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作物学报 ›› 2020, Vol. 46 ›› Issue (02): 179-193.doi: 10.3724/SP.J.1006.2020.91029

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦抗麦红吸浆虫基因标记的开发与验证

郝志明1,耿妙苗1,温树敏1,闫桂军2,王睿辉1,*(),刘桂茹1,*()   

  1. 1 河北农业大学 / 华北作物种质资源研究与利用教育部重点实验室, 河北保定 071000
    2 西澳大学农业与环境学院, 澳大利亚尼德兰兹 6009
  • 收稿日期:2019-04-08 接受日期:2019-09-26 出版日期:2020-02-12 网络出版日期:2019-10-16
  • 通讯作者: 王睿辉,刘桂茹
  • 作者简介:E-mail: haozhiming0730@hotmail.com
  • 基金资助:
    本研究由国家自然科学基金项目资助(31371617)

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-Hui1,*(),LIU Gui-Ru1,*()   

  1. 1 Hebei Agricultural University / North China Key Laboratory for Crop Germplasm Resources of Education Ministry, Baoding 071000, Hebei, China
    2 School of Agriculture and Environment, the University of Western Australia, Nedlands 6009, Australia
  • Received:2019-04-08 Accepted:2019-09-26 Published:2020-02-12 Published online:2019-10-16
  • Contact: Rui-Hui WANG,Gui-Ru LIU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31371617)

摘要:

麦红吸浆虫(Sitodiplosis mosellana Géhin)严重影响小麦产量和品质, 选育和使用抗虫品种是降低虫害损失最安全有效的途径, 利用抗虫性连锁或功能标记对提高小麦抗虫分子育种效率具有重要意义。在前期从转录组数据中挖掘到抗虫性主效QTL (QSm.hbau-4A) 6个相关差异基因的基础上, 依据这些基因序列中存在的InDel和SNP, 分别开发了2个EST标记和6个KASP标记, 并在抗虫性不同的一套重组近交系(RIL)和一套小麦品种中进行了标记的有效性验证。所开发的8个标记在抗、感虫小麦亲本间均表现出较好的多态性, 在RIL株系中的检测有效率均达到90%左右; 除E10-10外, 这些标记在供试高抗(56.3%~86.7%)和高感(85.7%~100.0%)小麦品种中的检测有效率均较高, 可用于小麦种质资源的抗虫性筛选。同时发现, 11个具备所有抗虫标记位点的抗虫小麦品种, 多为审定时间较早或已停止使用的品种, 这使得结合标记辅助选择等手段鉴定和创新小麦抗虫种质资源的工作日益紧迫。

关键词: 小麦, 麦红吸浆虫, 抗虫相关基因, 功能标记, SNP, EST, KASP

Abstract:

Orange wheat blossom midge (OWBM) (Sitodiplosis mosellana Géhin) has seriously reduced wheat production and processing quality. Breeding midge-resistant wheat has been considered the most effective way to reduce kernel losses caused by OWBM, and marker-assisted selection (MAS) strategy in crop breeding using linked or functional markers of target trait of interest is of great importance in improving breeding efficiency. Based on the InDels and SNPs within the sequences of six midge resistance-related genes discovered from transcriptome data in the previously mapped major QTL (QSm.hbau-4A) region, we developed and validated two EST and six KASP markers in a panel of recombinant inbred lines (RILs) and a panel of wheat cultivars with different OWBM resistance levels. These markers were polymorphic between the resistant and susceptible wheat parents, and approximately 90% of RIL lines showed the corresponding marker-based genotypes with their phenotypes. Except for E10-10, the other seven markers had higher detection efficiency in highly resistant (56.3%-86.7%) and in highly susceptible (85.7%-100.0%) wheat cultivars, thus making them applicable for screening midge-resistant wheat germplasm with locus QSm.hbau-4A. Among the eleven midge-resistant wheat cultivars with all resistance alleles for the seven markers developed, most were historical wheat cultivars, and rarely used in the present production, which suggests how to use old cultivars in wheat germplasm identification and enhancement on midge-resistance through MAS is urgent.

Key words: bread wheat (Triticum aestivum L.), Sitodiplosis mosellana, resistance-related genes, functional markers, SNP, EST, KASP

表1

供试小麦亲本与RIL株系的表型"

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

图1

6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。"

图2

EST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)。"

图3

KASP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。"

表2

EST标记和KASP标记在供试RIL株系中的标记基因型及比例"

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)

表3

供试小麦品种的表型"

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

表4

EST标记和KASP标记在供试小麦品种中的标记基因型及比例"

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)

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"

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"

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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