quality genes were low and the number of cultivars with high yellow pigment mass fraction was high in wheat cultivars released in Gansu province.

Key wordsGansu wheat; High molecular mass glutenin subunits; Quality genes; Molecular marker

Corresponding authorYANG Wenxiong,male,researcher.Research area:wheat breeding.E-mail:ywxm822@126.com

中圖分類號S512.1+2

文獻標志碼A

文章編號1004-1389(2016)03-0353-08

通信作者：楊文雄，男，研究員，主要從事小麥育種研究。E-mail:ywxm822@126.com

基金項目：國家自然科學基金(31560390)；公益性行業(yè)(農(nóng)業(yè))科研專項(201503125-1)；甘肅省小麥工程技術(shù)研究中心專項(144JTGA230)；國家農(nóng)業(yè)科技成果轉(zhuǎn)化資金(2014GB2G100140)。

收稿日期：2015-01-05修回日期：2015-04-30

網(wǎng)絡(luò)出版日期：2016-03-06

網(wǎng)絡(luò)出版地址：http://www.cnki.net/kcms/detail/61.1220.S.20160306.1610.012.html

第一作者：柳娜，女，助理研究員，從事小麥分子和常規(guī)育種研究。E-mail:592905658@qq.com