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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (02): 179-193.doi: 10.3724/SP.J.1006.2020.91029

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2020-02-12 Published:2019-10-16
  • Contact: Rui-Hui WANG,Gui-Ru LIU E-mail:wangrh@hebau.edu.cn;nxlgr@hebau.edu.cn
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31371617)

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

Table 1

Phenotypes for wheat parents and selected RIL lines"

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

Fig. 1

Sequence 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."

Fig. 2

Polyacrylamide 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)."

Fig. 3

KASP 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."

Table 2

Proportion of marker-based genotypes for EST and KASP markers in selected wheat RIL lines"

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)

Table 3

Phenotypes for selected wheat cultivars"

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

Table 4

Proportion of marker-based genotypes for EST and KASP markers in selected wheat cultivars (%)"

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)

Fig. 4

Marker-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"

Table 5

Phenotypes and marker-based genotypes in eleven wheat cultivars with the same resistant allele sets at seven marker loci"

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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