,1,*, 刘桂茹,1,*Development and validation of markers linked to genes resistant to Sitodiplosis mosellana in wheat
HAO Zhi-Ming1, GENG Miao-Miao1, WEN Shu-Min1, YAN Gui-Jun2, WANG Rui-Hui,1,*, LIU Gui-Ru,1,*通讯作者:
收稿日期:2019-04-8接受日期:2019-09-26网络出版日期:2019-10-16
| 基金资助: |
Received:2019-04-8Accepted:2019-09-26Online:2019-10-16
| Fund supported: |
作者简介 About authors
E-mail:haozhiming0730@hotmail.com。
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郝志明, 耿妙苗, 温树敏, 闫桂军, 王睿辉, 刘桂茹. 小麦抗麦红吸浆虫基因标记的开发与验证[J]. 作物学报, 2020, 46(2): 179-193. doi:10.3724/SP.J.1006.2020.91029
HAO Zhi-Ming, GENG Miao-Miao, WEN Shu-Min, YAN Gui-Jun, WANG Rui-Hui, LIU Gui-Ru.
麦红吸浆虫(Sitodiplosis mosellana Géhin)是严重威胁小麦生产的重要害虫, 吸浆虫以幼虫吸吮灌浆期小麦籽粒内含物, 使籽粒秕瘦、粒重降低, 更易受病原菌侵染, 导致小麦籽粒品质下降, 商品性受到严重影响。受害麦田产量损失在10%~60%之间, 严重时甚至颗粒无收[1,2]。在加拿大、美国、英国、波兰、德国等小麦主产国, 均有吸浆虫为害的报道[3,4,5,6,7,8]。在20世纪50年代和80年代, 我国小麦生产曾两度遭遇吸浆虫大面积为害, 损失巨大[9,10]。21世纪以来, 吸浆虫的危害依然严重。2013—2018年, 小麦主产区年均虫害发生面积超过2.0×106 hm2 (http://www. agri.gov.cn/), 成为影响华北、黄淮和西北等地小麦生产的重要虫害(https://www.natesc.org.cn/)。生产上, 可通过药物(农药)、农艺措施(深翻、轮作等)、生物(性引诱剂、天敌等)和抗虫品种防治麦红吸浆虫[4,11-12], 而以抗虫品种的选育和利用最为经济有效[13]。在20世纪, 西农6028等抗虫品种的使用对抑制虫害起到了关键作用, 而抗虫品种的缺乏也是当前我国麦田吸浆虫为害面积居高不下的重要原因[9]。但吸浆虫危害的隐蔽性、毁灭性以及小麦对吸浆虫抗性遗传的复杂性, 使得小麦抗虫育种进展缓慢[14,15]。传统的抗性育种单纯依赖于表型鉴定结果, 极易受环境和人为因素的影响, 而且费时费力, 抗虫品种的选育效率十分低下。分子标记辅助选择(marker-assisted selection, MAS)技术在小麦等主要作物抗性育种中的应用, 能够显著降低外界因素对抗虫鉴定结果的干扰, 也有利于实现作物抗虫性与农艺性状的同步改良, 进而提高育种效率[16,17]。开发与抗麦红吸浆虫基因/QTL紧密连锁的分子标记, 尤其是根据抗虫候选基因或抗虫基因序列上的突变位点开发成的目的基因标记(gene-targeted markers, GTMs)和功能标记(functional markers, FMs)[18,19,20,21,22], 不仅能够提高抗虫品种筛选及抗虫性选择的效率, 而且也有利于辅助实现不同来源抗虫基因的聚合, 使小麦的抗虫性更持久和稳定[23]。
Sm1是最早发现的小麦抗麦红吸浆虫基因[24], 位于2BS上SSR标记Xgwm210和Xbarc35与SCAR标记XWM1之间2.5 cM的遗传区间内[3]。Xbarc35和XWM1已在北美和英国的小麦抗虫性MAS育种中得以应用[25]。Kassa等[26]利用含有Sm1基因的小麦亲本组合构建的DH群体, 又发现了7个由单核苷酸多态性(SNP)位点开发的KASP标记与抗虫性紧密连锁, 可用于鉴定抗/感虫单倍型。Sm1也是当前加拿大、英国和美国的抗虫小麦品种中唯一被广泛利用的抗虫基因[27](http://midgetolerantwheat.ca/; http://www. bcpc.org/; https://www.usda.gov/)。过度依赖单一抗虫基因, 大大增加了新毒性吸浆虫生物型出现的风险, 也促使人们寻找新的抗虫基因资源[28]。
中国小麦品种可能携带了不同的麦红吸浆虫抗性基因。我国抗吸浆虫小麦品种多具有南大2419、西农6028和洛夫林10号等的血缘[9,14,29-32], 晋麦31、咸农151、武农99、郑州8号、冀麦23、冀麦24、河农215、荆麦66等抗虫品种的系谱中都或多或少携带有上述品种的血缘[9,14,30-31], 与国外抗虫小麦品种的系谱不同[3,24,27-28,33-34]。北美等地小麦的抗虫基因或QTL被定位于2B (Sm1)[24]和1A (QSm.mst-1A)[28]染色体上, 以Sm1为主要抗源。我们发现, 来自我国小麦品种的抗虫性主要被定位于4A染色体(QSm.hbau- 4A)[14,35]。Sm1基因的连锁标记(Xgwm210、Xbarc35和XWM1)虽已被应用于当地小麦抗虫品种的培育, 但这些标记在我国小麦品种中要么不具多态性, 要么检测结果与抗虫性不吻合。阿魏酸等多酚类物质曾被认为是小麦籽粒中主要的抗虫成分[36,37], 与Sm1基因的抗性有关[3,26]。然而, 我们测定了4A主效QTL近等基因系的阿魏酸含量, 未能发现这种差异的存在。咖啡酸-O-甲基转移酶(COMT)是阿魏酸合成途径中的关键酶[38] (https://www.kegg.jp/)。我们从吸浆虫胁迫下的小麦转录组数据中鉴定出3个COMT同源基因, 但在抗、感小麦亲本间均不存在SNP。上述研究表明, 我国小麦品种可能存在新的吸浆虫抗性遗传机制。鉴于此, 开发我国小麦种质中与抗虫位点紧密连锁甚至共分离的分子标记和功能标记, 不但能更好地满足我国抗虫性MAS育种的需要, 也有助于对我国小麦种质资源抗虫性的准确鉴定。
随着测序技术的改进、成本的降低, 以及多个物种参考基因组序列(包括中国春小麦参考基因组序列)的公开, 对基因组中大量序列变异和结构变异的挖掘成为可能, 对直接利用转录组数据或结合集群分离分析法(bulked segregant analysis, BSA)的思路进行标记开发变得更加便利[39]。王智兰等[40]对抗旱相关基因TaPP2Aa进行序列检测和分析, 根据该基因在不同小麦品种中存在的多态性位点开发了功能标记。Wu等[41]从转录组和芯片测序数据中挖掘到235个染色体特异性SNP位点, 并开发成KASP标记, 成功用于Yr26基因的精细定位。徐晓丹[42]利用BSR-Seq方法开发了SNP标记, 其中2个SNP标记对PmYBL和PmSGD检测的有效率达97%和100%。
在前期研究中, 我们采用BSR-Seq[43]方法和QTL-Seq分析[44]的思路, 结合基因功能注释信息及qRT-PCR表达谱分析, 从抗、感虫小麦样本(含双亲、极端混池、重组近交系、近等基因系)的转录组数据中, 获得了位于4AL主效QTL[14,35]区间的6个抗虫性相关差异基因, 其中基因TraesCS4A01G436100和TraesCS4A01G437800不仅在全部抗、感RIL株系及NIL株系间的表达水平差异显著, 而且显著富集于与抗虫性相关的异黄酮生物合成代谢通路(isoflavonoid biosynthesis pathway)中[45,46]。因此, 本研究以上述6个抗虫相关基因及其SNP和插入缺失位点(InDel)为基础, 设计和开发目的基因标记, 并利用抗、感虫小麦RIL系和小麦品种验证了这些标记在小麦吸浆虫抗性鉴定中的可用性。
1 材料与方法
1.1 植物材料及DNA提取
用于标记验证的植物材料包括小麦亲本6218 (感虫)、冀麦24 (抗虫), 从6218/冀麦24重组近交系(RIL)群体中选择的92个抗、感吸浆虫株系以及具有不同抗性水平的95个小麦品种。其中, RIL株系来自小麦亲本6218与冀麦24的F2代群体的单粒传自交后代, 经多年抗虫鉴定, 表型稳定。供试小麦材料分别在2014—2015年度、2015—2016年度和2016—2017年度种植于河北农业大学育种中心虫圃。每个材料种植1行, 每行40粒, 行长20 cm, 行距20 cm[14]。完全随机区组设计, 3次重复。供试材料的名称及表型见表1。采用CTAB法[47]提取各供试材料的基因组DNA。Table 1
表1
表1供试小麦亲本与RIL株系的表型
Table 1
| 小麦亲本及株系 Wheat parent and line | 抗性指数(RI) Resistance index | 抗虫等级 Classification | 小麦株系 Wheat line | 抗性指数(RI) Resistance index | 抗虫等级 Classification |
|---|---|---|---|---|---|
| 冀麦24 Jimai 24 | 0.0128-0.1233 | 1 | RIL-264 | 0.1007-0.2659 | 2 |
| 6218 | 2.9543-7.2714 | 5 | RIL-285 | 0.0064-0.2105 | 2 |
| RIL-7 | 0.0575-0.0622 | 1 | RIL-16 | 0.7300-1.3397 | 4 |
| RIL-12 | 0.0133-0.0336 | 1 | RIL-21 | 0.9122-1.4373 | 4 |
| RIL-18 | 0.0745-0.0817 | 1 | RIL-60 | 1.2009-1.2429 | 4 |
| RIL-23 | 0.0029-0.0330 | 1 | RIL-106 | 1.2142-1.2325 | 4 |
| RIL-38 | 0.1441-0.1886 | 1 | RIL-251 | 0.9603-1.4106 | 4 |
| RIL-64 | 0.0188-0.0380 | 1 | RIL-19 | 2.7085-2.8602 | 5 |
| RIL-73 | 0.0784-0.1409 | 1 | RIL-20 | 2.6327-3.1797 | 5 |
| RIL-91 | 0.0205-0.0818 | 1 | RIL-25 | 3.0783-4.3757 | 5 |
| RIL-115 | 0.0503-0.1358 | 1 | RIL-29 | 2.1227-2.5619 | 5 |
| RIL-134 | 0.0104-0.0394 | 1 | RIL-45 | 2.2811-2.7601 | 5 |
| RIL-156 | 0.0244-0.0253 | 1 | RIL-46 | 2.3158-6.3697 | 5 |
| RIL-169 | 0.0434-0.0569 | 1 | RIL-49 | 2.0398-2.6978 | 5 |
| RIL-170 | 0.0539-0.1697 | 1 | RIL-62 | 1.2193-2.6308 | 5 |
| RIL-175 | 0.0123-0.0731 | 1 | RIL-63 | 2.0683-2.5504 | 5 |
| RIL-186 | 0.0231-0.0467 | 1 | RIL-68 | 2.2674-4.9395 | 5 |
| RIL-194 | 0.1535-0.1781 | 1 | RIL-72 | 2.8236-3.1560 | 5 |
| RIL-214 | 0.0778-0.1384 | 1 | RIL-84 | 2.0781-2.8191 | 5 |
| RIL-223 | 0.0460-0.1956 | 1 | RIL-92 | 2.1872-4.3090 | 5 |
| RIL-249 | 0.0403-0.0848 | 1 | RIL-95 | 2.6088-2.6854 | 5 |
| RIL-253 | 0.0037-0.0109 | 1 | RIL-97 | 1.6879-2.1144 | 5 |
| RIL-259 | 0.0406-0.0534 | 1 | RIL-102 | 2.7594-2.9148 | 5 |
| RIL-274 | 0.0293-0.0953 | 1 | RIL-113 | 1.4552-2.8464 | 5 |
| RIL-283 | 0.0091-0.0658 | 1 | RIL-119 | 1.2420-3.1363 | 5 |
| RIL-13 | 0.1109-0.2648 | 2 | RIL-122 | 2.4698-2.6681 | 5 |
| RIL-28 | 0.1087-0.4394 | 2 | RIL-125 | 1.6834-2.1306 | 5 |
| RIL-39 | 0.2354-0.2983 | 2 | RIL-139 | 2.5142-3.2524 | 5 |
| RIL-44 | 0.2961-0.3166 | 2 | RIL-148 | 1.8636-5.5925 | 5 |
| RIL-54 | 0.0331-0.2245 | 2 | RIL-150 | 2.4328-3.1815 | 5 |
| RIL-56 | 0.0064-0.3427 | 2 | RIL-167 | 2.5747-2.9479 | 5 |
| RIL-69 | 0.1050-0.2124 | 2 | RIL-168 | 1.9348-3.3892 | 5 |
| RIL-78 | 0.0586-0.2337 | 2 | RIL-174 | 1.1562-2.6991 | 5 |
| RIL-107 | 0.0039-0.3020 | 2 | RIL-182 | 2.7128-3.3572 | 5 |
| RIL-155 | 0.0188-0.2217 | 2 | RIL-185 | 3.1085-4.9160 | 5 |
| RIL-158 | 0.2659-0.4615 | 2 | RIL-212 | 2.0868-2.6547 | 5 |
| RIL-164 | 0.1272-0.3710 | 2 | RIL-213 | 2.0094-3.0880 | 5 |
| RIL-180 | 0.3315-0.3891 | 2 | RIL-216 | 1.1651-1.7900 | 5 |
| RIL-183 | 0.0392-0.3963 | 2 | RIL-233 | 2.4937-2.6506 | 5 |
| RIL-196 | 0.0518-0.2096 | 2 | RIL-238 | 1.1102-3.0323 | 5 |
| RIL-197 | 0.1203-0.2525 | 2 | RIL-240 | 3.0399-6.1309 | 5 |
| RIL-218 | 0.1467-0.2391 | 2 | RIL-247 | 1.5939-2.1915 | 5 |
| RIL-219 | 0.1613-0.2189 | 2 | RIL-248 | 2.1621-4.1447 | 5 |
| RIL-226 | 0.3513-0.4673 | 2 | RIL-265 | 2.6932-2.9406 | 5 |
| RIL-241 | 0.2658-0.3099 | 2 | RIL-267 | 2.7879-4.7565 | 5 |
| RIL-244 | 0.0933-0.2738 | 2 | RIL-275 | 1.5796-2.3936 | 5 |
| RIL-260 | 0.0977-0.2581 | 2 | RIL-276 | 1.3032-2.7370 | 5 |
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1.2 供试小麦材料的抗虫性鉴定
利用抗性指数(resistance index, RI)评价供试小麦材料的抗虫性[14]。在小麦灌浆至乳熟期, 取每重复、每株系10~15个穗子, 密封于纸袋中, 带回室内计数籽粒上的虫子。根据存在的吸浆虫数目, 将待鉴定小麦籽粒抗性分为5级。0级为籽粒上无虫; 1级为籽粒上有虫1头; 2级为籽粒上有虫2头; 3级为籽粒上有虫3头; 4级为籽粒上的虫数≥4头。根据以下公式计算每个株系的估计损失率(L)[2]。L(%) = Σ(xf) / 4Σf × 100%
式中, x为相应籽粒的抗性级别, f为各级别的籽粒数目。
用每个株系的估计损失率(L)除以全部参试株系(品种)的平均估计损失率(ML), 计算出抗性指数(RI)来确定株系的抗性分级。当同一株系在不同重复间的鉴定结果存在较大差异时, 以最重重复数据进行L、RI值的估算[2]。基于RI值将供试小麦材料的抗性分为免疫(RI=0) (immune, I)、高抗(0.01≤RI<0.19) (highly resistant, HR)、中抗(0.20≤RI<0.49)(moderately resistant, MR)、低抗(0.50≤RI<0.99) (lowly resistant, LR)、感虫(1.00≤RI<1.50) (susceptible, S)和高感(RI≥1.50)(highly susceptible, HS)。
1.3 抗虫差异基因序列检测
以前期研究中获得的6个抗虫相关基因TraesCS4A01G436000、TraesCS4A01G436100、TraesCS4A01G436500、TraesCS4A01G437300、TraesCS4A01G437400和TraesCS4A01G437800进行抗虫标记的开发。根据与中国春参考序列(IWGSC RefSeq v1.0, https://wheat-urgi.versailles.inra.fr/)的比对结果, 提取上述基因的侧翼序列。用Primer5软件设计引物。用高保真酶在小麦亲本冀麦24和6218中扩增目的基因。10 μL PCR体系包括模板DNA 50 ng、正反引物各0.4 μmol L-1、1× Pfu PCR MasterMix (天根生化科技(北京)有限公司)。PCR条件为: (1) 94℃, 预变性3 min; (2) 94℃, 30 s; 55℃, 30 s; 72℃, 2 min; 共35个循环; (3) 72℃终延伸5 min。扩增产物经1%琼脂糖凝胶电泳分离, 回收目的条带, 送生工生物工程(上海)股份有限公司进行双向测序。将测序结果与中国春(IWGSCv1.0)和百农AK58 (v4.24)的基因组序列进行比对。1.4 抗虫标记的开发
1.4.1 EST (expressed sequence tag, 表达序列标签)标记 根据测序结果, 分析目标基因在小麦亲本间的InDel, 并根据InDel两侧翼各200 bp的序列设计引物。采用普通DNA聚合酶(2× Es Taq MasterMix)进行PCR扩增。10 μL PCR体系包括模板DNA 50 ng、正反引物各0.4 μmol L-1、1× Es Taq MasterMix (北京康为世纪生物科技有限公司)。反应程序为: (1) 94℃, 预变性5 min; (2) 94℃, 30 s; 57℃, 30 s; 72℃, 1 min; 共35个循环; (3) 72℃终延伸7 min。扩增产物在30%的聚丙烯酰胺凝胶上电泳, 银染显色。1.4.2 KASP (kompetitive allele-specific PCR, 竞争性等位特异PCR)标记 以抗虫相关基因在冀麦24 (高抗吸浆虫)与6218 (高感吸浆虫)、百农AK58 (高感吸浆虫)间均表现出多态性的SNP位点及其侧翼序列与小麦参考序列(IWGSCv1.0)进行比对, 挑选出在基因组中具有特异序列或其中一种SNP等位型(突变类型)在参考基因组中不存在的SNP, 进行KASP标记的设计与开发。提取选定SNP在参考基因组中的侧翼序列(>200 bp), 由艾吉析科技(上海)有限公司(LGC Science Shanghai Ltd.)进行引物的设计与合成, 在两条KASP正向引物序列的5′端加上FAM (6-carboxy-fluorescein)或HEX (Hexachlorofluorescein)荧光基团的接头序列。参照公司提供的程序进行KASP标记的分型试验。反应体系总体积为3 μL, 含KASP (V4.0) 2× Mastermix 1.5 μL, 引物mix (72×) 0.0417 μL, DNA模板50~100 ng左右。PCR为Touchdown反应程序, 94℃变性15 min; 94℃变性20 s, 68℃退火60 s, 每循环一次下降0.6℃, 10个循环; 94℃变性20 s, 62℃退火60 s, 26个循环。PCR扩增完毕后, 利用LGC系统的SNPline平台进行SNP分型检测, 利用Kraken (v16.3.16.16288)软件分析扫描数据。
2 结果与分析
2.1 基因序列分析
6个抗虫相关基因在抗、感小麦亲本冀麦24和6218中均能扩增到目的片段。除TraesCS4A01G436500外, 其他5个基因在抗、感小麦亲本中的序列与参考基因组序列(中国春和百农AK58)相符; TraesCS4A01G436500在抗虫亲本冀麦24的序列与参考基因组序列相符, 而在感虫亲本6218的序列与参考基因组序列差异较大。上述基因在抗、感小麦亲本间均存在SNP, 且都包含使氨基酸发生改变的非同义突变位点(图1)。由于6218和百农AK58均高感麦红吸浆虫, 因此那些同时存在于冀麦24与6218之间、冀麦24与百农AK58之间的多态性SNP, 被视为高可信度SNP。TraesCS4A01G436100基因中存在4个非同义突变SNP, 其中3个为可信度较高的SNP。TraesCS4A01G437400基因中存在6个非同义突变SNP, 其中4个为可信度较高的SNP。TraesCS4A01G437800存在1个高可信度的非同义突变的SNP。TraesCS4A01G437300存在SNP和插入/缺失(InDel)各1个, 其中抗虫品种(冀麦24)中存在一个12 bp的插入, 而感虫品种(系)(6218和百农AK58)则缺失了这12 bp。由于未能获得TraesCS4A01G436500在感虫亲本(6218)中的扩增序列, 故根据该基因在抗虫亲本冀麦24与感虫品种百农AK58间存在的2个SNP位点进行标记开发。TraesCS4A01G436000在抗、感小麦亲本中分别存在1个4 bp和1 bp的插入, 由于这种插入可能导致其后的读码框发生改变(移码突变), 因此重新预测了该基因在抗、感虫小麦亲本中的所有开放阅读框(https://web.expasy.org/), 分别获得在抗、感小麦亲本中使编码蛋白仍具有丝氨酸蛋白酶抑制剂(SPI)结构域的编码序列, 发现在编码区序列上存在11个非同义突变位点, 其中1个为可信度较高的SNP; 此外, 该基因在冀麦24的基因下游处存在一个29 bp的插入, 而在感虫亲本则缺失了这29 bp的序列。
图1
新窗口打开|下载原图ZIP|生成PPT图16个差异基因在抗(冀麦24)、感(6218)小麦亲本及中国春参考序列之间的比对结果
蓝色或红色碱基代表非同义突变的SNP位点或插入缺失, 其中蓝色碱基为可信度较高的SNPs, 并以箭头标注。
Fig. 1Sequence alignments of the six differentially expressed genes from wheat parents, ‘6218’ (susceptible) and ‘Jimai 24’ (resistant) and the Chinese Spring reference genome
Red or blue font letters represent non-synonymous SNPs and InDels present in the genes. Blue letters with arrowheads represent the SNPs with high confidence.
以这些高可信度的SNP位点及其侧翼序列与小麦参考基因组序列(IWGSCv1.0)的比对结果, 对基因组特异性或突变等位型特异性(即仅在目的基因序列中的特定碱基位置上具有抗虫亲本的突变类型, 而在参考基因组中全部同源序列对应的碱基位点上均不存在该突变类型)的SNP, 进行KASP标记开发。根据比对结果, 选择基因TraesCS4A01G436100上存在的2个特异性位点, TraesCS4A01G437400、TraesCS4A01G437800、TraesCS4A01G436000和TraesCS4A01G436500上存在的各1个特异性位点, 共6个可信度较高的特异性SNP用于KASP标记的开发, 选择基因TraesCS4A01G437300和TraesCS4A01G436000序列中存在的InDel, 用于EST标记的开发。
2.2 抗虫标记的开发及多态性检测
根据上述基因中存在的SNP和InDel, 分别设计出6个KASP标记(K3-1-1、K3-7-1、K3-7-3、K3-16-1、K10-10-6和K10-13-x)和2个EST标记(E1-2和E10-10), 用这些标记检测了抗、感虫小麦亲本、RIL株系和小麦品种。2个EST标记在小麦亲本间均表现出多态性, 其中E1-2在抗、感虫亲本中分别扩增出173 bp和161 bp的片段, E10-10分别扩增出211 bp和182 bp的片段(图2)。6个KASP标记在抗、感虫小麦亲本间亦具有多态性(图3)。标记K3-7-1、K3-7-3、K3-16-1和K10-10-6在小麦亲本中均为纯合分型, 在抗虫亲本中分别为A:A、T:T、T:T和G:G, 在感虫亲本中分别为C:C、G:G、A:A和A:A。K3-1-1和K10-13-x在抗虫小麦亲本中为杂合分型(分别为T:G和C:G), 在感虫亲本中表现纯合分型(均为G:G)。将K3-1-1和K10-13-x的2条正向引物序列与小麦参考基因组序列(IWGSCv1.0)比对后发现, 感虫位点对应序列在参考基因组中均存在多个匹配(数据未列出)。因此, 在抗、感虫小麦亲本中, 这2个标记均能检测出感虫等位型; 而抗虫位点对应序列与参考基因组间不存在3′末端匹配, 即存在于抗虫小麦品种冀麦24中的突变型位点在参考基因组中不存在, 因此在不具有该突变的材料中无法检测到抗虫标记位点。综上, 这些标记可用于检测小麦品种或株系中4A染色体抗虫QTL。图2
新窗口打开|下载原图ZIP|生成PPT图2EST标记E1-2 (A)和E10-10 (B)在抗、感虫小麦亲本及部分RIL系中的扩增结果
M: 分子量marker (pBR322/Mst I酶切片段); 1: 感虫小麦亲本6218; 2: 抗虫小麦亲本冀麦24; 3~14: 感虫株系(RIL-16、RIL-19、RIL-20、RIL-21、RIL-25、RIL-29、RIL-45、RIL-102、RIL-113、RIL-119、RIL-125和RIL-139); 15~26: 抗虫株系(RIL-7、RIL-12、RIL-18、RIL-23、RIL-64、RIL-73、RIL-91、RIL-115、RIL-134、RIL-156、RIL-169和RIL-170)。
Fig. 2Polyacrylamide gel electrophoresis profiles for EST markers E1-2 (A) and E10-10 (B) in susceptible, or resistant-wheat parent, and selected RIL lines
M: Molecular marker (restriction fragments of plasmid pBR322 digested with Mst I endonuclease); 1: Susceptible wheat parent ‘6218’; 2: Resistant parent ‘Jimai 24’; 3-14: Susceptible lines (RIL-16, RIL-19, RIL-20, RIL-21, RIL-25, RIL-29, RIL-45, RIL-102, RIL-113, RIL-119, RIL-125, and RIL-139); 15-26: Resistant lines (RIL-7, RIL-12, RIL-18, RIL-23, RIL-64, RIL-73, RIL-91, RIL-115, RIL-134, RIL-156, RIL-169, and RIL-170).
图3
新窗口打开|下载原图ZIP|生成PPT图3KASP标记在抗、感虫小麦亲本、92个RIL系和95个小麦品种中的分型结果
红色点, HEX基因型; 蓝色点, FAM基因型; 绿色点, 杂合基因型; 粉色点, 缺失; 黑色点, 对照. 图中A~F分别为KASP标记K3-1-1、K3-7-1、K3-7-3、K3-16-1、K10-10-6和K10-13-x的分型结果。
Fig. 3KASP assays in wheat parents, 92 RIL lines, and 95 wheat cultivars
Red dots represent the HEX-type allele; blue dots, the FAM-type allele; green dots, the heterozygous-type allele; pink dots, undetected; black dots, the NTC (non-template control); A to F refer to genotypes for K3-1-1, K3-7-1, K3-7-3, K3-16-1, K10-10-6, and K10-13-x, respectively.
2.3 EST、KASP标记在小麦抗、感虫RIL系中的检测结果
用这些标记检测了47个抗虫(RI = 0.0330~ 0.4673)、45个感虫(RI = 1.2325~6.3697) RIL系(表1)。所有KASP标记和EST标记在抗、感虫RIL株系中的检测结果(带型或分型)与相应抗、感小麦亲本的吻合度较高。KASP标记能够在85%以上的抗虫株系中检测出抗虫标记位点(其中标记K3-1-1和K10-13-x为杂合分型), 在95%以上的感虫株系中检测出感虫标记位点(表2)。两个EST标记, 均能够在85%以上的抗虫株系中扩增出与抗虫亲本一致的带型, 在超过90%的感虫株系中扩增出与感虫亲本一致的带型(表2)。如果忽略个别标记在个别株系中的缺失情况(标记K3-1-1、K3-7-1、K3-7-3和K10-10-6均分别在2个RIL株系中缺失, 标记K10-13-x在4个RIL株系中存在缺失), KASP标记在所检测RIL株系间的抗、感分型结果完全一致, 说明这些标记均应位于同一个染色体交换区段内, 并且可能与抗虫基因紧密连锁或共分离。Table 2
表2
表2EST标记和KASP标记在供试RIL株系中的标记基因型及比例
Table 2
| 标记 Marker | 物理位置a Physical location a (bp) | 抗虫RILs各标记基因型及比例 Marker-based genotypes and their ratio for resistant RIL lines (%) | 感虫RILs各标记基因型及比例 Marker-based genotypes and their ratio for susceptible RIL lines (%) | |||||
|---|---|---|---|---|---|---|---|---|
| A | H | B | A | H | B | |||
| E1-2 | 707248439 | 40 (85.1) | 0 (0.0) | 7 (14.9) | 2 (4.4) | 0 (0.0) | 43 (95.6) | |
| E10-10 | 705763221 | 40 (85.1) | 0 (0.0) | 7 (14.9) | 4 (8.9) | 0 (0.0) | 41 (91.1) | |
| K3-7-1 | 705889579 | 39 (86.7) | 0 (0.0) | 6 (13.3) | 2 (4.4) | 0 (0.0) | 43 (95.6) | |
| K3-7-3 | 705890140 | 39 (86.7) | 0 (0.0) | 6 (13.3) | 2 (4.4) | 0 (0.0) | 43 (95.6) | |
| K10-10-6 | 705763509 | 39 (86.7) | 0 (0.0) | 6 (13.3) | 2 (4.4) | 0 (0.0) | 43 (95.6) | |
| K3-16-1 | 707251252 | 41 (87.2) | 0 (0.0) | 6 (12.8) | 2 (4.4) | 0 (0.0) | 43 (95.6) | |
| K3-1-1 | 707477512 | 0 (0.0) | 39 (86.7) | 6 (13.3) | 0 (0.0) | 2 (4.4) | 43 (95.6) | |
| K10-13-x | 706095841 | 0 (0.0) | 39 (88.6) | 5 (11.4) | 0 (0.0) | 2 (4.6) | 42 (95.5) | |
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2.4 EST、KASP标记在不同抗性小麦品种中的检测结果
利用95个不同抗性的小麦品种为材料(表3), 对上述标记的适用性进行了检测。标记E1-2能够在37.2%的抗虫小麦品种中扩增出纯合抗虫带型, 在80%的感虫品种中扩增出纯合感虫带型(表4)。由于E1-2在感虫小麦品种中的检测有效率较高(为80.0%), 因此该标记对具有抗虫标记位点小麦品种的鉴定准确率为80%左右。在对小麦品种进行检测时, 则只要在测试品种中检测出抗虫带型, 那么该品种为抗虫品种(即具有QSm.hbau-4A抗虫位点)的可能性在80%左右, 可作为筛选抗虫种质资源的标记。而标记E10-10在抗、感虫小麦品种中所检测出的抗、感基因型所占比例相近(表4), 不能区分抗、感虫小麦品种, 因此该标记不能用于鉴定供试小麦品种的抗虫性。Table 3
表3
表3供试小麦品种的表型
Table 3
| 小麦品种 Wheat cultivar | 抗性指数(RI) Resistance index | 抗虫等级 Classification | 小麦品种 Wheat cultivar | 抗性指数(RI) Resistance index | 抗虫等级 Classification |
|---|---|---|---|---|---|
| 小偃81 Xiaoyan 81 | 0.0039-0.0073 | 1 | 邯麦12号 Hanmai 12 | 0.3599-0.8138 | 3 |
| 晋麦47 Jinmai 47 | 0.0026-0.0155 | 1 | 河农6425 Henong 6425 | 0.1632-0.8449 | 3 |
| 河农6049 Henong 6049 | 0.0000-0.0235 | 1 | 周黑麦1号 Zhouheimai 1 | 0.6180-0.8651 | 3 |
| 石麦12号 Shimai 12 | 0.0197-0.0468 | 1 | 中麦12 Zhongmai 12 | 0.3425-0.8812 | 3 |
| 衡优18 Hengyou 18 | 0.0442-0.0513 | 1 | 藁优9618 Gaoyou 9618 | 0.0978-0.9218 | 3 |
| 河农58-3 Henong 58-3 | 0.0300-0.0770 | 1 | 石麦14号 Shimai 14 | 0.9019-0.9743 | 3 |
| 石新828 Shixin 828 | 0.0013-0.0862 | 1 | 科农199 Kenong 199 | 0.8349-1.0414 | 4 |
| 河农4198 Henong 4198 | 0.0602-0.1087 | 1 | 良星99 Liangxing 99 | 0.3466-1.1006 | 4 |
| 西农6028 Xinong 6028 | 0.0015-0.1089 | 1 | 石家庄10号 Shijiazhuang 10 | 0.0074-1.1150 | 4 |
| 河农215 Henong 215 | 0.0900-0.1214 | 1 | 石4185 Shi 4185 | 0.3530-1.1329 | 4 |
| PH82-2-2 | 0.0423-0.1223 | 1 | 邢麦7号 Xingmai 7 | 0.6757-1.1334 | 4 |
| 科农1093 Kenong 1093 | 0.0678-0.1338 | 1 | 观35 Guan 35 | 0.7848-1.1727 | 4 |
| 矮丰1号 Aifeng 1 | 0.0009-0.1345 | 1 | 长6878 Chang 6878 | 0.1776-1.1764 | 4 |
| 晋麦33 Jinmai 33 | 0.0632-0.1395 | 1 | 石家庄8号 Shijiazhuang 8 | 1.1628-1.2792 | 4 |
| 中农28 Zhongnong 28 | 0.1412-0.1509 | 1 | 藁优9908 Gaoyou 9908 | 0.1786-1.2974 | 4 |
| 丰产2号 Fengchan 2 | 0.0302-0.1567 | 1 | 济麦20 Jimai 20 | 0.6224-1.3508 | 4 |
| 南大2419 Nanda 2419 | 0.0012-0.1645 | 1 | 衡0628 Heng 0628 | 1.1138-1.3548 | 4 |
| 济麦22 Jimai 22 | 0.0636-0.1806 | 1 | 河农826 Henong 826 | 1.0058-1.3817 | 4 |
| 中麦155 Zhongmai 155 | 0.1302-0.2029 | 2 | 轮选061 Lunxuan 061 | 0.8530-1.5171 | 5 |
| 晋麦79 Jinmai 79 | 0.0115-0.2049 | 2 | 北京0045 Beijing 0045 | 0.7773-1.7354 | 5 |
| 河农822 Henong 822 | 0.0567-0.2088 | 2 | 农大399 Nongda 399 | 0.6830-1.7594 | 5 |
| 陕229 Shaan 299 | 0.1115-0.2167 | 2 | 衡95观26 Heng 95 guan 26 | 1.2248-1.7931 | 5 |
| 汶农14 Wennong 14 | 0.1723-0.2213 | 2 | 衡4444 Heng 4444 | 1.6026-1.8759 | 5 |
| 邯麦9号 Hanmai No.9 | 0.0259-0.2524 | 2 | 衡7228 Heng 7228 | 0.8897-1.8930 | 5 |
| 临汾3050 Linfen 3050 | 0.0146-0.2557 | 2 | 邢麦6号 Xingmai 6 | 1.6227-1.9706 | 5 |
| 白硬冬2号 Baiyingdong 2 | 0.2241-0.2673 | 2 | 周麦23 Zhoumai 23 | 1.1381-1.9960 | 5 |
| 良星66 Liangxing 66 | 0.1759-0.2731 | 2 | 石新618 Shixin 618 | 0.8939-1.9989 | 5 |
| 婴泊700 Yingbo 700 | 0.1775-0.2770 | 2 | NC2 | 1.4307-2.0183 | 5 |
| 河农9206 Henong 9206 | 0.2792-0.2844 | 2 | 衡4399 Heng 4399 | 1.7993-2.1132 | 5 |
| 冀麦23 Jimai 23 | 0.0296-0.2923 | 2 | 衡4338 Heng 4338 | 2.0068-2.1404 | 5 |
| 师栾02-1 Shiluan 02-1 | 0.0307-0.3112 | 2 | 周麦22 Zhoumai 22 | 1.6367-2.2851 | 5 |
| 冀5579 Ji 5579 | 0.0469-0.3598 | 2 | 冀糯200 Jiru 200 | 0.0244-2.2972 | 5 |
| 陕225 Shaan 225 | 0.1824-0.3881 | 2 | 河农7106 Henong 7106 | 0.3960-2.4059 | 5 |
| 石麦21号 Shimai 21 | 0.3801-0.4025 | 2 | 邯麦14 Hanmai 14 | 2.4645-2.6210 | 5 |
| 中麦175 Zhongmai 175 | 0.0873-0.4178 | 2 | 沧麦6005 Cangmai 6005 | 0.5190-2.6750 | 5 |
| 冀5265 Ji 5265 | 0.2441-0.4259 | 2 | 郑麦9694 Zhengmai 9694 | 0.4956-2.7112 | 5 |
| 科农213 Kenong 213 | 0.2667-0.4381 | 2 | 沧麦119 Cangmai 119 | 1.0727-3.1996 | 5 |
| 石新539 Shixin 539 | 0.1862-0.4457 | 2 | 百农AK58 Bainong AK58 | 3.0456-3.2033 | 5 |
| 保麦10号 Baomai 10 | 0.1057-0.4679 | 2 | 沧麦028 Cangmai 028 | 2.5945-3.3725 | 5 |
| 烟农23 Yannong 23 | 0.0583-0.4747 | 2 | 周麦16 Zhoumai 16 | 1.5595-3.9194 | 5 |
| 石家庄11号 Shijiazhuang 11 | 0.3940-0.4798 | 2 | 烟优361 Yanyou 361 | 4.1998-4.7174 | 5 |
| 轮选987 Lunxuan 987 | 0.4651-0.4932 | 2 | 临汾6035 Linfen 6035 | 1.5158-4.9971 | 5 |
| 洛麦21 Luomai 21 | 0.5366-0.5377 | 3 | 冀6358 Ji 6358 | 2.8190-5.1615 | 5 |
| 农大3432 Nongda 3432 | 0.1076-0.5399 | 3 | 沧6003 Cang 6003 | 2.9203-5.2570 | 5 |
| 石麦16号 Shimai 16 | 0.5887-0.6046 | 3 | 周麦18 Zhoumai 18 | 5.9047-19.5165 | 5 |
| 晶白麦1号 Jingbaimai 1 | 0.3560-0.6230 | 3 | 中国春 Chinese Spring | — | — |
| 河农827 Henong 827 | 0.1498-0.6978 | 3 | 咸农39 Xiannong 39 | — | — |
| 河农5290 Henong 5290 | 0.6108-0.7991 | 3 |
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Table 4
表4
表4EST标记和KASP标记在供试小麦品种中的标记基因型及比例
Table 4
| 标记 Marker | 抗虫品种的标记基因型及比例 Marker-based genotypes and their ratios for resistant wheat cultivars | 中间型品种的标记基因型及比例 Marker-based genotypes and their ratios for lowly resistant wheat cultivars | 感虫品种的标记基因型及比例 Marker-based genotypes and their ratios for susceptible wheat cultivars | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| A | H | B | A | H | B | A | H | B | |||
| E1-2 | 16 (37.2) | 7 (16.3) | 20 (46.5) | 3 (25.0) | 0 (0.0) | 9 (75.0) | 5 (12.5) | 3 (7.5) | 32 (80.0) | ||
| E10-10 | 37 (84.1) | 0 (0.0) | 7 (15.9) | 9 (75.0) | 1 (8.3) | 2 (16.7) | 30 (75.0) | 1 (2.5) | 9 (22.5) | ||
| K3-7-1 | 12 (30.8) | 0 (0.0) | 27 (69.2) | 1 (8.3) | 0 (0.0) | 11 (91.7) | 3 (7.7) | 0 (0.0) | 36 (92.3) | ||
| K3-7-3 | 12 (31.6) | 0 (0.0) | 26 (68.4) | 1 (9.1) | 0 (0.0) | 10 (90.9) | 3 (7.7) | 0 (0.0) | 36 (92.3) | ||
| K10-10-6 | 15 (37.5) | 0 (0.0) | 25 (62.5) | 2 (16.7) | 0 (0.0) | 10 (83.3) | 5 (12.8) | 0 (0.0) | 34 (87.2) | ||
| K3-16-1 | 20 (62.5) | 1 (3.1) | 11 (34.4) | 3 (25.0) | 0 (0.0) | 9 (75.0) | 8 (20.5) | 1 (2.6) | 30 (76.9) | ||
| K3-1-1 | 1 (2.5) | 13 (32.5) | 26 (65.0) | 0 (0.0) | 1 (8.3) | 11 (91.7) | 0 (0.0) | 3 (7.7) | 36 (92.3) | ||
| K10-13-x | 0 (0.0) | 15 (38.5) | 24 (61.5) | 0 (0.0) | 1 (11.1) | 8 (88.9) | 0 (0.0) | 5 (20.8) | 19 (79.2) | ||
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KASP标记K3-7-1、K3-7-3和K10-10-6在超过30% (分别为30.8%、31.6%和37.5%)的抗虫小麦品种中检测到纯合抗虫标记位点, 在85%以上(分别为92.3%、92.3%和87.2%)的感虫小麦品种中检测到纯合感虫标记位点(表4)。这说明, 如果能在品种中检测到这些抗虫标记位点, 则该供试品种为抗虫品种(即具有QSm.hbau-4A抗虫位点)的可能性高于85%、为感虫品种(基因型与表型不相符)的概率低于15%, 因此这3个标记可以较好地筛选具有QSm.hbau-4A位点的抗虫小麦种质。
标记K3-16-1, 能够在62.5%的抗虫品种中检测出纯合抗虫标记位点, 在76.9%的感虫品种中检测出纯合感虫标记位点(表4), 检测有效率为70.4%, 因此该标记亦可作为品种抗虫性的筛选标记。
标记K3-1-1和K10-13-x在抗虫小麦亲本中为杂合分型。分型结果表明, 2个标记能够在超过30% (分别为35.0%和38.5%)的抗虫小麦品种中检测到抗虫标记位点, 在超过75% (分别为92.3%和79.2%)的感虫品种中检测到纯合感虫标记位点(表4), 即, 如果K3-1-1和K10-13-x检测到抗虫标记位点, 则该供试品种为抗虫品种(即具有QSm.hbau-4A抗虫位点)的可能性在75%以上, 而为感虫品种(基因型与表型不相符)的概率不到10%和25%。因此标记K3-1-1比K10-13-x能更好地检测出具有QSm.hbau-4A位点的抗虫小麦种质。
进一步分析7个标记E1-2、K3-1-1、K3-7-1、K3-7-3、K3-16-1、K10-10-6和K10-13-x在各不同抗性小麦品种的分型结果(不计检测缺失的品种)表明, 这些标记在高抗小麦品种中的平均检测有效率为64.7% (56.3%~86.7%), 在中抗品种中的平均检测有效率为19.1% (13.0%~41.2%), 在感虫品种中为66.7% (50.0%~75.0%), 在高感品种中为94.1% (85.7%~100.0%) (图4)。同样, 如果只考虑高抗和高感两类极端类型品种, 则这7个标记对抗性品种的平均检测有效率为64.7% (56.3%~86.7%), 低于对抗虫RIL系的检测有效率(平均86.8%, 范围85.1%~ 88.6%); 对感虫品种的平均检测有效率为94.1% (85.7%~100.0%), 与对感虫RIL系的检测有效率接近(平均95.5%, 范围95.5%~95.6%)(图4)。标记在RIL系间检测率较高, 与RIL系具有较为一致的遗传背景密不可分, 而标记在高抗RIL系间、高抗品种间的检测有效率低于对应高感RIL系和高感品种的事实, 再次说明小麦对麦红吸浆虫抗性的数量遗传特征。
图4
新窗口打开|下载原图ZIP|生成PPT图4抗(47个)、感(45个)虫小麦RIL株系、高抗品种(19个)、中抗品种(24个)、低抗品种(12个)、感虫品种(12个)和高感品种(28个)中的标记基因型所占比例
Fig. 4Marker-based genotypes and their proportions for resistant- (47) or susceptible- (45) RIL lines, highly resistant- (19), moderately resistant- (24), lowly resistant- (12), susceptible- (12), and highly susceptible- (28) wheat cultivars
根据3个检测率较高的标记K3-1-1、K3-7-1和K3-7-3在95个小麦品种中的分型结果可以看出, 这3个标记在11个抗虫小麦品种(RI = 0.0235~0.4798)中的分型与抗虫亲本冀麦24的分型完全一致(表5), 这11个品种分别是中农28 (1932年引进种质)、西农6028 (1956年审定, 下同)、丰产2号(1968)、晋麦33 (1985)、冀麦23 (1986)、河农215 (与冀麦23、冀麦24互为姊妹系)、邯麦9号(2003)、石家庄11号(2003)、石麦12号(2004)、河农4198(2005)和河农6049 (2008)。
Table 5
表5
表5在7个抗性标记位点上完全一致的11个小麦品种的抗虫性及其标记基因型
Table 5
| 小麦品种名称 Wheat cultivar name | 抗性指数(RI) Resistance index | 抗虫等级 Classification | E1-2a | K10-10-6 | K3-7-1 | K3-7-3 | K10-13-x | K3-16-1 | K3-1-1 |
|---|---|---|---|---|---|---|---|---|---|
| 河农6049 Henong 6049 | 0.0000-0.0235 | 1 | a | G:G | A:A | T:T | C:G | T:T | T:G |
| 石麦12号 Shimai 12 | 0.0197-0.0468 | 1 | a | G:G | A:A | T:T | C:G | T:T | T:G |
| 河农4198 Henong 4198 | 0.0602-0.1087 | 1 | a | G:G | A:A | T:T | C:G | T:T | T:G |
| 西农6028 Xinong 6028 | 0.0015-0.1089 | 1 | a | G:G | A:A | T:T | C:G | T:T | T:G |
| 晋麦33 Jinmai 33 | 0.0632-0.1395 | 1 | a | G:G | A:A | T:T | C:G | T:T | T:G |
| 中农28 Zhongnong 28 | 0.1412-0.1509 | 1 | a | G:G | A:A | T:T | C:G | T:T | T:G |
| 丰产2号 Fengchan 2 | 0.0302-0.1567 | 1 | a | G:G | A:A | T:T | C:G | T:T | T:T |
| 河农215 Henong 215 | 0.0502-0.2069 | 2 | a | G:G | A:A | T:T | C:G | T:T | T:G |
| 邯麦9号 Hanmai 9 | 0.0259-0.2524 | 2 | a | G:G | A:A | T:T | C:G | T:T | T:G |
| 冀麦23 Jimai 23 | 0.0296-0.2923 | 2 | a | G:G | A:A | T:T | C:G | T:T | T:G |
| 石家庄11号 Shijiazhuang 11 | 0.3940-0.4798 | 2 | a | G:G | A:A | T:T | C:G | T:T | T:G |
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综合EST和KASP标记的检测结果, 共有20个小麦品种[包括14个抗虫品种, 5个感虫或中间型(低抗)品种, 1个无表型鉴定结果的品种(咸农39)]都被检测出具有较多的抗虫标记位点(分型与带型同抗虫亲本冀麦24), 且这20份品种在任何标记位点上均未被检测出感虫标记位点, 因此说明这些材料在4AL抗虫标记位点QSm.hbau-4A附近的基因组区段可能是相同的。而在一些感虫小麦品种被检测出具有文中所有抗虫位点的现象, 也间接说明了基因组内可能有与4AL抗虫主效位点QSm.hbau-4A互作的基因(如抑制QSm.hbau-4A效应的上位基因等)存在。
3 讨论
3.1 功能标记的检测有效性
功能标记是一类可通过对个体基因型的间接检测来反应目标性状(表型)的分子标记, 理论上对表型的检测率应达到或接近100%, 能够准确检测作物种质资源是否含有目的基因[18,20,22]。但由于影响表型变异的基因或功能位点可能不止一个, 实际开发出的功能标记不一定均能在全部参试材料中获得基因型与相应的表型完全一致的结果, 这对复杂性状尤为明显。伍玲等用Lr34/Yr18/Pm38位点的功能标记csLV34[48]和cssfr1、cssfr2、cssfr3、cssfr4、cssfr5[49], 在273份CIMMYT来源的小麦材料中获得高达96.7%的标记检测有效率; 但将这些标记用于检测中国小麦地方品种时却发现, 25.8%的标记阳性品种却表现完全感病[50]。张帆等以抗旱相关基因TaNRX开发出的4个显性互补标记, 虽然能够对我国150份小麦品种的种子萌发期抗旱性进行检测, 但仍有6%的弱抗旱性材料被检测为抗旱基因型, 2%的抗旱性较强材料被检测为弱抗旱基因型[51]。在用功能标记检测小麦多酚氧化酶(PPO)、面粉黄色素含量时, 也发现功能标记检测与表型不完全一致的现象[52,53,54,55]。这些现象可归因于目的位点上其他变异的存在(包括互作位点)影响到目的基因的表达[50,51], 同时也不能排除品种遗传背景差异和环境因素的影响[56]。本研究基于转录组数据挖掘到的抗吸浆虫差异基因, 开发出8个抗虫性基因标记(2个EST和6个KASP标记)。这些标记在遗传背景较近的RIL系中能够达到很高的检测有效率(~90%), 在遗传背景差异较大的小麦品种中, 这些标记(不包括E10-10)在感虫品种中的检测有效率(76.9%~92.3%)远高于在抗虫品种中的检测有效率(30.8%~62.5%), 这说明抗虫位点(QSm.hbau- 4A)之外的其他抗虫基因、互作因子[50,51]和遗传背景的差异均可能对标记的检测效率产生影响。因此, 本研究开发的这7个标记可应用于选择小麦种质资源QSm.hbau-4A位点的抗虫等位基因型, 为提高小麦吸浆虫抗性的重要手段。同时, 鉴定、开发小麦基因组中存在的其他遗传因子及标记, 对于揭示小麦抗吸浆虫分子机制和提高小麦抗虫分子标记的检测效率也将十分重要。3.2 抗虫相关标记的应用前景及展望
本研究将RNA-Seq技术和BSA分析思路应用于小麦抗吸浆虫差异基因发掘及标记开发工作, 从多组抗、感吸浆虫小麦材料转录组数据中发掘的差异基因, 经qRT-PCR、KEGG Pathway及基因功能注释等分析过程, 初步证实了6个差异基因可能与抗虫性相关; 并根据这些基因在小麦抗、感亲本之间存在的序列差异, 开发出8个标记, 能够较好地分辨抗/感RIL株系。其中7个标记K3-1-1、K3-7-1、K3-7-3、K10-10-6、E1-2、K10-13-x和K3-16-1在供试感虫小麦品种中的检测有效率较高(76.9%~92.3%, 表4), 如果只考虑高感品种, 检测有效率则更高(85.7%~ 100.0%, 图4)。理论上说, 感虫品种不应携带任何抗虫等位基因, 如果在某些感虫品种中存在抗性位点, 那么该位点就不能完全准确预测品种的抗虫性。因此, 能够准确预测小麦品种抗虫性、并可应用于MAS育种中的标记, 应在感虫品种中的检测有效率高(尽可能达到100%)。本研究中, 标记K3-1-1、K3-7-1和K3-7-3在供试抗虫小麦品种中的检测有效率为30%左右(30.8%~35.0%), 在供试感虫小麦品种中的检测有效率超过90% (为92.3%), 如果用这3个标记从小麦品种中检测出全部抗虫标记位点, 则该品种为抗虫品种(具有QSm.hbau-4A)的可能性应超过90%, 因此这3个标记可较好地作为筛选具有QSm.hbau-4A抗虫位点小麦种质的标记。同时, 从图4也可看出, 小麦品种的吸浆虫抗性越好, 所用标记的抗虫等位型(resistant alleles)所占比例相对越高, 因此这3个标记可以很好地用于品种是否具有QSm.hbau-4A抗虫位点的检测, 这对我们鉴别抗虫种质以及早代育种材料的抗虫性具有重要意义。本研究中, 供试小麦品种中有11个抗虫品种被检测到携带了这3个标记的全部抗虫等位基因(表5), 这些品种多为生产上已经停止推广的老品种, 比较近的品种其审定时间也在10年以上, 因此如何更有效地使用比较老的小麦种质创新小麦抗虫资源, 任务十分迫切。这种基于混池转录组测序(BSR-Seq)并结合生物信息学工具、qRT-PCR、基因测序和遗传定位信息等的方法, 不但可快速挖掘用于开发标记的候选基因及功能位点, 提升潜在功能标记开发速度, 还能缩小差异基因范围、提升标记与目标性状紧密相关的几率。虽然我们目前的工作尚未实现对小麦抗虫主效QTL的图位克隆, 但利用该方法所获得潜在候选基因的抗虫相关标记, 在不同作图群体中被证明是有用的, 不仅能被用于对小麦种质资源抗虫性的辅助鉴定, 同时可对小麦抗吸浆虫主效QTL的精细定位和图位克隆提供重要帮助。
4 结论
基于6个小麦抗吸浆虫相关基因序列中存在的差异位点, 成功设计并开发了8个基因标记, 这些标记在抗、感小麦RIL株系间的分型结果与对应株系的抗虫性水平相符度在90%左右, 在不同小麦品种中的检测有效率变异较大。标记K3-1-1、K3-7-1和K3-7-3能够从超过30% (30.8%~35.0%)抗虫小麦品种中检测到抗虫等位型, 从90% (92.3%)以上的感虫品种中检测到感虫等位型, 对于那些均能检测到抗虫标记等位型的小麦品种, 其具有4AL抗虫主效位点QSm.hbau-4A的可能性超过90% (为92.3%), 可用于小麦抗虫性种质资源筛选和分子标记辅助育种。此外, 包含全部抗虫等位标记的11个抗虫小麦品种, 多已或即将停止推广, 这为如何有效利用老品种的抗虫性创新小麦新种质提出了十分迫切的要求。致谢:
感谢中国农业科学院杨欣明研究员提供了部分小麦种质, 感谢中国农业科学院作物科学研究所贾继增研究员、赵光耀研究员提供了百农AK58 (v4.24)参考基因组序列并进行的相关分析, 感谢本课题组张力菁、章悦等在抗虫鉴定等方面的大力帮助。参考文献 原文顺序
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DOI:10.1007/s11032-004-5041-2URL [本文引用: 4]
DOI:10.1002/ps.1307URLPMID:17078014 [本文引用: 2]
Field-trapping experiments with synthetic 2,7-nonadiyl dibutyrate, the female-produced sex pheromone of the orange wheat blossom midge, Sitodiplosis mosellana (Géhin), demonstrated that pheromone traps were highly attractive to males and caught very few non-target organisms. Different formulations of pheromone were tested to identify the optimum release rate and dispenser type for use in pheromone traps in the UK. Key findings were that racemic pheromone was as effective as enantiomerically pure (2S,7R)-2,7-nonadiyl dibutyrate, that release rates higher than 0.5 microg day(-1) were not necessary and that the optimal formulation was a 1 mg pheromone loading in a rubber septum. Pheromone traps gave a reliable indication of peak midge emergence, onset of flight and abundance of midges throughout the season. A strong correlation between maximum trap catch and crop infestation levels was obtained.
[本文引用: 1]
[本文引用: 1]
DOI:10.1007/s10343-010-0227-5URL [本文引用: 1]
The intensity of thrips and wheat blossom midges (WBM) infestations in twelve wheat cultivars was evaluated at the Plant Breeding Station, Silstedt, central Germany in 2008 & 2009 growing crop seasons. The research aimed at selecting the least infested cultivar to be profitably used in the forthcoming cultivation. Infestation levels were studied in flowering and milky stages (GS 65 and 73) of each cultivar in every single-spikelet in sample of 10 ears in both years.
There were significant differences in thrips and (WBM) densities among different cultivars in both years. Thrips numbers were the highest in Turkis, Global and Esket cultivars, while the lowest values were recorded in Robigus, Brompton and Carenius. The results showed that the highest WBM infestation was observed in Turkis, Tommi and Potenzial; on the other hand the lowest WBM infestation was found in some insect resistant cultivars (Brompton, Skalmeje, Robigus, Welford and Glasgow). The infested ears were positively correlated with the numbers of WBM among cultivars. The obtained results would give a good guide for choosing the proper cultivars which proved highly resistant to their specific pests.
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[本文引用: 1]
DOI:10.7606/j.issn.1009-1041.2003.02.064URL [本文引用: 4]
本文综述了20世纪后50年以来，国内外有关小麦品种对麦红吸浆虫抗虫性鉴定的常用方法、数学模型鉴定法以及较适用的定级标准；概括介绍了避害性、形态抗虫性和生化抗虫性三个方面的研究情况；指出多层次、综合性抗性遗传和蛋白带存在与诱导抗虫性有关；介绍了与抗虫性密切相关的性状，初步认为遗传机制属数量遗传，不可能是单基因遗传，起码是寡基因遗传；介绍了抗麦红吸浆虫种质资源创新应采取双轮回选择杂交系语法，有目的地将不同抗虫基因逐代积累，选育了一批抗性亲本和新品种。
DOI:10.7606/j.issn.1009-1041.2003.02.064URL [本文引用: 4]
本文综述了20世纪后50年以来，国内外有关小麦品种对麦红吸浆虫抗虫性鉴定的常用方法、数学模型鉴定法以及较适用的定级标准；概括介绍了避害性、形态抗虫性和生化抗虫性三个方面的研究情况；指出多层次、综合性抗性遗传和蛋白带存在与诱导抗虫性有关；介绍了与抗虫性密切相关的性状，初步认为遗传机制属数量遗传，不可能是单基因遗传，起码是寡基因遗传；介绍了抗麦红吸浆虫种质资源创新应采取双轮回选择杂交系语法，有目的地将不同抗虫基因逐代积累，选育了一批抗性亲本和新品种。
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麦红吸浆虫Sitodiplosis mosellana是我国的一种重要农业害虫， 以幼虫危害小麦正在发育的籽粒， 可造成小麦严重减产， 甚至绝收。该害虫具有虫体小， 滞育时间长， 为害隐蔽等特点。近些年来， 受全球气候变化、 耕作制度改变、 小麦品种更换、 人类活动等多种因素的影响， 麦红吸浆虫在我国的发生危害情况发生了很大变化， 出现了北扩东移的现象。麦红吸浆虫主要分布在我国的北方麦区， 发生为害具有隐蔽性、 间歇性、 局部性和暴发性的特点。这种害虫的发生危害受虫源基数、 生态因子、 农业生产措施及人类活动等多种因素的影响。进入21世纪后， 麦红吸浆虫在我国的发生范围发生了很大的变化， 且主要分布在43°N以南到27°N以北的冬小麦主产区。有关麦红吸浆虫滞育的多态性、 小麦对麦红吸浆虫的抗性机理、 抗性品种的选育和天敌资源的开发等方面的研究将是今后的主要研究方向； 未来仍需加强对麦红吸浆虫滞育的分子机制、 发生危害规律、 预测预报、 综合防治和寄主植物麦红吸浆虫天敌三级营养关系等方面研究。本综述可为今后了解麦红吸浆虫在我国的发生危害规律、 预测预报及综合防治等提供参考。
URL [本文引用: 1]
麦红吸浆虫Sitodiplosis mosellana是我国的一种重要农业害虫， 以幼虫危害小麦正在发育的籽粒， 可造成小麦严重减产， 甚至绝收。该害虫具有虫体小， 滞育时间长， 为害隐蔽等特点。近些年来， 受全球气候变化、 耕作制度改变、 小麦品种更换、 人类活动等多种因素的影响， 麦红吸浆虫在我国的发生危害情况发生了很大变化， 出现了北扩东移的现象。麦红吸浆虫主要分布在我国的北方麦区， 发生为害具有隐蔽性、 间歇性、 局部性和暴发性的特点。这种害虫的发生危害受虫源基数、 生态因子、 农业生产措施及人类活动等多种因素的影响。进入21世纪后， 麦红吸浆虫在我国的发生范围发生了很大的变化， 且主要分布在43°N以南到27°N以北的冬小麦主产区。有关麦红吸浆虫滞育的多态性、 小麦对麦红吸浆虫的抗性机理、 抗性品种的选育和天敌资源的开发等方面的研究将是今后的主要研究方向； 未来仍需加强对麦红吸浆虫滞育的分子机制、 发生危害规律、 预测预报、 综合防治和寄主植物麦红吸浆虫天敌三级营养关系等方面研究。本综述可为今后了解麦红吸浆虫在我国的发生危害规律、 预测预报及综合防治等提供参考。
DOI:10.1007/s10530-008-9324-0URL [本文引用: 1]
Wheat midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), was first detected as early as 1901 in western Canada. The first major outbreak in Saskatchewan was recorded in 1983. Today wheat midge infests much of the wheat-growing area of Manitoba, Saskatchewan and North Dakota (USA), and is beginning to invade Alberta and Montana (USA). In 1984, Saskatchewan wheat midge populations were found to be parasitized by the egg-larval parasitoid, Macroglenes penetrans (Kirby) (Hymenoptera). Through the successful implementation of conservation techniques, this parasitoid now controls an average of 31.5% of the wheat midge across Saskatchewan. Estimated value of the parasitoid, due to reduction in insecticide costs in Saskatchewan alone, was estimated to be in excess of $248.3 million in the 1990s. The environmental benefits of not having to apply this amount of chemical insecticide are a bonus. To minimize the economic and ecological impact of S. mosellana today, wheat producers in western Canada have access to one of the most comprehensive management programs of any insect pest of field crops. Forecasts and risk warnings, monitoring tools, cultural control, agronomic practices, chemical control, biological control and plant resistance are all available for producers to manage wheat midge.
DOI:10.1007/s10340-010-0325-2URL [本文引用: 1]
Population densities of wheat ear insects infesting different winter wheat varieties (n = 50) were estimated during 2008 and 2009 seasons near Halle, central Germany. The research was aimed at identifying wheat varieties most resistant to wheat ear insect pests. Two methods were used to evaluate the degree of insect infestations in different wheat ear varieties. Wheat ears were dissected when kernels were in Zadoks stage 73 and examined using a binocular microscope to count the number of spikelets and infested kernels, and to identify the insect pests present. In addition, white water traps were placed on the soil underneath each variety to collect mature larvae of wheat blossom midges (WBMs) as an indicator of potential crop risk for the next year. There were significant differences in the number of thrips and WBM infesting wheat ears among varieties in both years. Thrips numbers were the highest in Akratos, Limes and Ritmo varieties in 2008 and in Michigan Amber, Elegant and Kontrast in 2009. Thrips were the lowest in Thuareg in 2008 and Robigus varieties in 2009. The results showed that the highest WBM infestation level was observed in Michigan Amber in both years. The lowest WBM infestations were found in Turkis, Cubus, Capo, Welford and Robigus in both years. The number of infested kernels was positively correlated with WBM among varieties. In the water traps, the highest numbers of WBM larvae were recorded in Saladin and Bussard in 2008 and Orlando, Julius and Glasgow varieties in 2009. The lowest values were recorded in Victo, Enorm, Robigus and Welford varieties in both years. The results provide a guide for selecting winter wheat varieties with resistance to these wheat ear pests.
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DOI:10.7606/j.issn.1009-1041.2011.06.010URL [本文引用: 1]
为更深入地揭示小麦抗麦红吸浆虫品种（系）的遗传多样性，从而为进一步选育抗虫品种提供依据，在对田间虫圃1 562份小麦品种(系)损失率鉴定的基础上，取47份年度间鉴定抗性结果较为一致的材料，利用表型和SSR标记，进行遗传多样性分析。这些抗麦红吸浆虫品种农艺性状表现出较大的差异，表型聚类在遗传距离为0.68处将供试材料分为6个类群。19对SSR标记在47份不同抗性品种中检测到104个等位基因，能够将所有品种区分开来，每对引物可以检测到3～8个等位基因，平均5.47个。47个小麦品种间遗传距离为0.40～0.95，平均为0.71。SSR标记聚类分析在遗传距离为0.74处将供试材料分为6大类群。Mental测验结果表明，表型同基因型距离矩阵间存在显著正相关(r=0.76,P<0.05)。抗虫品种晋麦65号单独聚为一类，同其余品种具有较远的亲缘关系，可作为新的抗源用于抗虫育种，并在吸浆虫发生地块推广种植。
DOI:10.7606/j.issn.1009-1041.2011.06.010URL [本文引用: 1]
为更深入地揭示小麦抗麦红吸浆虫品种（系）的遗传多样性，从而为进一步选育抗虫品种提供依据，在对田间虫圃1 562份小麦品种(系)损失率鉴定的基础上，取47份年度间鉴定抗性结果较为一致的材料，利用表型和SSR标记，进行遗传多样性分析。这些抗麦红吸浆虫品种农艺性状表现出较大的差异，表型聚类在遗传距离为0.68处将供试材料分为6个类群。19对SSR标记在47份不同抗性品种中检测到104个等位基因，能够将所有品种区分开来，每对引物可以检测到3～8个等位基因，平均5.47个。47个小麦品种间遗传距离为0.40～0.95，平均为0.71。SSR标记聚类分析在遗传距离为0.74处将供试材料分为6大类群。Mental测验结果表明，表型同基因型距离矩阵间存在显著正相关(r=0.76,P<0.05)。抗虫品种晋麦65号单独聚为一类，同其余品种具有较远的亲缘关系，可作为新的抗源用于抗虫育种，并在吸浆虫发生地块推广种植。
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DOI:10.11869/j.issn.100-8551.2014.11.1963URL [本文引用: 2]
功能标记是根据与表型紧密相关的功能基因内部特定区域多态性基序开发出来的一种新型分子标记.由于功能标记直接来源于基因内部的功能性基序,因此,此类标记可以对不同遗传背景下目标等位基因的有无作直接、快速的判定.本文在3种DNA分子标记基于植物中的开发、应用特点比较论述的基础上,重点介绍了功能标记的开发特点,最后探讨功能标记作为一种辅助育种手段在禾谷类作物常规育种中的应用及其开发前景.
DOI:10.11869/j.issn.100-8551.2014.11.1963URL [本文引用: 2]
功能标记是根据与表型紧密相关的功能基因内部特定区域多态性基序开发出来的一种新型分子标记.由于功能标记直接来源于基因内部的功能性基序,因此,此类标记可以对不同遗传背景下目标等位基因的有无作直接、快速的判定.本文在3种DNA分子标记基于植物中的开发、应用特点比较论述的基础上,重点介绍了功能标记的开发特点,最后探讨功能标记作为一种辅助育种手段在禾谷类作物常规育种中的应用及其开发前景.
DOI:10.1007/s001220100570URL [本文引用: 1]
We developed a simple marker technique called sequence-related amplified polymorphism (SRAP) aimed for the amplification of open reading frames (ORFs). It is based on two-primer amplification. The primers are 17 or 18 nucleotides long and consist of the following elements. Core sequences, which are 13 to 14 bases long, where the first 10 or 11 bases starting at the 5′ end, are sequences of no specific constitution (”filler” sequences), followed by the sequence CCGG in the forward primer and AATT in the reverse primer. The core is followed by three selective nucleotides at the 3′ end. The filler sequences of the forward and reverse primers must be different from each other and can be 10 or 11 bases long. For the first five cycles the annealing temperature is set at 35°C. The following 35 cycles are run at 50°C. The amplified DNA fragments are separated by denaturing acrylamide gels and detected by autoradiography. We tested the marker technique in a series of recombinant inbred and doubled-haploid lines of Brassica oleracea L. After sequencing, approximately 45% of the gel-isolated bands matched known genes in the Genbank database. Twenty percent of the SRAP markers were co-dominant, which was demonstrated by sequencing. Construction of a linkage map revealed an even distribution of the SRAP markers in nine major linkage groups, not differing in this regard to AFLP markers. We successfully tagged the glucosinolate desaturation gene BoGLS-ALK with these markers. SRAPs were also easily amplified in other crops such as potato, rice, lettuce, Chinese cabbage (Brassica rapa L.), rapeseed (Brassica napus L.), garlic, apple, citrus, and celery. We also amplified cDNA isolated from different tissues of Chinese cabbage, allowing the fingerprinting of these sequences.
DOI:10.13560/j.cnki.biotech.bull.1985.2016.11.003URL [本文引用: 2]
功能标记是根据与表型紧密相关的功能基因内部特定区域的多态性序列,利用关联分析、表达谱分析、RNA干扰和QTL作图等方法开发而出的一种新型显性分子标记,此类标记可以对不同遗传背景下目标等位基因的有无作直接、快速的判定。简单介绍了功能标记的概念及特点,着重探讨功能标记作为一种辅助育种手段在小麦育种中的应用及其开发前景,以期为相关分子标记的开发提供参考。
DOI:10.13560/j.cnki.biotech.bull.1985.2016.11.003URL [本文引用: 2]
功能标记是根据与表型紧密相关的功能基因内部特定区域的多态性序列,利用关联分析、表达谱分析、RNA干扰和QTL作图等方法开发而出的一种新型显性分子标记,此类标记可以对不同遗传背景下目标等位基因的有无作直接、快速的判定。简单介绍了功能标记的概念及特点,着重探讨功能标记作为一种辅助育种手段在小麦育种中的应用及其开发前景,以期为相关分子标记的开发提供参考。
DOI:10.1016/j.tplants.2003.09.010URLPMID:14607101 [本文引用: 1]
Different approaches (including association studies) have recently been adopted for the functional characterization of allelic variation in plants and to identify sequence motifs affecting phenotypic variation. We propose the term 'functional markers' for DNA markers derived from such functionally characterized sequence motifs. Functional markers are superior to random DNA markers such as RFLPs, SSRs and AFLPs owing to complete linkage with trait locus alleles. We outline the definition, development, application and prospects of functional markers.
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DOI:10.1046/j.1439-0523.2002.745267.xURL [本文引用: 3]
DOI:10.1094/PDIS-10-16-1421-REURLPMID:30682944 [本文引用: 1]
Genetic control of resistance to Fusarium head blight (FHB) is quantitative, making phenotypic selection difficult. Genetic markers to resistance are helpful to select favorable genotypes. This study was conducted to determine if Fhb1 and Fhb5 present in the Sumai 3 source of FHB resistance occur in Sumai 3-derived North American spring wheat cultivars and to understand the appropriateness of using markers to select for the favorable alleles at these loci in breeding. Sumai 3-derived parents Alsen, ND3085, ND744, Carberry, and Glenn were used in crosses to generate 14 doubled haploid breeding populations. The parents and progeny were genotyped with five Fhb1 and three Fhb5 microsatellite markers. Progeny were selected based on performance relative to parents and other control cultivars in FHB nurseries near Portage la Prairie and Carman, MB. χ2 and t test analyses were performed on marker and FHB data. The χ2 test frequently determined the proportion of lines carrying molecular variants associated with FHB resistance increased following nursery selection for FHB. Similarly, the t test regularly demonstrated that selection for FHB resistance lowered the mean level of disease associated with resistant marker haplotypes. The study affirmed FHB resistance sources Alsen, Carberry, ND3085, and ND744 have Fhb1 and Fhb5 loci like Sumai 3, but no evidence was found that Glenn carries Fhb1 and Fhb5 resistance alleles. The results justified use of Fhb1 and Fhb5 markers for marker assisted selection in populations derived from Alsen, Carberry, ND3085, and ND744, but not Glenn. Combined or individual application of Xgwm493 and Xgwm533 in selection of genotypes carrying Fhb1, and Xgwm150, Xgwm304, and Xgwm595 for Fhb5 will enhance FHB resistance in wheat.
DOI:10.1007/s00122-016-2720-4URLPMID:27160855 [本文引用: 2]
SNP markers were developed for the OWBM resistance gene Sm1 that will be useful for MAS. The wheat Sm1 region is collinear with an inverted syntenic interval in B. distachyon. Orange wheat blossom midge (OWBM, Sitodiplosis mosellana Géhin) is an important insect pest of wheat (Triticum aestivum) in many growing regions. Sm1 is the only described OWBM resistance gene and is the foundation of managing OWBM through host genetics. Sm1 was previously mapped to wheat chromosome arm 2BS relative to simple sequence repeat (SSR) markers and the dominant, sequence characterized amplified region (SCAR) marker WM1. The objectives of this research were to saturate the Sm1 region with markers, develop improved markers for marker-assisted selection (MAS), and examine the synteny between wheat, Brachypodium distachyon, and rice (Oryza sativa) in the Sm1 region. The present study mapped Sm1 in four populations relative to single nucleotide polymorphisms (SNPs), SSRs, Diversity Array Technology (DArT) markers, single strand conformation polymorphisms (SSCPs), and the SCAR WM1. Numerous high quality SNP assays were designed that mapped near Sm1. BLAST delineated the syntenic intervals in B. distachyon and rice using gene-based SNPs as query sequences. The Sm1 region in wheat was inverted relative to B. distachyon and rice, which suggests a chromosomal rearrangement within the Triticeae lineage. Seven SNPs were tested on a collection of wheat lines known to carry Sm1 and not to carry Sm1. Sm1-flanking SNPs were identified that were useful for predicting the presence or absence of Sm1 based upon haplotype. These SNPs will be a major improvement for MAS of Sm1 in wheat breeding programs.
DOI:10.3198/jpr2011.06.0329crcURL [本文引用: 2]
'Fieldstar' (Reg. No. CV-1067, PI 663950) (Registration. No. 6328 by the Plant Variety Registration Office, Plant Production Division, Seed Section, Canadian Food Inspection Agency, Agriculture and Agri-Food Canada [AAFC]) hard red spring wheat (Triticum aestivum L.) was developed and released by the Cereal Research Centre of AAFC and meets the end-use quality specifications of the Canada western red spring wheat market class. Fieldstar is a partial backcross derivative of 'McKenzie', with 'Clark' as the donor of the Sm1 gene for midge resistance, which produces a product that reduces the palatability of developing seeds to wheat midge larvae (Sitodiplosis mosellana Gehin). Fieldstar is adapted to the eastern wheat growing regions of the Canadian prairies as determined by the Central Bread Wheat Cooperative Registration Test in 2004, 2005, and 2006. For registration testing, the performance of Fieldstar was estimated using the varietal blend Fieldstar VB, which consisted of 90% Fieldstar and 10% 'Waskada'. The grain yield of Fieldstar was similar to that of the highest-yielding checks, McKenzie and 'Superb', and expressed resistance to leaf rust (caused by Puccinia triticina Eriks.) and stem rust (caused by P. graminis Pers.:Pers. f. sp. tritici Eriks. E. Henn.) and intermediate resistance to common bunt [caused by Tilletia tritici (Bjerk.) R. Wolff and T. laevis Kuhn in Rabenh.], loose smut [caused by Ustilago tritici (Pers.) Rostr.], and Fusarium head blight [FHB; caused by Fusarium graminearum Schwabe; teleomorph Gibberella zeae (Schwein.) Petch]. Fieldstar was released because of its combination of high yield, resistance to wheat midge, and intermediate resistance to FHB.
DOI:10.1111/pbr.2011.130.issue-1URL [本文引用: 3]
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DOI:10.7606/j.issn.1009-1041.2003.03.106URL [本文引用: 1]
对小麦品种进行麦红吸浆虫抗性鉴定及抗性机制研究是培育抗虫品种、对麦红吸浆虫实行综合治理的基础工作。本文综述了20世纪50年代以来国内外对小麦品种进行麦红吸浆虫抗性鉴定的大田自然感虫鉴定法、虫圃人工接虫鉴定法、室内鉴定法、数学模型鉴定法，以及目前抗性鉴定所应用的等级评价标准和已取得的抗性鉴定结果；详细介绍了小麦品种抗麦红吸浆虫的形态抗虫机制、避虫机制以及生化抗虫机制中营养物质和次生代谢物质与品种抗虫性的关系。大量研究结果表明，小麦品种对麦红吸浆虫的抗性主要表现在抗性品种以其独特的穗形特征及避害性来阻止其产卵或取食，或通过产生有毒的次生物质或营养上的欠缺使其取食后不能正常发育而死亡。本文还对今后的研究趋势进行了展望。
DOI:10.7606/j.issn.1009-1041.2003.03.106URL [本文引用: 1]
对小麦品种进行麦红吸浆虫抗性鉴定及抗性机制研究是培育抗虫品种、对麦红吸浆虫实行综合治理的基础工作。本文综述了20世纪50年代以来国内外对小麦品种进行麦红吸浆虫抗性鉴定的大田自然感虫鉴定法、虫圃人工接虫鉴定法、室内鉴定法、数学模型鉴定法，以及目前抗性鉴定所应用的等级评价标准和已取得的抗性鉴定结果；详细介绍了小麦品种抗麦红吸浆虫的形态抗虫机制、避虫机制以及生化抗虫机制中营养物质和次生代谢物质与品种抗虫性的关系。大量研究结果表明，小麦品种对麦红吸浆虫的抗性主要表现在抗性品种以其独特的穗形特征及避害性来阻止其产卵或取食，或通过产生有毒的次生物质或营养上的欠缺使其取食后不能正常发育而死亡。本文还对今后的研究趋势进行了展望。
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DOI:10.1007/BF00051118URL [本文引用: 1]
DOI:10.4039/Ent132591-5URL [本文引用: 1]
DOI:10.1007/s12298-019-00662-8URLPMID:31168238 [本文引用: 1]
Sunn pest is one of the most destructive insects in the western and central parts of Asia causing severe reductions of wheat yield and flour quality. Therefore, an effort was undertaken to find effective resistance by analyzing genetic variation among 25 wheat genotypes artificially infested in field-cages as well as using start codon targeted (SCoT) polymorphism and inter- retrotransposon amplified polymorphism (IRAP) markers. High variation was revealed amongst genotypes with Sunn pest resistance characteristics including Bayat, Bezostaya, Sayson, Line93, Line120, Rashagol, Golsepi and AarasGolsoor, which were classified as resistant to moderately resistant. SCoTs and IRAPs were determined as efficient markers for studying genetic diversity. The non-parametric Kruskal-Wallis test was conducted to evaluate the effect of specific SCoT and IRAP amplicons on Sunn pest resistance characteristics for wheat genotypes. The stepwise regression analysis exhibited seven informative SCoTs and IRAPs explaining the highest resistance characteristics variation ranging from 25.7-50.1 to 17.6-40.1, respectively. The relationship between resistance of genotypes and informative SCoT and IRAP amplicons was found based on canonical discriminant analysis showing the capacity of informative markers for functional marker selection method and screening the wheat germplasms for Sunn pest resistance characteristics.
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DOI:10.1023/A:1005412309735URL [本文引用: 1]
Larvae of the wheat midge, Sitodiplosis mosellana (Géhin) feed on the surface of wheat seeds for about 10 days beginning when pollination occurs. A few wheats have a high level of antibiotic resistance to the larvae, which suppresses their growth and development. Nearly all larvae develop successfully on susceptible wheats. Analysis by HPLC of seed extracts produced by alkaline hydrolysis revealed rapid changes in the levels of p-coumaric and ferulic acids during early seed development. Seed infested by wheat midge larvae showed induced changes in the dynamics of these phenolic acids. The most resistant wheats had a higher constitutive level and a more rapid induction of ferulic acid than susceptible wheats. Levels of ferulic acid exceeding 0.35
g/g fresh weight were associated with a high mortality of newly hatched larvae. In one wheat line, resistance also was associated with induced production of p-coumaric acid. The induction of ferulic acid was similar in wheat from the laboratory and field, except in one resistant wheat that produced higher levels in the field. In ripe seeds, resistant and susceptible wheats had similar levels of phenolic acids.DOI:10.1021/jf010027hURLPMID:11513628 [本文引用: 1]
The concentration of ferulic acid (FA), the major phenolic acid in the wheat kernel, was found to differ significantly in the mature grain of six wheat cultivars known to have a range of tolerance to the orange wheat blossom midge (Sitodiplosis mosellana). Differences in FA content were correlated with floret infestation level of the cultivars. The wheat cultivars ranked similarly in FA content at the four locations where they were tested, despite a significant effect of environment. Ferulic acid was synthesized mainly during the early stages of grain filling but at different rates among cultivars. Ferulic acid was concentrated primarily in the shorts and bran fractions in an insoluble-bound form. A high correlation was obtained between FA contents as determined by GLC, fluorometry, UV, and colorimetry. The colorimetric procedure was modified as a qualitative, simple, and rapid test for identifying midge-resistant wheat and evaluated in several field trials. The method should provide a rapid tool in the preliminary screening of experimental lines in the development of midge-resistant wheat cultivars.
DOI:10.1155/2013/423189URLPMID:23431288 [本文引用: 1]
Caffeic acid o-methyltransferase (COMT) is one of the important enzymes controlling lignin monomer production in plant cell wall synthesis. Analysis of the genome sequence of the new grass model Brachypodium distachyon identified four COMT gene homologs, designated as BdCOMT1, BdCOMT2, BdCOMT3, and BdCOMT4. Phylogenetic analysis suggested that they belong to the COMT gene family, whereas syntenic analysis through comparisons with rice and sorghum revealed that BdCOMT4 on Chromosome 3 is the orthologous copy of the COMT genes well characterized in other grass species. The other three COMT genes are unique to Brachypodium since orthologous copies are not found in the collinear regions of rice and sorghum genomes. Expression studies indicated that all four Brachypodium COMT genes are transcribed but with distinct patterns of tissue specificity. Full-length cDNAs were cloned in frame into the pQE-T7 expression vector for the purification of recombinant Brachypodium COMT proteins. Biochemical characterization of enzyme activity and substrate specificity showed that BdCOMT4 has significant effect on a broad range of substrates with the highest preference for caffeic acid. The other three COMTs had low or no effect on these substrates, suggesting that a diversified evolution occurred on these duplicate genes that not only impacted their pattern of expression, but also altered their biochemical properties.
[本文引用: 1]
[本文引用: 1]
DOI:10.3864/j.issn.0578-1752.2011.12.001URL [本文引用: 1]
【目的】开发小麦抗旱相关蛋白磷酸酶结构亚基基因TaPP2Aa的功能标记并作图，为分子标记辅助选择抗逆育种提供依据。【方法】测序得到普通小麦及其野生近缘种TaPP2Aa的基因序列，分析其SNP位点差异，设计3对基因组特异引物和6对基因组内等位基因特异引物，利用中国春缺四体对TaPP2Aa进行染色体定位，利用RIL群体（Opata 85×W7984）和DH群体（旱选10号×鲁麦14）进行该基因的功能标记作图。【结果】TaPP2Aa定位于小麦第5同源染色体群上；TaPP2Aa-B位于RIL群体5B的标记区间Xwg909—Xgwm67，与2个标记的遗传距离分别为4.0 cM和3.6 cM，在DH群体5B染色体的标记区间Xgwm234—WMC363，与WMC363的遗传距离为7.5 cM；在2个遗传群体中与标记Xgwm67的距离分别为3.6 cM和11.4 cM。TaPP2Aa-D位于RIL群体染色体5D的标记区间Xcmwg770—Xbarc205，遗传距离分别为9.8 cM和10.0 cM。【结论】确定了TaPP2Aa所在的染色体位置，通过与DH和RIL 2个遗传作图群体中已有的抗逆主效QTL进行对比分析，明确了TaPP2Aa与小麦抗逆性状QTL具有遗传连锁关系，开发的功能标记可用于小麦抗逆性状的分子标记辅助选择。
DOI:10.3864/j.issn.0578-1752.2011.12.001URL [本文引用: 1]
【目的】开发小麦抗旱相关蛋白磷酸酶结构亚基基因TaPP2Aa的功能标记并作图，为分子标记辅助选择抗逆育种提供依据。【方法】测序得到普通小麦及其野生近缘种TaPP2Aa的基因序列，分析其SNP位点差异，设计3对基因组特异引物和6对基因组内等位基因特异引物，利用中国春缺四体对TaPP2Aa进行染色体定位，利用RIL群体（Opata 85×W7984）和DH群体（旱选10号×鲁麦14）进行该基因的功能标记作图。【结果】TaPP2Aa定位于小麦第5同源染色体群上；TaPP2Aa-B位于RIL群体5B的标记区间Xwg909—Xgwm67，与2个标记的遗传距离分别为4.0 cM和3.6 cM，在DH群体5B染色体的标记区间Xgwm234—WMC363，与WMC363的遗传距离为7.5 cM；在2个遗传群体中与标记Xgwm67的距离分别为3.6 cM和11.4 cM。TaPP2Aa-D位于RIL群体染色体5D的标记区间Xcmwg770—Xbarc205，遗传距离分别为9.8 cM和10.0 cM。【结论】确定了TaPP2Aa所在的染色体位置，通过与DH和RIL 2个遗传作图群体中已有的抗逆主效QTL进行对比分析，明确了TaPP2Aa与小麦抗逆性状QTL具有遗传连锁关系，开发的功能标记可用于小麦抗逆性状的分子标记辅助选择。
DOI:10.1007/s00122-018-3092-8URLPMID:29666883 [本文引用: 1]
NGS-assisted super pooling emerging as powerful tool to accelerate gene mapping and haplotype association analysis within target region uncovering specific linkage SNPs or alleles for marker-assisted gene pyramiding. Conventional gene mapping methods to identify genes associated with important agronomic traits require significant amounts of financial support and time. Here, a single nucleotide polymorphism (SNP)-based mapping approach, RNA-Seq and SNP array assisted super pooling analysis, was used for rapid mining of a candidate genomic region for stripe rust resistance gene Yr26 that has been widely used in wheat breeding programs in China. Large DNA and RNA super-pools were genotyped by Wheat SNP Array and sequenced by Illumina HiSeq, respectively. Hundreds of thousands of SNPs were identified and then filtered by multiple filtering criteria. Among selected SNPs, over 900 were found within an overlapping interval of less than 30?Mb as the Yr26 candidate genomic region in the centromeric region of chromosome arm 1BL. The 235 chromosome-specific SNPs were converted into KASP assays to validate the Yr26 interval in different genetic populations. Using a high-resolution mapping population (&gt;?30,000 gametes), we confined Yr26 to a 0.003-cM interval. The Yr26 target region was anchored to the common wheat IWGSC RefSeq v1.0 and wild emmer WEWSeq v.1.0 sequences, from which 488 and 454?kb fragments were obtained. Several candidate genes were identified in the target genomic region, but there was no typical resistance gene in either genome region. Haplotype analysis identified specific SNPs linked to Yr26 and developed robust and breeder-friendly KASP markers. This integration strategy can be applied to accelerate generating many markers closely linked to target genes/QTL for a trait of interest in wheat and other polyploid species.
[本文引用: 1]
[本文引用: 1]
DOI:10.1371/journal.pone.0036406URLPMID:22586469 [本文引用: 1]
Bulked segregant analysis (BSA) is an efficient method to rapidly and efficiently map genes responsible for mutant phenotypes. BSA requires access to quantitative genetic markers that are polymorphic in the mapping population. We have developed a modification of BSA (BSR-Seq) that makes use of RNA-Seq reads to efficiently map genes even in populations for which no polymorphic markers have been previously identified. Because of the digital nature of next-generation sequencing (NGS) data, it is possible to conduct de novo SNP discovery and quantitatively genotype BSA samples by analyzing the same RNA-Seq data using an empirical Bayesian approach. In addition, analysis of the RNA-Seq data provides information on the effects of the mutant on global patterns of gene expression at no extra cost. In combination these results greatly simplify gene cloning experiments. To demonstrate the utility of this strategy BSR-Seq was used to clone the glossy3 (gl3) gene of maize. Mutants of the glossy loci exhibit altered accumulation of epicuticular waxes on juvenile leaves. By subjecting the reference allele of gl3 to BSR-Seq, we were able to map the gl3 locus to an ≈ 2 Mb interval. The single gene located in the ≈ 2 Mb mapping interval whose expression was down-regulated in the mutant pool was subsequently demonstrated to be the gl3 gene via the analysis of multiple independent transposon induced mutant alleles. The gl3 gene encodes a putative myb transcription factor, which directly or indirectly affects the expression of a number of genes involved in the biosynthesis of very-long-chain fatty acids.
DOI:10.1111/tpj.12105URL [本文引用: 1]
The majority of agronomically important crop traits are quantitative, meaning that they are controlled by multiple genes each with a small effect (quantitative trait loci, QTLs). Mapping and isolation of QTLs is important for efficient crop breeding by marker-assisted selection (MAS) and for a better understanding of the molecular mechanisms underlying the traits. However, since it requires the development and selection of DNA markers for linkage analysis, QTL analysis has been time-consuming and labor-intensive. Here we report the rapid identification of plant QTLs by whole-genome resequencing of DNAs from two populations each composed of 2050 individuals showing extreme opposite trait values for a given phenotype in a segregating progeny. We propose to name this approach QTL-seq as applied to plant species. We applied QTL-seq to rice recombinant inbred lines and F2 populations and successfully identified QTLs for important agronomic traits, such as partial resistance to the fungal rice blast disease and seedling vigor. Simulation study showed that QTL-seq is able to detect QTLs over wide ranges of experimental variables, and the method can be generally applied in population genomics studies to rapidly identify genomic regions that underwent artificial or natural selective sweeps.
DOI:10.1016/0305-1978(80)90029-0URL [本文引用: 1]
DOI:10.1021/np50048a030URL [本文引用: 1]
DOI:10.1073/pnas.81.24.8014URLPMID:6096873 [本文引用: 1]
Spacer-length (sl) variation in ribosomal RNA gene clusters (rDNA) was surveyed in 502 individual barley plants, including samples from 50 accessions of cultivated barley, 25 accessions of its wild ancestor, and five generations of composite cross II (CCII), an experimental population of barley. In total, 17 rDNA sl phenotypes, made up of 15 different rDNA sl variants, were observed. The 15 rDNA sl variants comprise a complete ladder in which each variant differs in length from adjacent variants by approximately equal to 115 nucleotide pairs. Studies of four rDNA sl variants in an F2 population showed that these variants are located at two unlinked loci, Rrn1 and Rrn2, each with two codominant alleles. Using wheat-barley addition lines, we determined that Rrn1 and Rrn2 are located on chromosomes 6 and 7, respectively. The nonrandom distribution of sl variants between loci suggests that genetic exchange occurs much less frequently between than within the two loci, which demonstrates that Rrn1 and Rrn2 are useful as new genetic markers. Frequencies of rDNA sl phenotypes and variants were monitored over 54 generations in CCII. A phenotype that was originally infrequent in CCII ultimately became predominant, whereas the originally most frequent phenotype decreased drastically in frequency, and all other phenotypes originally present disappeared from the population. We conclude that the sl variants and/or associated loci are under selection in CCII.
DOI:10.1007/s00122-006-0406-zURL [本文引用: 1]
Wheat expressed sequence tags (wESTs) were identified in a genomic interval predicted to span the Lr34/Yr18 slow rusting region on chromosome 7DS and that corresponded to genes located in the syntenic region of rice chromosome 6 (between 2.02 and 2.38Mb). A subset of the wESTs was also used to identify corresponding bacterial artificial chromosome (BAC) clones from the diploid D genome of wheat (Aegilops tauschii). Conservation and deviation of micro-colinearity within blocks of genes were found in the D genome BACs relative to the orthologous sequences in rice. Extensive RFLP analysis using the wEST derived clones as probes on a panel of wheat genetic stocks with or without Lr34/Yr18 revealed monomorphic patterns as the norm in this region of the wheat genome. A similar pattern was observed with single nucleotide polymorphism analysis on a subset of the wEST derived clones and subclones from corresponding D genome BACs. One exception was a wEST derived clone that produced a consistent RFLP pattern that distinguished the Lr34/Yr18 genetic stocks and well-established cultivars known either to possess or lack Lr34/Yr18. Conversion of the RFLP to a codominant sequence tagged site (csLV34) revealed a bi-allelic locus, where a variant size of 79bp insertion in an intron sequence was associated with lines or cultivars that lacked Lr34/Yr18. This association with Lr34/Yr18 was validated in wheat cultivars from diverse backgrounds. Genetic linkage between csLV34 and Lr34/Yr18 was estimated at 0.4cM
DOI:10.1007/s00122-009-1097-zURL [本文引用: 1]
The locus Lr34/Yr18/Pm38 confers partial and durable resistance against the devastating fungal pathogens leaf rust, stripe rust, and powdery mildew. In previous studies, this broad-spectrum resistance was shown to be controlled by a single gene which encodes a putative ATP-binding cassette transporter. Alleles of resistant and susceptible cultivars differed by only three sequence polymorphisms and the same resistance haplotype was found in the three independent breeding lineages of Lr34/Yr18/Pm38. Hence, we used these conserved sequence polymorphisms as templates to develop diagnostic molecular markers that will assist selection for durable multi-pathogen resistance in breeding programs. Five allele-specific markers (cssfr1–cssfr5) were developed based on a 3bp deletion in exon 11 of the Lr34-gene, and one marker (cssfr6) was derived from a single nucleotide polymorphism in exon 12. Validation of reference genotypes, well characterized for the presence or absence of the Lr34/Yr18/Pm38 resistance locus, demonstrated perfect diagnostic values for the newly developed markers. By testing the new markers on a larger set of wheat cultivars, a third Lr34 haplotype, not described so far, was discovered in some European winter wheat and spelt material. Some cultivars with uncertain Lr34 status were re-assessed using the newly derived markers. Unambiguous identification of the Lr34 gene aided by the new markers has revealed that some wheat cultivars incorrectly postulated as having Lr34 may possess as yet uncharacterised loci for adult plant leaf and stripe rust resistance.
URL [本文引用: 3]
【目的】明确CIMMYT观察圃273个小麦品种(系)在慢病基因Lr34/Yr18/Pm38位点的等位变异类型及其对条锈病、叶锈病和白粉病的抗性,进一步验证抗病基因功能标记的有效性。【方法】利用Lr34/Yr18/Pm38紧密连锁的STS标记csLV34和基于该基因第11外显子(exon 11)等位变异开发的5对功能标记cssfr1—cssfr5检测新引进的273个CIMMYT小麦品种(系),同时在成都和北京分别对其进行田间条锈病和白粉病的抗性鉴定,并结合CIMMYT的田间条锈病和叶锈病抗性鉴定结果进行分析。【结果】STS标记csLV34与5对功能标记cssfr1—cssfr5检测结果的一致性为96.7%;在273份CIMMYT材料中有43份材料含有Lr34/Yr18/Pm38,在不同地点对条锈病、叶锈病和白粉病具有不同程度的抗病性。 【结论】功能标记cssfr1—cssfr5可准确鉴定Lr34/Yr18/Pm38位点exon 11中的等位变异,cssfr3、cssfr4、cssfr5可用于含有Lr34/Yr18/Pm38材料杂交后代的分子标记辅助选择。
URL [本文引用: 3]
【目的】明确CIMMYT观察圃273个小麦品种(系)在慢病基因Lr34/Yr18/Pm38位点的等位变异类型及其对条锈病、叶锈病和白粉病的抗性,进一步验证抗病基因功能标记的有效性。【方法】利用Lr34/Yr18/Pm38紧密连锁的STS标记csLV34和基于该基因第11外显子(exon 11)等位变异开发的5对功能标记cssfr1—cssfr5检测新引进的273个CIMMYT小麦品种(系),同时在成都和北京分别对其进行田间条锈病和白粉病的抗性鉴定,并结合CIMMYT的田间条锈病和叶锈病抗性鉴定结果进行分析。【结果】STS标记csLV34与5对功能标记cssfr1—cssfr5检测结果的一致性为96.7%;在273份CIMMYT材料中有43份材料含有Lr34/Yr18/Pm38,在不同地点对条锈病、叶锈病和白粉病具有不同程度的抗病性。 【结论】功能标记cssfr1—cssfr5可准确鉴定Lr34/Yr18/Pm38位点exon 11中的等位变异,cssfr3、cssfr4、cssfr5可用于含有Lr34/Yr18/Pm38材料杂交后代的分子标记辅助选择。
DOI:10.3724/SP.J.1006.2014.00029URL [本文引用: 3]
抗旱相关基因的挖掘和分子标记开发对选育抗旱小麦品种有重要意义。采用同源克隆、电子克隆、RACE技术和生物信息学分析手段，获得了普通小麦硫氧还蛋白(Trx)超家族一个新基因(TaNRX)的全长cDNA序列(GenBank登录号为KC890769)，包含2015 bp，其中开放阅读框1734 bp；预测编码577个氨基酸，分子量为63.79 kD，含3个Trx活性功能区，其中2个存在典型的Cys-X1-X2-Cys结构，具有催化氧化还原反应的活性。TaNRX基因被定位在小麦的5B染色体短臂上，包含4个外显子和3个内含子。比较该基因在两组极端相对发芽率品种中的序列差异，发现第1个内含子差异明显。基于差异位点开发了4个显性互补标记，并用其检测150份小麦品种(系)。检测到TaNRX基因在普通小麦中至少存在2种与抗旱相关的等位变异，分别是TaNRX-a和TaNRX-b；TaNRX-a基因型的品种(系)平均相对发芽率显著高于TaNRX-b基因型(P<0.01)，说明开发的标记可被用于小麦抗旱性鉴定筛选。
DOI:10.3724/SP.J.1006.2014.00029URL [本文引用: 3]
抗旱相关基因的挖掘和分子标记开发对选育抗旱小麦品种有重要意义。采用同源克隆、电子克隆、RACE技术和生物信息学分析手段，获得了普通小麦硫氧还蛋白(Trx)超家族一个新基因(TaNRX)的全长cDNA序列(GenBank登录号为KC890769)，包含2015 bp，其中开放阅读框1734 bp；预测编码577个氨基酸，分子量为63.79 kD，含3个Trx活性功能区，其中2个存在典型的Cys-X1-X2-Cys结构，具有催化氧化还原反应的活性。TaNRX基因被定位在小麦的5B染色体短臂上，包含4个外显子和3个内含子。比较该基因在两组极端相对发芽率品种中的序列差异，发现第1个内含子差异明显。基于差异位点开发了4个显性互补标记，并用其检测150份小麦品种(系)。检测到TaNRX基因在普通小麦中至少存在2种与抗旱相关的等位变异，分别是TaNRX-a和TaNRX-b；TaNRX-a基因型的品种(系)平均相对发芽率显著高于TaNRX-b基因型(P<0.01)，说明开发的标记可被用于小麦抗旱性鉴定筛选。
URL [本文引用: 1]
【目的】利用分子标记研究中国冬小麦品种PPO基因的等位变异及其与PPO活性的关系，为小麦PPO活性的分子标记辅助选择（MAS）奠定基础。【方法】利用PPO基因的功能标记PPO18、PPO29和PPO16对中国4个麦区的冬小麦品种（系）（试验Ⅰ）和山东省常用亲本（试验Ⅱ）共311份进行2A和2D染色体上PPO等位基因Ppo-A1a、Ppo-A1b、Ppo-D1a和Ppo-D1b的检测。【结果】PPO18在等位基因Ppo-A1a（高PPO）和Ppo-A1b（低PPO）中分别扩增685 bp和876 bp的片段，PPO16和PPO29在Ppo-D1a（低PPO）和Ppo-D1b（高PPO）两个等位基因中分别扩增713 bp和490 bp的片段。311份品种（系）中Ppo-A1a、Ppo-A1b、Ppo-D1a和Ppo-D1b等位基因的频率分别为41.8%、58.2%、59.8%和40.2%，PPO活性在同一基因的两个等位基因间差异均达显著水平（P＜0.05）；两个PPO基因的等位基因组合类型分布频率为：Ppo-A1a/Ppo-D1a（27.3%）、Ppo-A1a/Ppo-D1b（14.5%）、Ppo-A1b/Ppo-D1a（32.5%）和Ppo-A1b/Ppo-D1b（25.7%），彼此差异均达显著水平（P＜0.05）。各麦区间PPO活性基因分布存在明显差异，Ppo-A1a基因在长江中下游冬麦区和西南冬麦区分布较多，频率分别为60.7%和56.0%；Ppo-A1b基因在北部冬麦区和黄淮冬麦区分布较高，频率分别为65.6%和60.3%；而Ppo-D1a基因在北部冬麦区、黄淮冬麦区、长江中下游冬麦区和西南冬麦区分布明显高于Ppo-D1b基因频率，分别为65.6%、53.6%、75.0%和76.0%。【结论】PPO18、PPO16和PPO29标记的检测方法简单、稳定性好，所检测的等位变异类型能有效反映品种的PPO活性值。中国冬麦区品种低PPO活性基因分布频率相对较多。因此，根据PPO基因类型组配亲本，并利用PPO基因功能标记在育种早代筛选低PPO活性的基因型，可促进面制品色泽的改良。
URL [本文引用: 1]
【目的】利用分子标记研究中国冬小麦品种PPO基因的等位变异及其与PPO活性的关系，为小麦PPO活性的分子标记辅助选择（MAS）奠定基础。【方法】利用PPO基因的功能标记PPO18、PPO29和PPO16对中国4个麦区的冬小麦品种（系）（试验Ⅰ）和山东省常用亲本（试验Ⅱ）共311份进行2A和2D染色体上PPO等位基因Ppo-A1a、Ppo-A1b、Ppo-D1a和Ppo-D1b的检测。【结果】PPO18在等位基因Ppo-A1a（高PPO）和Ppo-A1b（低PPO）中分别扩增685 bp和876 bp的片段，PPO16和PPO29在Ppo-D1a（低PPO）和Ppo-D1b（高PPO）两个等位基因中分别扩增713 bp和490 bp的片段。311份品种（系）中Ppo-A1a、Ppo-A1b、Ppo-D1a和Ppo-D1b等位基因的频率分别为41.8%、58.2%、59.8%和40.2%，PPO活性在同一基因的两个等位基因间差异均达显著水平（P＜0.05）；两个PPO基因的等位基因组合类型分布频率为：Ppo-A1a/Ppo-D1a（27.3%）、Ppo-A1a/Ppo-D1b（14.5%）、Ppo-A1b/Ppo-D1a（32.5%）和Ppo-A1b/Ppo-D1b（25.7%），彼此差异均达显著水平（P＜0.05）。各麦区间PPO活性基因分布存在明显差异，Ppo-A1a基因在长江中下游冬麦区和西南冬麦区分布较多，频率分别为60.7%和56.0%；Ppo-A1b基因在北部冬麦区和黄淮冬麦区分布较高，频率分别为65.6%和60.3%；而Ppo-D1a基因在北部冬麦区、黄淮冬麦区、长江中下游冬麦区和西南冬麦区分布明显高于Ppo-D1b基因频率，分别为65.6%、53.6%、75.0%和76.0%。【结论】PPO18、PPO16和PPO29标记的检测方法简单、稳定性好，所检测的等位变异类型能有效反映品种的PPO活性值。中国冬麦区品种低PPO活性基因分布频率相对较多。因此，根据PPO基因类型组配亲本，并利用PPO基因功能标记在育种早代筛选低PPO活性的基因型，可促进面制品色泽的改良。
DOI:10.1007/s00122-007-0660-8URL [本文引用: 1]
Phytoene synthase (Psy), a critical enzyme in the carotenoid biosynthetic pathway, demonstrated high association with the yellow pigment (YP) content in wheat grain. Characterization of Psy genes and the development of functional markers for them are of importance for marker-assisted selection in wheat breeding. In this study, the full-length genomic DNA sequence of a Psy gene (Psy-A1) located on chromosome 7A, was characterized by in silico cloning and experimental validation. The cloned Psy-A1 comprises six exons and five introns, 4,175bp in total, and an ORF of 1,284bp. A co-dominant marker, YP7A, was developed based on polymorphisms of two haplotypes of Psy-A1, yielding 194 and 231-bp fragments in cultivars with high and low YP content, respectively. The marker YP7A was mapped on chromosome 7AL using an RIL population from cross PH82-2/Neixing 188, and a set of Chinese Spring nullisomic–tetrasomic lines and ditelosomic line 7AS. Psy-A1, co-segregating with the STS marker YP7A, was linked to SSR marker Xwmc809 on chromosome 7AL with a genetic distance of 5.8cM, and explained 20–28% of the phenotypic variance for YP content across three environments. A total of 217 Chinese wheat cultivars and advanced lines were used to validate the association between the polymorphic band pattern and grain YP content. The results showed that the functional marker YP7A was closely related to grain YP content and, therefore, could be used in wheat breeding programs targeting of YP content for various wheat-based products.
DOI:10.7606/j.issn.1009-1041.2009.05.007URL [本文引用: 1]
为了给新疆小麦品种面粉色泽改良提供理论依据，利用黄色素含量基因(Psy A1)的功能标记YP7A检测了247份新疆小麦品种（包括农家品种、国内外引进品种和20世纪60年代以来的自育品种）Psy A1等位基因Psy A1a（高黄色素含量）和Psy A1b（低黄色素含量）的分布情况，探讨了Psy A1等位基因与面粉黄色素含量和黄度b*值的关系。结果表明，新疆小麦品种中Psy A1a和Psy A1b基因型的频率分别为91.9%和8.1%，以高黄色素含量的Psy A1a基因型为主。新疆小麦农家品种、引进品种和自育品种Psy A1a基因型的分布频率分别为100%、81.3%和95.3%，外来品种的引进和利用对降低新疆小麦品种黄色素含量起到了积极作用。新疆小麦品种面粉黄色素含量和黄度b*值普遍偏高，冬小麦品种面粉黄色素含量及黄度b*值高于春小麦品种。分析表明Psy A1a和Psy A1b两种基因型的面粉黄色素含量和黄度b*值平均值间的差异达到极显著水平。总之，新疆小麦品种面粉色泽改良应该以培育低黄色素含量的Psy A1b基因型为重点，同时可以直接利用黄色素含量基因(Psy A1)的功能标记YP7A来提高育种效率。
DOI:10.7606/j.issn.1009-1041.2009.05.007URL [本文引用: 1]
为了给新疆小麦品种面粉色泽改良提供理论依据，利用黄色素含量基因(Psy A1)的功能标记YP7A检测了247份新疆小麦品种（包括农家品种、国内外引进品种和20世纪60年代以来的自育品种）Psy A1等位基因Psy A1a（高黄色素含量）和Psy A1b（低黄色素含量）的分布情况，探讨了Psy A1等位基因与面粉黄色素含量和黄度b*值的关系。结果表明，新疆小麦品种中Psy A1a和Psy A1b基因型的频率分别为91.9%和8.1%，以高黄色素含量的Psy A1a基因型为主。新疆小麦农家品种、引进品种和自育品种Psy A1a基因型的分布频率分别为100%、81.3%和95.3%，外来品种的引进和利用对降低新疆小麦品种黄色素含量起到了积极作用。新疆小麦品种面粉黄色素含量和黄度b*值普遍偏高，冬小麦品种面粉黄色素含量及黄度b*值高于春小麦品种。分析表明Psy A1a和Psy A1b两种基因型的面粉黄色素含量和黄度b*值平均值间的差异达到极显著水平。总之，新疆小麦品种面粉色泽改良应该以培育低黄色素含量的Psy A1b基因型为重点，同时可以直接利用黄色素含量基因(Psy A1)的功能标记YP7A来提高育种效率。
DOI:10.7606/j.issn.1009-1041.2011.01.009URL [本文引用: 1]
为了改良小麦加工品质、提高小麦育种效率，利用位于7AL和7BL染色体上与黄色素含量相关的八氢番茄红素合酶（Phytoene synthase，PSY）基因Psy A1的标记YP7A、YP7A 2，基因Psy B1的标记YP7B 1、YP7B 2、YP7B 3、YP7B 4，以及位于2AL染色体上的多酚氧化酶（Polyphenol oxidase，PPO）活性基因Ppo A1 的标记PPO18，对221份冬小麦品种（系）进行黄色素含量和多酚氧化酶活性基因的等位变异检测。结果表明: (1) 在所检测的小麦品种（系）中，含低黄色素含量等位基因Psy A1b的材料78份，频率为35.3%；含低黄色素含量等位基因Psy B1b 的材料117份，频率为52.9%；含高黄色素含量等位基因Psy B1c的材料31份，频率为14.0%；含Psy B1d等位基因的材料4份，频率为1.8%；未发现携带Psy B1e的材料。（2）含低PPO活性等位基因Ppo A1b的材料119份，频率为53.8%。(3) 在221份材料中，黄色素含量和多酚氧化酶活性基因型皆符合中国面条和馒头加工品质要求的品种仅25份，聚合多个优良基因的小麦品质改良工作急需加强。本实验使用的标记均为基因特异性功能标记，重复性好，准确率高，可有效地应用于小麦品质改良的分子标记辅助选择。
DOI:10.7606/j.issn.1009-1041.2011.01.009URL [本文引用: 1]
为了改良小麦加工品质、提高小麦育种效率，利用位于7AL和7BL染色体上与黄色素含量相关的八氢番茄红素合酶（Phytoene synthase，PSY）基因Psy A1的标记YP7A、YP7A 2，基因Psy B1的标记YP7B 1、YP7B 2、YP7B 3、YP7B 4，以及位于2AL染色体上的多酚氧化酶（Polyphenol oxidase，PPO）活性基因Ppo A1 的标记PPO18，对221份冬小麦品种（系）进行黄色素含量和多酚氧化酶活性基因的等位变异检测。结果表明: (1) 在所检测的小麦品种（系）中，含低黄色素含量等位基因Psy A1b的材料78份，频率为35.3%；含低黄色素含量等位基因Psy B1b 的材料117份，频率为52.9%；含高黄色素含量等位基因Psy B1c的材料31份，频率为14.0%；含Psy B1d等位基因的材料4份，频率为1.8%；未发现携带Psy B1e的材料。（2）含低PPO活性等位基因Ppo A1b的材料119份，频率为53.8%。(3) 在221份材料中，黄色素含量和多酚氧化酶活性基因型皆符合中国面条和馒头加工品质要求的品种仅25份，聚合多个优良基因的小麦品质改良工作急需加强。本实验使用的标记均为基因特异性功能标记，重复性好，准确率高，可有效地应用于小麦品质改良的分子标记辅助选择。
DOI:10.7606/j.issn.1009-1041.2010.02.012URL [本文引用: 1]
为了明确遗传和环境因素对小麦品质的影响，选用6个有代表性的冬小麦品种（豫麦34、藁麦8901、豫麦49、豫麦70、洛阳8716和豫麦50）在河南省五个纬度点（32°N~36°N）种植，研究了品种遗传因素、环境因素与冬小麦品质性状的关系。结果表明，遗传因素对硬度、出粉率、耐揉指数、断裂时间的影响较大，而环境因素对灰分、沉淀值、容重有显著影响。由南向北随着纬度的升高，灰分、沉淀值呈减少趋势，容重呈增加趋势。小麦不同品质性状受气候因子的影响程度不同，其中硬度、灰分、沉淀值、容重均与小麦生育后期5月份的主要气象因子呈显著相关。因此，小麦生育后期的田间管理应充分考虑气象因素，以改善小麦品质。
DOI:10.7606/j.issn.1009-1041.2010.02.012URL [本文引用: 1]
为了明确遗传和环境因素对小麦品质的影响，选用6个有代表性的冬小麦品种（豫麦34、藁麦8901、豫麦49、豫麦70、洛阳8716和豫麦50）在河南省五个纬度点（32°N~36°N）种植，研究了品种遗传因素、环境因素与冬小麦品质性状的关系。结果表明，遗传因素对硬度、出粉率、耐揉指数、断裂时间的影响较大，而环境因素对灰分、沉淀值、容重有显著影响。由南向北随着纬度的升高，灰分、沉淀值呈减少趋势，容重呈增加趋势。小麦不同品质性状受气候因子的影响程度不同，其中硬度、灰分、沉淀值、容重均与小麦生育后期5月份的主要气象因子呈显著相关。因此，小麦生育后期的田间管理应充分考虑气象因素，以改善小麦品质。
