大豆引进种质抗胞囊线虫病、抗花叶病毒病和耐盐基因型鉴定及优异等位基因聚合种质筛选

叶俊华 杨启台 刘章雄 郭 勇 李英慧 关荣霞 邱丽娟,*



大豆引进种质抗胞囊线虫病、抗花叶病毒病和耐盐基因型鉴定及优异等位基因聚合种质筛选

叶俊华1,2杨启台2,3刘章雄2郭 勇2李英慧2关荣霞2邱丽娟2,*

1东北农业大学农学院, 黑龙江哈尔滨 150030;2国家农作物基因资源与遗传改良重大科学工程 / 农业部种质资源利用重点实验室 / 中国农业科学院作物科学研究所, 北京 100081;3蚌埠医学院生物科学系, 安徽蚌埠 233000

我国从美国、俄罗斯、日本等26个国家或地区共引进大豆种质3218份, 仅对部分种质进行了大豆胞囊线虫病(Soybean cyst nematode, SCN)、大豆花叶病毒病(Soybean mosaic virus, SMV)和盐敏感性的抗性鉴定, 但基因型的系统分析尚未见报道。本研究针对大豆抗胞囊线虫病3个基因(、、)和耐盐基因()开发KASP标记5个, 结合与大豆花叶病毒抗性相关的1个SCAR标记(SCN11), 对1489份大豆引进种质进行基因型鉴定。结果表明, 具有优异等位基因的种质共1084份; 携带3个位点优异等位基因的种质19份, 包括抗胞囊线虫病3个位点(、、)叠加(Peking型)种质3份, 聚合抗胞囊线虫病基因和抗花叶病毒病标记7份, 聚合抗胞囊线虫病和耐盐基因2份, 聚合抗胞囊线虫病、抗花叶病毒病和耐盐基因7份; 携带4个位点优异等位基因的种质9份, 包括聚合抗胞囊线虫病基因和抗花叶病毒病标记6份, 聚合抗胞囊线虫病和耐盐基因2份, 聚合抗胞囊线虫病、抗花叶病毒病和耐盐7份; 携带5个位点优异等位基因8份, 聚合了抗胞囊线虫病、抗花叶病毒病和耐盐优异等位变异。在这些携带优异等位变异的种质中, 44份已由前人证明具有相应的抗性。携带3个或3个以上优异等位基因的36份种质中, 有52.78%种质的一种或两种特性已被报道。在不携带抗性优异等位变异的种质中, 93份已证明有耐盐性或对SMV3号株系抗性, 这些种质可能存在新的抗性(等位)基因。本研究利用高通量分子标记筛选出的携带抗病、抗逆优异等位基因的种质为我国大豆资源表型鉴定、抗源的快速筛选及利用提供理论依据和新思路。

大豆种质; 分子标记; KASP技术; 基因型鉴定

引进种质在我国大豆品种改良中发挥了重要作用, 通过对1923¾1995年间育成和推广的651份大豆品种统计, 有224份品种可追溯到46个国外种质[1]。其中包括绥农14、合丰25等优良品种。绥农14拥有国外品种十胜长叶、Amsoy的血缘, 具有良好的遗传基础和农艺特征, 曾获得国家科技进步二等奖; 合丰25具有早熟、高产稳产、广适、抗逆性强等特点, 曾连续13年保持全国大豆品种播种面积第一的记录。分子标记遗传多样性分析结果表明, 美国和日本的品种与中国品种间存在明显遗传差异, 是重要的优异基因来源, 有利于改善我国大豆育种遗传基础狭窄的问题[2-5]。系统比较分析发现, 引进种质株型好、抗倒伏、抗病性较好, 对提高大豆产量、增强抗病、抗逆性、改善品质具有重要意义[6]。因此, 对引进种质的研究将促进其有效利用[7]。

大豆种质的鉴定评价是合理利用的前提。传统的种质鉴定是表型鉴定, 其鉴定不仅耗时费力, 鉴定结果准确性易受到环境条件的影响。如大豆胞囊线虫(soybean cyst nematode, SCN)鉴定至少在30 d以上[8]且鉴定数量有限。由于抗病、抗逆性鉴定条件及人力物力的限制, 难以对庞大数目的种质进行系统鉴定。因此, 自“八五”以来, 我国对引进国外大豆种质抗SCN、抗SMV (soybean mosaic virus)和耐盐性的鉴定数目不到总数的10%。

大豆基因组测序的完成促进了重要性状基因定位和克隆, 促进了基于抗病、耐逆性状的分子标记鉴定。抗大豆胞囊线虫方面, 已克隆SCN抗性的主效基因和, 发现了微效基因[9]。Kadam等[10]利用KASPar (Kompetitive Allele-Specific PCR)技术明确了95份大豆种质的、及其他QTL基因型。史学晖等[11]对105份种质鉴定发现, Rhg4-389鉴定的抗性等位变异对抗病种质的选择效率为97.1%。在抗大豆花叶病毒方面, Zheng等[12]定位了东北最强株系的抗性基因, 开发并验证了与SMV紧密连锁的SCAR标记——SCN11。利用SCN11对中品95-5117的12份系谱材料进行检测, 抗性条带的选择效率约为63.6%[13]。在耐盐方面, Guan等[14]图位克隆了大豆耐盐基因, 从53份大豆种质中鉴定出9种单倍型, 包括2种耐盐单倍型和7种盐敏感单倍型, 通过对172份微核心种质及12份美国大豆种质进行鉴定, 单倍型H1对耐盐性的选择效率为91.9%[14]。

为了加快大豆优异种质鉴定效率, 本研究开发了大豆抗胞囊线虫病3个基因(、、)和耐盐基因()的KASP标记, 并利用与大豆花叶病毒病抗性相关的SCAR标记(SCN11), 对1489份引进种质进行了分析, 鉴定出携带优异等位基因且已具有抗性的种质, 同时, 筛选出少量携带优异等位基因的种质, 为表型高效鉴定提供了依据, 发掘出具有抗性但不携带优异等位基因的种质, 是新抗性(等位)基因挖掘的重要材料。本研究结果为大豆新基因发掘和新品种培育提供了材料和技术支撑。

1 材料与方法

1.1 试验材料

本研究利用引进大豆种质共1489份, 分别来自亚洲、欧洲和美洲。其中以来自美洲(包括美国、加拿大和巴西3个国家)的种质数目最多, 达935份, 来自欧洲(包括俄罗斯、瑞典、德国等13个国家)的有371份, 来自亚洲(包括日本、泰国、韩国等6个国家或地区)的种质共91份, 解放前留下的大豆种质共27份, 另有65份无来源记载。

1.2 大豆基因组DNA的提取与质量检测

将参试种质每份播种5粒, 收集新鲜叶片。在TECAN液体自动化工作站平台进行大豆基因组DNA提取, 以96孔板为单位, 用改良的CTAB法[15]从大豆叶片中提取基因组DNA。提取后的DNA, 每96孔板抽检12个样品, 进行琼脂糖电泳检测, 并取2 µL DNA在BioTeK Synergy HIM上测定OD260、OD230值以及DNA浓度质检。

1.3 标记的开发与鉴定

大豆SCN抗性及耐盐相关的5个SNP位点采用KASP标记进行检测。根据选取的5个SNP位点及其侧翼序列(表1), 用Primer软件设计3¢末端PCR扩增引物,m值在55~65°C之间。每个SNP位点设计两条SNP特异性引物和一条通用引物[16]。标记的验证与检测在DouglasArray Tape平台上进行, 在SOELLEX高通量PCR水浴中完成PCR反应。PCR反应体系5 μL, 包括2 μL模板DNA (浓度约20 ng L–1)、2.5 μL 2×Master Mix (LGC Genomics, Hoddeston, UK)、0.07 μL Primer Mix、0.43 μL ddH2O。反应程序为94°C热激15 min; 94°C变性20 s, 65°C退火和延伸60 s, 10个循环, 每循环降低0.8°C; 94°C变性20 s, 57°C退火和延伸60 s, 26个循环。使用ARAYA荧光阅读仪读取荧光信号, 由Krake软件根据读取结果按照分型明确、NTC (无样品阴性对照)无特异性扩增的原则进行样品SNP分型。

与大豆花叶病毒抗性相关的位点用SCAR标记[12]进行检测。以基因组DNA为模板, 反应体系10 μL, 包括50 ng基因组DNA 2 μL、10×PCR缓冲液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 (全式金生物技术有限公司)、ddH2O 5.4 μL。PCR在ABI (Applied Biosystems, 美国)公司的PCR扩增热循环仪上进行, 反应程序为95°C预变性5 min; 95°C变性30 s, 59°C退火30 s, 72°C延伸70 s, 34个循环; 最后72°C延伸8 min, 于4°C保存。采用浓度为1.5%的琼脂糖凝胶电泳, 130V电压下电泳25 min, 经EB染色后在紫外灯下观察结果, 记录结果。

表1 抗SCN、抗SMV和耐盐相关标记的类型及引物序列

FAM和HEX分别代表引物为FAM标签序列和HEX标签序列; 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 数据分析

使用Microsoft Excel 2007进行等位基因分布频率计算。

2 结果与分析

2.1 5个位点的优异等位基因频率分析

利用抗大豆胞囊线虫病基因(、、)和耐盐基因的KASP标记对引进种质的分型结果显示, 两种纯合基因型和杂合基因型呈明显的簇状分离(图1-A~E), 证明了开发KASP标记的有效性。通过PCR扩增检测结果可以看出, 大豆花叶病毒病抗性相关标记SCN将参试种质分为扩增片段980 bp的抗病优异等位基因和扩增片段1070 bp的感病等位基因(图1-F)。标记和源自同一个位点(), 共同决定了耐盐单倍型H1, 记作。用6个分子标记对1489份引进大豆种质鉴定, 除去分型结果不明确的种质, 对有效鉴定的种质分析结果显示, 功能标记rhg1、Rhg4-389、SCN3-11、SALT3优异等位基因的频率较低(分别为0.022、0.025、0.049和0.170), 而连锁标记SCN11的优异等位基因频率较高(0.645) (表2)。

2.2 5个位点抗性种质的来源分布规律

各位点的抗性种质来源分布显示, SCN主效位点、的抗性种质主要来自美洲, 分别为24份和30份; 少量来自亚洲, 分别为4份和2份。微效基因的抗性种质多数(37份)来自美洲, 但部分种质(16份)来自欧洲。耐盐单倍型的大豆种质中来自美洲的最多(53.19%), 其次是欧洲(31.06%), 亚洲最少(4.68%)。SCAR标记SCN11鉴定出的抗SMV种质960份, 占总数的64.47%, 且美洲(700份)多于欧洲(147份)和亚洲(57份) (图2)。

图1 KASP和SCAR标记对部分种质基因型检测结果

A~E: 每个圆点各对应一份检测种质。红色或蓝色表示纯合基因型; 绿色表示杂合基因型; 粉色表示检测无信号或信号较弱; 紫色表示有信号但无明确分型; 黑色代表NTC, 即无模板对照。F: M是DL2000; 1、3为感SMV基因型; 2、4为抗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个抗性标记对引进种质的分型结果

1)R/T表示抗病或耐盐, S表示感病或盐敏感。

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

图2 5个位点抗性等位基因或单倍型分布及来源

2.3 基于优异等位基因的优异种质筛选

按表型特性进行分析, 筛选出仅具有单一性状抗性(单抗)的种质共907份, 包括抗SCN种质21份、抗SMV种质796份、耐盐种质90份。筛选出具有2种抗性（双抗）种质160份, 兼抗SCN和SMV种质32份; 抗SCN且耐盐的种质13份; 抗SMV且耐盐的种质115份。筛选出同时具有3种抗性(三抗)种质17份。

按基因型进行分析, 鉴定出携带优异等位基因的种质1084份, 包括具有单位点优异等位基因种质901份, 以SCN11标记筛选出的种质数目最多; 携带2个位点优异等位基因种质147份, 抗SCN双位点叠加种质最少, 而耐盐和抗SMV的基因聚合种质数量最多(表3); 携带3个或3个以上优异等位基因的种质36份, 其中有聚合3个位点的种质19份、聚合4个位点的种质9份、聚合5个位点的种质8份(表4)。

3 讨论

3.1 基于分子标记的基因型检测为提高种质鉴定和利用效率创造了条件

20世纪40年代以来, 我国多次引入国外大豆种质[5], 目前引进大豆种质已超过3000余份, 并保存在国家种质库[19]。20世纪80年代, 我国开始对大豆种质的农艺性状、抗病性及抗逆性进行评价, 而美国已从种质中筛选出了抗病、抗逆及抗虫种质[6]。由于基于表型鉴定筛选种质受到人力、财力、物力的影响, 鉴定结果受环境影响很大, 鉴定种质的数量非常有限。迄今为止, 仅对不超过500份引入种质的抗病性、抗逆性等性状进行了不完全鉴定。

表3 5个位点对引进种质的筛选

表4 携带3个或3个以上抗性等位基因的36份种质

(续表4)

+表示该种质来自泰国;#表示该种质来源不详; 其余种质均来自美国;*表示该种质此表型特性已有报道

+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状相关的分子标记对种质进行基因型鉴定的报道较少。例如，利用大豆灰斑病抗性基因连锁的3个SSR标记, 对45份地方种质进行检测, 鉴定效率分别为72.7%、81.8%、83.3%[20]；用大豆白粉病基因连锁标记Sat_366和Sat_393检测2个杂交分离F2群体, 鉴定效率分别为92.7%和60.3%[21]。用大豆黄种皮基因紧密连锁的标记ls1-22对355分析大豆核心种质等材料，鉴定选择效率为76.23%[21]。利用色素合成基因的4个功能标记分析272份种质, 对茸毛色的检测效率在80%以上[22]。利用与生育期相关的SSR标记Satt431、Satt215和Satt557对47份黄淮育成品种进行鉴定, 对熟期组MGIII和MGV的选择效率分别为83.33%和90.00%[23]。本研究选用的功能标记Rhg4-389及单倍型的鉴定效率在90%以上, 连锁标记SCN11的鉴定效率在60%以上, 揭示了在分子水平上直接对目的性状进行选择的可能性。通过高通量基因型鉴定明确了1489份引入种质的3个性状基因型, 鉴定种质数量占引进种质总数近50%, 选择出具有优异等位基因的种质, 其中已证明的有耐盐种质20份, 抗SCN种质21份, 抗SMV3号小种的种质12份, 可直接利用[25]。携带3个或3个以上抗性等位基因的优异种质36份, 虽然有33份表型尚需进一步验证, 但提高了筛选优异种质的目标性。本研究除了抗大豆花叶病毒SCAR标记SCN11外, 抗SCN和耐盐基因开发了可进行高通量检测的KASP标记, 降低了检测成本, 为上万份种质资源的高效发掘与利用提供了新思路。

3.2 KASP技术在大豆基因型鉴定中的应用

KASP技术是由LGC公司(http://www.lgcgroup. com/)设计和创制, 与芯片技术同属于高通量的SNP测序技术。与下一代测序(NGS)技术相比, 高通量SNP检测具有快速高效、敏感度高、使用方便、结果可靠、价格低廉等优势。芯片技术适用于对100到1 000 000个以上的SNP进行检测, 对少量SNP进行检测时, KASP技术具有经济高效的优势[26]。与基于芯片的Illumina GoldenGate相比, KASP技术分型的错误率(0.7%~1.6%)低于芯片GoldeGate平台(2.0%~2.4%), 且用于分子标记辅助回交选择时, 使用KASP技术分型将比使用其他高通量平台节省7.9%~46.1%的费用[27]。KASP技术以价格低廉、高效灵活性[28], 已在水稻[29]、玉米[30-31]、小麦[32-36]、蚕豆[37]等作物的研究中应用。在大豆研究中，Patil等[38]开发了基于种子组成性状相关等位基因KASP标记, 并应用于明确突变来源和基因定位。Patil等[39]还对耐盐基因多个与结构变异相关的SNP进行KASP检测, 验证了这些SNP与表型的高度相关。Pham等[40]定位来自PI594891和PI594774中抗灰斑病的2个候选基因, 开发KASP标记用于检测群体基因型与表型间的关联。本研究利用抗胞囊线虫病基因和耐盐基因开发的5个KASP标记, 系统检测了1489份引进种质基因型，占引进种质的46.2%，开创了我国大豆种质重要性状基因型快速鉴定的新局面。

3.3 优异等位基因种质的发掘为遗传育种提供了材料支撑

利用目的基因或与目的基因紧密连锁的分子标记可聚合多个优异等位基因。Maroof等[41]利用SSR标记对SMV感病大豆Essex的近等基因系中含有位点进行检测, 发现、和可通过两基因和三基因等位基因聚合可产生抗性, 但则表现出晚期易感。Wang等[42]用SMV抗性基因、、连锁的10个SSR标记对杂交后代群体检测, 并对标记聚合株系进行SMV抗性评价, F7代选出抗21个SMV菌株的纯合株系5个。分子标记辅助选择不仅应用于聚合同一性状的多个基因, 还可以聚合多个性状的多个基因。Kumar等[43]通过分子辅助回交育种将抗细菌性枯萎病基因和抗稻瘿蚊的基因、聚合到水稻恢复系RPHR-1005; Hur等[44]利用标记鉴定了BC4F6群体, 筛选出聚合水稻细菌枯萎病基因和及与耐冷QTL的株系ABL132-36。姚姝等[45]通过对杂交分离后代进行分子标记辅助选择, 创制出聚合水稻抗稻瘟病基因、和低直链淀粉含量基因的新品系“南粳0051”。

分子标记辅助选择在多基因聚合育种的成功应用, 提高了育种效率, 但在种质资源基因型鉴定方面鲜见报道。本研究检测的5个位点涉及抗SCN、抗SMV和耐盐3个性状, 筛选出携带抗SCN的3个位点优异等位基因(Peking型)种质19份, 其中15份具有抗SCN特性[46-50], 包括Centennial、Franklin、Forrest等; 用耐盐基因的2个标记筛选出235份种质, 其中20份已证明有耐盐性[51], 包括Altona、Mansoy、Baekun Kong、Lee68等; 用抗SMV标记筛选出960份大豆种质, 其中12份已证明表现为抗SMV3号株系[52-53], 包括L88-8440、L82-951、L84-2112、Columbia和L93-3327。除此之外, 仍有81份种质具有耐盐性, 但经标记检测不属于的耐盐单倍型H1, 推断少数种质[14]可能为耐盐单倍型H2, 还可能存在影响耐盐性的新位点; 有5份引进种质经SCN11检测为SMV感病型, 却表现为抗SMV3号株系, 包括L88-8431[54]、L78-379[55]、L83-4744 (内部资源)、L83-4483 (内部资源)和新八达2号[56], 一个原因可能是标记与基因间发生了交换, 这也是功能标记比连锁标记检测效率高的原因, 另一个原因可能是存在控制SMV抗性的新位点。有6份抗SCN种质(Bedford、Yale、Fayette、PI 209332、Cartter、Bell)在SCN三个位点鉴定结果显示均为感病基因型, 与它们的实际抗性表型不符[57-62], 其原因是这些种质为PI88788型, 在的另一处发生变异所致[63]。与现有资源相比, 新增可能抗SCN的种质4份, 可能具有耐盐特性的种质215份, 可能抗SMV的种质948份。携带3个或3个以上抗性等位基因的36份种质中, 其中16份种质的一种或两种特性已被报道, 且可能存在新特性; 17份种质尚未有3种特性的鉴定报道, 可能存在抗SCN、SMV或耐盐性。

本研究基于主要性状已知基因(、、、)开发的功能标记鉴定出的优异种质数目较少, 分别占鉴定种质总量的2.24%、2.50%、4.87%和17.03%; 而基于主要性状紧密连锁的标记(SCN11)鉴定出的种质数目较多, 占鉴定种质总量的64.47%。这是由于基因的连锁标记的遗传效应值依赖于该标记与基因的连锁紧密程度[64], 而功能标记的遗传效应具有可靠性和普适性[65], 能够准确地检测目的基因, 因此, 基于功能基因筛选的优异种质效率高于与基因连锁的标记。然而, 无论是用功能基因标记还是连锁标记, 一旦选择出优异种质, 其分子标记的选择效率都会提高, 可应用于分子标记辅助选择育种。

4 结论

抗病耐逆性是与大豆产量、品质相关的重要性状, 而通过分子标记进行基因型鉴定是进行优异种质筛选的有效手段。本研究鉴定出携带至少1种优异等位基因的种质1084份, 44份已由前人证明相应的抗性; 携带3个或3个以上优异等位基因的种质有36份, 其中52.78%种质的一种或两种优异特性已被报道。在不携带抗性优异等位变异的种质中, 93份具有耐盐性或SMV3号株系抗性报道, 这些种质可能存在新的抗性(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

邱丽娟, E-mail: qiulijuan@caas.cn

E-mail: yejunhua1994@qq.com

本研究由国家重点研发计划项目(2016YFD0100304, 2016YFD0100201)和中国农业科学院科技创新工程项目资助。

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