中国农业科学 ›› 2018, Vol. 51 ›› Issue (24): 4633-4646.doi: 10.3864/j.issn.0578-1752.2018.24.005
汪文娟(),苏菁,杨健源,韦小燕,陈凯玲,陈珍,陈深,朱小源(
)
收稿日期:
2018-06-22
接受日期:
2018-08-09
出版日期:
2018-12-16
发布日期:
2018-12-16
基金资助:
WANG WenJuan(),SU Jing,YANG JianYuan,WEI XiaoYan,CHEN KaiLing,CHEN Zhen,CHEN Shen,ZHU XiaoYuan(
)
Received:
2018-06-22
Accepted:
2018-08-09
Online:
2018-12-16
Published:
2018-12-16
摘要:
目的 分析源于广8 A杂交稻组合的稻瘟病菌(Magnaporthe oryzae)无毒基因型,为华南不同主栽抗病水稻品种的合理布局提供参考依据。 方法 利用抗稻瘟病单基因系,对稻瘟病菌单孢分离菌株进行致病型测定;并根据已克隆的且与稻瘟病菌致病性相关的8个无毒基因功能性分子标记进行分离菌株的无毒基因型分析。选取分离自广8 A杂交稻组合的27个稻瘟病菌单孢菌株,提取各菌株的菌丝体DNA作为模板,进行PCR扩增及琼脂糖凝胶电泳检测。并对其中的7个稻瘟病菌菌株进行相应无毒基因全长或CDS区域的PCR产物测序,将测序序列与相应无毒菌株的无毒基因序列进行比较分析。 结果 所测定的菌株对其中的5个单基因系IRBLkh-K3(Pikh)、IRBLb-B(Pib)、IRBLz5-CA(Pi2)、NIL-e1(Pi50)和IRBL9-W(Pi9)表现高的无毒性频率(>85%),所有菌株对单基因系IRBL9-W(Pi9)和NIL-e1(Pi50)都表现无毒;这些菌株对其中的7个单基因系表现较低的无毒性频率(<20%),包括IRBLks-F5(Piks)、IRBLa-A(Pia)和IRBL19-A(Pi19)等,表明分离自广8 A杂交稻组合的稻瘟病菌对不同的单基因系表现出不同的致病型。所有菌株都被检测出含无毒基因AvrPi9和AvrPik,并可检测到无毒基因AvrPik 的2种类型:AvrPik-D型和AvrPik-E型,但未检测出无毒基因Avr1-CO39、AvrPii和AvrPia。在部分菌株中能检测到无毒基因AvrPiz-t、AvrPib和AvrPita预期大小的产物,但在所检测的菌株中均以较低的频率与不同的突变类型出现。对GD13-621、GD14-349与GD15-291等7个菌株分别进行相应无毒基因的PCR产物测序,AvrPi9、AvrPita及AvrPiz-t的基因序列与其各自无毒菌株的无毒基因序列完全一致,表明分离菌株中这3个无毒基因的基因结构相对稳定。测序菌株的AvrPib基因序列与已报道的AvrPib无毒菌株的序列在基因的上游区域存在2个SNP的差异及3个连续碱基的插入,具有较丰富的多样性;6个测序菌株的AvrPik基因序列高度一致,仅在编码区存在1个SNP的差异即136(C/A),该位点SNP的变异导致氨基酸的错义突变46(H/N),表现为菌株携带AvrPik-D或AvrPik-E型。 结论 在华南主栽品种广8 A杂交稻组合上分离的稻瘟病菌中AvrPi9和AvrPik分布较为广泛,在上述品种感病区域,可选用含Pi9和Pik的抗病品种作为轮换种植。
汪文娟,苏菁,杨健源,韦小燕,陈凯玲,陈珍,陈深,朱小源. 源于广8 A杂交稻组合的稻瘟病菌无毒基因型分析[J]. 中国农业科学, 2018, 51(24): 4633-4646.
WANG WenJuan,SU Jing,YANG JianYuan,WEI XiaoYan,CHEN KaiLing,CHEN Zhen,CHEN Shen,ZHU XiaoYuan. Analysis of Magnaporthe oryzae Avirulent Genes in the Infected Hybrid Rice Combinations Derived from a Sterile Line of Guang 8 A[J]. Scientia Agricultura Sinica, 2018, 51(24): 4633-4646.
表1
41个稻瘟病菌菌株及其来源"
采集地区 Sampling location | 采集品种 Sampling cultivar | 菌株编号 Strain number | 采集地区 Sampling location | 采集品种 Sampling cultivar | 菌株编号 Strain number | |
---|---|---|---|---|---|---|
河源 Heyuan | 广8优165 Guang8you165 | GD12-535 | 河源 Heyuan | 广8优169 Guang8you169 | GD15-259 | |
阳江 Yangjiang | 广8优169 Guang8you169 | GD12-716 | 河源 Heyuan | 广8优169 Guang8you169 | GD15-270 | |
阳江 Yangjiang | 广8优169 Guang8you169 | GD12-721 | 河源 Heyuan | 广8优165 Guang8you165 | GD15-291 | |
阳江 Yangjiang | 广8优169 Guang8you169 | GD12-730 | 河源 Heyuan | 广8优2168 Guang8you2168 | GD15-304 | |
河源 Heyuan | 广8优169 Guang8you169 | GD13-493 | 河源 Heyuan | 广8优165 Guang8you165 | GD15-315 | |
阳江 Yangjiang | 广8优169 Guang8you169 | GD13-621 | 河源 Heyuan | 广8优188 Guang8you188 | GD15-316 | |
阳江 Yangjiang | 广8优169 Guang8you169 | GD13-626 | 云浮 Yunfu | 广8优169 Guang8you169 | GD15-436 | |
茂名 Maoming | 广8优169 Guang8you169 | GD13-659 | 阳江 Yangjiang | 广8优169 Guang8you169 | GD15-506 | |
阳江 Yangjiang | 广8优188 Guang8you188 | GD13-3009 | 阳江 Yangjiang | 广8优2168 Guang8you2168 | GD15-520 | |
阳江 Yangjiang | 广8优2168 Guang8you2168 | GD13-3024 | 阳江 Yangjiang | 广8优169 Guang8you169 | GD15-523 | |
河源 Heyuan | 广8优2168 Guang8you2168 | GD14-070 | 阳江 Yangjiang | 广8优169 Guang8you169 | GD15-530 | |
云浮 Yunfu | 广8优169 Guang8you169 | GD14-298 | 阳江 Yangjiang | 广8优169 Guang8you169 | GD15-542 | |
阳江 Yangjiang | 广8优169 Guang8you169 | GD14-349 | 阳江 Yangjiang | 广8优165 Guang8you165 | GD15-543 | |
阳江 Yangjiang | 广8优169 Guang8you169 | GD14-366 | 茂名 Maoming | 广8优169 Guang8you169 | GD15-586 | |
阳江 Yangjiang | 广8优169 Guang8you169 | GD14-368 | 河源 Heyuan | 广8优165 Guang8you165 | GD16-246 | |
阳江 Yangjiang | 广8优169 Guang8you169 | GD14-372 | 河源 Heyuan | 广8优188 Guang8you188 | GD16-272 | |
阳江 Yangjiang | 广8优165 Guang8you165 | GD14-376 | 河源 Heyuan | 广8优165 Guang8you165 | GD16-280 | |
阳江 Yangjiang | 广8优169 Guang8you169 | GD14-381 | 阳江 Yangjiang | 广8优169 Guang8you169 | GD16-327 | |
阳江 Yangjiang | 广8优165 Guang8you165 | GD14-401 | 阳江 Yangjiang | 广8优2168 Guang8you2168 | GD16-334 | |
河源 Heyuan | 广8优2168 Guang8you2168 | GD15-023 | 从化 Conghua | 广8优169 Guang8you169 | GD16-3071 | |
韶关 Shaoguan | 广8优金占 Guang8youjinzhan | GD15-236 |
表2
本研究中用于检测无毒基因的引物"
引物 Primer | 引物序列 Sequence (5′-3′) | 预期片段大小 Expected fragment size (bp) | 目的 Purpose |
---|---|---|---|
Avr1-CO39 F | TGCCGCATTTTGCTAACCG | 994 | 检测Avr1-CO39启动子及CDS To detect Avr1-CO39 promoter and CDS |
Avr1-CO39 R | GCGAATCCATAGACAAGGAC | ||
AvrPita F | CAGGCATACATTGGAGAGCC | 1549 | 检测AvrPita启动子及CDS To detect AvrPita promoter and CDS |
AvrPita R | CCCTCCATTCCAACACTAAC | ||
AvrPii F | GGTAGATATCCGCTGACTGG | 839 | 检测AvrPii启动子及CDS To detect AvrPii promoter and CDS |
AvrPii R | ACTGTCCGCCGCTCGTTTGG | ||
AvrPi9 F | ATGCAGTTCTCTCAGATCCTC | 342 | 检测AvrPi9 CDS To detect AvrPi9 CDS |
AvrPi9 R | CTACCAGTGCGTCTTTTCGAC | ||
AvrPiz-t F | GTTGCGATTATGATCCGTCG | 1144 | 检测AvrPiz-t启动子及CDS To detect AvrPiz-t promoter and CDS |
AvrPiz-t R | GTACTCTAGCAAACGACCGG | ||
AvrPia F | CAGAGAAACGGACTTGGAGG | 1220 | 检测AvrPia启动子及CDS To detect AvrPia promoter and CDS |
AvrPia R | GGTATACACGTACGGTAGGG | ||
AvrPik-CDE F | TCCTGCTGCTAACTCCATTC | ~1000 | 检测AvrPik-CDE型启动子及CDS To detect the type of AvrPik-CDE promoter and CDS |
AvrPik-CDE R | GGGTACAGGAATACCAGG | ||
AvrPik-D F | ACCCTAACTTTTTCGACC | 286 | 检测AvrPik-D型CDS To detect the type of AvrPik-D CDS |
AvrPik-D R | TCAACCAAGCGTAAACCTCG | ||
AvrPib F | ATGCCGACAATGCGAGGTAT | 1598 | 检测AvrPib启动子及CDS To detect AvrPib promoter and CDS |
AvrPib R | GGACAAGGGAGGCAAATCTAAC |
表3
不同菌株对25个抗稻瘟单基因系的致病型"
菌株 Strain | IRBLs单基因系对菌株的反应 Reactions of IRBLs to the strains | LTH | ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | ||
Pia | Pii | Piks | Pik | Pikp | Pikh | Piz | Piz5 | Pizt | Pita | Pib | Pit | Pish | Pi1 | Pi3 | Pi5 | Pi7 | Pi9 | Pi12 | Pi19 | Pikm | Pi20 | Pita2 | Pi11 | Pi50 | ||
GD12-535 | S | S | S | S | S | R | S | S | R | R | R | S | S | S | S | S | S | R | S | S | S | S | R | S | R | S |
GD13-3009 | S | R | S | R | R | R | R | R | S | S | R | R | R | S | R | R | R | R | S | S | R | S | S | S | R | S |
GD13-3024 | S | S | S | R | R | S | R | R | R | S | R | S | S | S | S | S | R | R | S | S | S | S | S | S | R | S |
GD13-493 | S | S | S | R | S | R | S | R | S | S | R | S | S | S | S | S | S | R | S | S | S | S | R | S | R | S |
GD13-621 | S | R | S | R | R | R | S | R | R | S | R | S | R | R | S | R | R | R | S | S | R | S | R | S | R | S |
GD13-659 | S | S | S | R | S | R | S | R | S | S | R | S | S | R | S | S | S | R | S | S | S | S | S | S | R | S |
GD14-070 | S | S | S | R | R | R | R | R | S | S | R | S | S | S | S | S | R | R | S | S | R | S | S | S | R | S |
GD14-349 | S | S | S | R | R | R | R | R | R | R | R | S | S | R | S | S | R | R | S | S | R | S | R | S | R | S |
GD14-366 | S | R | S | R | R | R | R | R | R | R | R | S | R | S | S | S | R | R | S | R | S | R | S | S | R | S |
GD14-368 | S | R | S | S | R | S | R | R | R | R | S | S | R | R | S | S | S | R | S | R | S | S | S | S | R | S |
GD14-376 | S | S | S | S | S | S | S | R | R | S | R | S | S | S | S | S | S | R | S | S | S | S | S | S | R | S |
GD14-401 | S | R | S | R | R | R | R | R | S | S | R | S | S | S | S | S | R | R | S | S | S | S | S | S | R | S |
GD15-23 | S | S | S | S | S | R | R | R | S | S | R | S | S | S | S | S | S | R | S | S | S | S | S | S | R | S |
GD15-259 | S | S | S | S | S | R | R | R | S | S | R | S | S | S | S | S | S | R | S | S | S | S | S | S | R | S |
GD15-270 | S | S | S | S | S | R | S | R | R | S | R | S | S | S | S | R | S | R | S | S | S | S | S | S | R | S |
GD15-291 | S | S | S | R | R | R | S | R | R | S | R | S | S | S | S | S | R | R | S | S | S | S | S | S | R | S |
GD15-315 | R | R | R | R | S | R | R | R | R | R | R | S | R | R | R | R | R | R | R | R | R | R | S | R | R | S |
GD15-436 | S | S | S | S | S | R | R | R | S | S | R | S | S | S | S | R | S | R | S | S | S | S | S | S | R | S |
GD15-506 | S | S | S | S | S | S | S | R | R | S | S | S | R | S | S | S | S | R | S | S | S | R | R | R | R | S |
GD15-523 | S | S | S | R | S | R | S | R | S | S | S | S | S | S | S | S | S | R | S | S | S | S | S | S | R | S |
GD15-530 | S | R | S | R | R | R | R | S | S | R | R | S | S | R | S | S | S | R | S | S | S | S | S | S | R | S |
GD15-586 | S | R | S | R | S | R | R | S | S | S | R | S | S | R | S | S | S | R | S | S | S | S | S | S | R | S |
GD16-272 | R | R | S | R | S | R | R | R | R | R | R | S | R | R | S | R | R | R | S | R | R | R | R | S | R | S |
GD16-280 | R | S | R | R | S | R | R | R | R | R | R | S | S | S | R | R | S | R | S | R | S | R | R | S | R | S |
GD16-3071 | S | S | S | S | S | R | S | R | R | S | R | S | S | S | S | S | S | R | S | S | S | R | R | R | R | S |
GD16-327 | S | S | S | R | S | R | S | R | S | S | S | S | S | R | S | R | S | R | S | S | S | S | S | S | R | S |
GD16-334 | S | S | S | S | S | R | R | R | S | S | R | S | S | S | S | S | S | R | S | S | S | S | S | S | R | S |
无毒基因频率 The frequency of avirulent genes (%) | 11.1 | 33.3 | 7.4 | 63.0 | 37.0 | 85.2 | 59.3 | 88.9 | 51.9 | 29.6 | 85.2 | 3.7 | 25.9 | 33.3 | 11.1 | 29.6 | 37.0 | 100.0 | 3.7 | 18.5 | 22.2 | 22.2 | 29.6 | 11.1 | 100.0 |
表4
稻瘟病菌菌株无毒基因单倍型与致病型的比较"
菌株 Strain | 对Pi9的反应 Reactions to Pi9 | 对Pik不同等位基因的反应 Reactions to different Pik alleles | 对Pii的反应 Reactions to Pii | 对Piz-t的反应 Reactions to Piz-t | 对Pita的反应 Reactions to Pita | 对Pib的反应 Reactions to Pib | 对Pia和Pi-CO39的反应 Reactions to Pia and Pi-CO39 | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
IRBL9 -W | AVR单倍型 AVR haplotype | IRBLk -Ka | IRBLkp -K60 | IRBLkh -K3 | IRBL1 -CL | IRBL7 -M | IRBLkm -Ts | AVR单倍型 AVR haplotype | IRBLi -F5 | AVR单倍型 AVR haplotype | IRBLzt -T | AVR单倍型 AVR haplotype | IRBLta -K1 | AVR单倍型 AVR haplotype | IRBLb -B | AVR单倍型 AVR haplotype | IRBLa -A | AVR单倍型 AVR haplotype | ||
Avr-Pia | Avr1-CO39 | |||||||||||||||||||
GD12-535 | R | + | S | S | R | S | S | S | D | S | - | R | - | R | - | R | - | S | - | - |
GD13-3009 | R | + | R | R | R | S | R | R | D | R | - | S | - | S | - | R | - | S | - | - |
GD13-3024 | R | + | R | R | S | S | R | S | - | S | - | R | + | S | + | R | - | S | - | - |
GD13-493 | R | + | R | S | R | S | S | S | - | S | - | S | - | S | + | R | - | S | - | - |
GD13-621 | R | + | R | R | R | S | R | R | D | R | - | R | + | S | + | R | + | S | - | - |
GD13-659 | R | + | R | S | R | S | S | S | - | S | - | S | - | S | - | R | - | S | - | - |
GD14-349 | R | + | R | R | R | S | R | R | D | S | - | R | - | R | + | R | - | S | - | - |
GD14-366 | R | + | R | R | R | S | R | S | D | R | - | R | - | R | - | R | - | S | - | - |
GD14-368 | R | + | S | R | S | S | S | S | - | R | - | R | - | R | - | S | - | S | - | - |
GD14-376 | R | + | S | S | S | S | S | S | - | S | - | R | + | S | - | R | - | S | - | - |
GD14-401 | R | + | R | R | R | S | R | S | D | R | - | S | - | S | - | R | - | S | - | - |
GD14-70 | R | + | R | R | R | S | R | R | D | S | - | S | - | S | - | R | - | S | - | - |
GD15-23 | R | + | S | S | R | S | S | S | - | S | - | S | - | S | - | R | - | S | - | - |
GD15-259 | R | + | S | S | R | S | S | S | - | S | - | S | - | S | - | R | - | S | - | - |
GD15-270 | R | + | S | S | R | S | S | S | - | S | - | R | + | S | - | R | - | S | - | - |
GD15-291 | R | + | R | R | R | S | R | S | D | S | - | R | + | S | - | R | + | S | - | - |
GD15-315 | R | + | R | S | R | S | R | R | D | R | - | R | - | R | - | R | - | R | - | - |
GD15-436 | R | + | S | S | R | S | S | S | - | S | - | S | - | S | - | R | - | S | - | - |
GD15-506 | R | + | S | S | S | S | S | S | - | S | - | R | + | S | - | S | + | S | - | - |
GD15-523 | R | + | R | S | R | S | S | S | - | S | - | S | - | S | - | S | - | S | - | - |
GD15-530 | R | + | R | R | R | S | S | S | D | R | - | S | - | R | - | R | - | S | - | - |
GD15-586 | R | + | R | S | R | S | S | S | - | R | - | S | - | S | - | R | - | S | - | - |
GD16-272 | R | + | R | S | R | S | R | R | D | R | - | R | - | R | - | R | - | R | - | - |
GD16-280 | R | + | R | S | R | S | S | S | D | S | - | R | - | R | - | R | - | R | - | - |
GD16-3071 | R | + | S | S | R | S | S | S | - | S | - | R | + | S | - | R | + | S | - | - |
GD16-327 | R | + | R | S | R | S | S | S | - | S | - | S | - | S | - | S | - | S | - | - |
GD16-334 | R | + | S | S | R | S | S | S | - | S | - | S | - | S | - | R | - | S | - | - |
表5
41个菌株对11个抗稻瘟病单基因系的致病性"
采集品种 Sampling cultivar | 菌株 Strain | 11个单基因系对菌株的反应 Reactions of 11 blast-resistant lines to the strains | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | ||
Pi9 | Pi2 | Piz | Pikh | Pi1 | Pikp | Pita2 | Pish | Pii | Piz-t | Pi50 | ||
广8优165 Guang8you165 | GD12-535 | R | R | S | R | R | S | R | S | S | R | R |
广8优169 Guang8you169 | GD12-716 | R | R | R | R | R | R | R | R | S | S | R |
广8优169 Guang8you169 | GD12-721 | R | R | R | R | R | S | S | S | S | S | R |
广8优169 Guang8you169 | GD12-730 | R | R | S | R | R | S | S | S | S | S | R |
广8优169 Guang8you169 | GD13-493 | R | R | S | R | R | S | R | S | S | S | R |
广8优169 Guang8you169 | GD13-621 | R | R | R | R | R | R | R | R | R | R | R |
广8优169 Guang8you169 | GD13-626 | R | R | R | R | R | R | S | R | S | R | R |
广8优169 Guang8you169 | GD13-659 | R | S | S | R | R | S | S | S | S | S | R |
广8优188 Guang8you188 | GD13-3009 | R | R | S | R | R | R | S | S | S | S | R |
广8优2168 Guang8you2168 | GD13-3024 | R | R | R | R | R | R | S | R | R | S | R |
广8优2168 Guang8you2168 | GD14-070 | R | R | R | R | R | S | S | S | S | S | R |
广8优169 Guang8you169 | GD14-298 | R | R | R | R | R | R | R | R | R | R | R |
广8优169 Guang8you169 | GD14-349 | R | R | S | R | R | R | R | S | S | S | R |
广8优169 Guang8you169 | GD14-366 | R | S | R | R | S | R | S | R | R | S | R |
广8优169 Guang8you169 | GD14-368 | R | S | R | R | R | R | S | R | R | R | R |
广8优169 Guang8you169 | GD14-372 | R | S | R | R | S | S | S | R | R | S | R |
广8优165 Guang8you165 | GD14-376 | R | R | S | R | R | S | S | R | S | R | R |
广8优169 Guang8you169 | GD14-381 | R | R | R | R | R | R | S | S | S | S | R |
广8优165 Guang8you165 | GD14-401 | R | S | R | R | R | R | S | S | S | S | R |
广8优2168 Guang8you2168 | GD15-023 | R | R | S | R | R | S | S | S | S | S | R |
广8优金占 Guang8youjinzhan | GD15-236 | R | S | R | R | R | S | S | R | R | S | R |
广8优169 Guang8you169 | GD15-259 | R | S | S | R | R | S | S | S | S | S | R |
广8优169 Guang8you169 | GD15-270 | R | R | S | S | R | S | S | S | R | R | R |
广8优165 Guang8you165 | GD15-291 | R | S | S | R | R | R | R | R | R | S | R |
广8优2168 Guang8you2168 | GD15-304 | R | R | R | R | R | R | S | S | S | R | R |
广8优165 Guang8you165 | GD15-315 | R | S | R | R | R | R | S | R | R | S | R |
广8优188 Guang8you188 | GD15-316 | R | S | R | R | R | R | R | R | R | S | R |
广8优169 Guang8you169 | GD15-436 | R | R | S | R | R | S | S | S | S | S | R |
广8优169 Guang8you169 | GD15-506 | R | R | R | R | R | S | S | R | R | S | R |
广8优2168 Guang8you2168 | GD15-520 | R | R | S | R | R | S | S | S | R | S | R |
广8优169 Guang8you169 | GD15-523 | R | R | R | R | R | S | S | S | S | S | R |
广8优169 Guang8you169 | GD15-530 | R | S | R | R | R | R | R | R | R | R | R |
广8优169 Guang8you169 | GD15-542 | R | R | R | R | R | R | S | S | R | S | R |
广8优165 Guang8you165 | GD15-543 | R | R | R | R | R | R | R | R | R | S | R |
广8优169 Guang8you169 | GD15-586 | R | S | R | R | R | S | S | S | S | S | R |
广8优165 Guang8you165 | GD16-246 | R | S | S | R | R | R | R | S | S | S | S |
广8优188 Guang8you188 | GD16-272 | R | R | R | R | R | R | R | R | S | S | R |
广8优165 Guang8you165 | GD16-280 | R | R | R | R | R | R | R | R | R | R | R |
广8优169 Guang8you169 | GD16-327 | R | R | S | R | R | S | S | S | S | S | R |
广8优2168 Guang8you2168 | GD16-334 | R | R | S | R | R | S | S | S | S | S | R |
广8优169 Guang8you169 | GD16-3071 | R | R | S | R | S | S | R | S | S | R | R |
无毒基因频率 The frequency of avirulent genes (%) | 100.00 | 68.29 | 58.54 | 97.56 | 92.68 | 51.22 | 34.15 | 43.90 | 41.46 | 26.83 | 97.56 |
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