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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (7): 977-987.doi: 10.3724/SP.J.1006.2018.00977

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

Fine Mapping and Candidate Gene Analysis of Rice Blast Resistance Gene Pi47

Xiang-Yi XIAO,Xue-Tao SHI,Hao-Wen SHENG,Jin-Ling LIU(),Ying-Hui XIAO()   

  1. Agronomy College of Hunan Agricultural University / Hunan Provincial Key Laboratory of Rice and Rapeseed Breeding for Disease Resistance / Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China, Changsha 410128, Hunan, China
  • Received:2017-11-21 Accepted:2018-03-26 Online:2018-07-10 Published:2018-04-27
  • Contact: Jin-Ling LIU,Ying-Hui XIAO E-mail:liujinling@hunau.edu.cn;xiaoyh@hunau.edu.cn
  • Supported by:
    This study was supported by the National Key Research and Development Program (2016YFD0101100), the National Natural Science Foundation of China (31171834), and the Science and Technology Major Project of Hunan Province (2015NK1001-2).

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Abstract:

Continuous mapping and cloning new blast resistance genes provided an important approach for revealing the molecular mechanism of resistance to rice disease and breeding new varieties resistant to rice blast. Previously, the blast resistance gene Pi47 was mapped between SSR markers RM224 and RM2956 on chromosome 11, from a broad-spectrum and durable resistance native cultivar Xiangzi 3150 in Hunan province. In this study, the fine mapping and candidate gene prediction were performed, showing that Pi47 was mapped in an interval of 0.24 cM between CAPS markers S32 and K33 with 171.2 kb on Nipponbare reference genome, and further narrowed in an interval of 67.8 kb between markers SC12 and K33, through background screening to Pi47 monogenic lines and susceptible parent CO39 with six STS markers. Eight genes were predicted in this region on the reference genome, among them two encoded NBS-LRR resistance-like proteins, which probably were Pi47 functional candidate genes. A blast resistance spectrum evaluation, using Pi47 monogenic lines and four near-isogenic lines with the Pik allelic gene Pik, Pikm, Pikh, and Pikp across Pi47 region, revealed that Pi47 shared different resistance spectra with these four alleles. These results shed light on further molecular cloning of Pi47, and the molecular marker will be useful for molecular maker assisted selection to breed new resistant cultivars.

Key words: rice, rice blast, resistance gene, Pi47, fine mapping

Table 1

Screening of Pi47 monogenic lines in CO39/Xiangzi 3150 RILs"

株系
Line		 接种菌株193-1-1的抗性表型
Phenotype inoculated with isolate 193-1-1		 Pi47连锁标记
Markers linked to Pi47		 Pi48连锁标记
Markers linked to Pi48
RM206 RM224 RM5926 RM3102 RM1337 RM7102
CX18 R + + + - - -
CX36 R + + + - - -
CX40 R + + + - - -
CX52 R + + + - - -
CX66 R + + + - - -
CX67 R + + + - - -
CX70 R + + + - - -
CX147 R + + + - - -
CX152 R + + + - - -
CX155 R + + + - - -
CX166 R + + + - - -
CX279 R + + + - - -

Table 2

Segregation analysis of resistance to rice blast for each F2 population"

F2群体
F2 population		 抗病单株
Resistant individuals		 感病单株
Susceptible individuals		 作图群体株数
Individual number of mapping population		 卡方值
χ2 for 3:1 ratio
CX18/CO39 F2 1040 329 308 0.68
CX52/CO39 F2 1116 340 247 2.21
CX155/CO39 F2 1512 310 0 62.0
CX279/CO39 F2 3820 1221 1132 1.66
总数 Total / / 1687 /

Table 3

Number of recombinants identified with molecular marker in the three F2 populations"

群体
Population		 感病单株数目
No. of susceptible individuals		 重组子分布 Distribution of recombinants
RM224 S11, S4, S32 S10 K33 K107 K134 RM5926
CX279/CO39F2 1132 20 4 0 1 1 8 56
CX18/CO39F2 308 5 2 0 0 0 1 8
CX52/CO39F2 247 2 1 0 0 0 1 11
合计 Total 1687 27 7 0 1 1 10 75

Fig. 1

Genetic and physical map construction of Pi47 locusA: the genetic map of Pi47 locus. The middle crude black line is the Pi47 locus on chromosome 11, the letter above the line is the molecular makers used in gene mapping, and the number below is the genetic distance between two adjacent markers. B: the number of recombinant of corresponding markers used in Pi47 fine mapping. The numbers from top to bottom are recombinants detected in CX52/CO39 (n=247), CX18/CO39 (n=308), CX279/CO39 (n=1132) F2 mapping population, respectively. C: the physical map of Pi47 locus in japonica rice Nipponbare reference genome. Each crude line represents one BAC clone with its clone number under the line; the crossing points are the position of markers used in fine mapping. D: final physical region of Pi47 fine mapping within 171.2 kb between S32 and S10 marks. E: genetic background analysis of Pi47 single gene lines with molecular marker."

Table 4

Molecular markers used for Pi47 fine mapping"

标记
Marker		 引物顺序
Primer sequence (5°-3°)		 物理位置
Physical position		 扩增大小
Expect size		 酶
Enzyme		 标记类型
Marker type
RM224 F: TCGATCGATCTTCACGAGG
R: TGCTATAAAAGGCATTCGGG		 27673251
-27673372		 122 / SSR
S11 F: TCTTTGAACAAGAGGACCAG
R: AATTACCGAATTACTTAGCG		 27824419
-27825439		 1021 Mva I or Hinf I CAPS
S4 F: CGAATGATGACGAGGACCCG
R: ATCAGGCACGGCAGCAGAAG		 27827786
-27828843		 1058 Hinf I or Rsa I CAPS
S32 F: GCCCAGTAACCGTAGGAGTC
R: TTTGATGAGCGGCAAAGTAG		 27835604
-27836295		 692 Msp I CAPS
S10 F: AGAGCAAGAAGGGCACGGTAT
R: AGAATGGTCCTCCTGACATCG		 27992866
-27993970		 1105 Rsa I CAPS
K33 F: TTTGGTGCTCCTCTTACGGG
R: TCGGAGTTCACAGAGCCAAG		 28005690
-28006842		 1153 Hinf I CAPS
K107 F: ACATCAATGGCTACAACT
R: TGCTAACGGTGCTGGTAT		 28012753
-28012940		 184/188 / STS
K134 F: GATGGCGAGATGGTTGTC
R: GCCTTTGAGATAGGGATTGC		 28163563
-28163742		 180/168 / STS
RM5926 F: TAGGTCCATCCAAATCTCGATCC
R: TGGCAGAGGAGATTAGAGTAATACGG		 28957564
-28957858		 295 / SSR
S6 F: TCAACCGAACGCAAGAATCAC
R: GGCCCTCCAGCTCATCTACCT		 27841529
-27842431		 903 / STS
SC1 F: CCCCACAATCCCACGAGAC
R: CGGGCCGACAATCCAATAC		 27848711
-27849635		 925 / STS
SC3 F: TTTCCCACCGATGACCCAG
R: GCTCGCCATCCTTCCCTTT		 27879005
-27879906		 902 / STS
SC6 F: TATTGGTAAGTGGTAACGGGTGA
R: GCAGATTCGAGGAGGAAGG		 27891111
-27892550		 1440 / STS
SC7 F: TGGCGTGGCGGTTTGGTAG
R: GCTGGTTGGGCATGGGTTG		 27909896
-27910602		 707 / STS
SC12 F: GGGCAACAAGCGAGCCATAA
R: GTGGTGAGGCAGCGGAACAG		 27939030
-27940473		 1444 / STS

Table 5

Candidate gene prediction between SC12 and K33 in Pi47 locus"

基因名称
Gene name		 推测功能
Putative functions
LOC_Os11g46150 Expressed protein
LOC_Os11g46180 Transposon protein
LOC_Os11g46190 Transposon protein
LOC_Os11g46200 Leucine rich repeat family protein, expressed
LOC_Os11g46210 NB-ARC domain containing protein, expressed
LOC_Os11g46220 Hypothetical protein
LOC_Os11g46230 Tetratricopeptide repeat domain containing protein
LOC_Os11g46240 Retrotransposon protein

Table 6

Sequence similarity of cloned genes at the DNA and protein levels between Pi47-1 and Pik sites"

基因 Gene Pik1-KA Pi7-1 Piks-1 Pik-2 Pikp-1 Pikm1-TS Pi1-5
Pi47-1 DNA 0.997 0.975 0.998 0.997 0.975 0.998 0.997
Pi47-1蛋白 Pi47-1 protein 0.987 0.947 0.993 0.989 0.946 0.994 0.995

Table 7

Sequence similarity of cloned genes at the DNA and protein levels between Pi47-2 and Pik sites"

基因 Gene Pik2-KA Pi7-2 Piks-2 Pik-1 Pikp-2 Pikm2-TS Pi1-6
Pi47-2 DNA 0.999 0.998 0.999 0.999 0.998 0.999 1.000
Pi47-2蛋白 Pi47-2 protein 0.999 0.996 0.999 0.999 0.996 0.999 1.000

Fig. 2

Phylogenetic analysis of Pi47 and cloned genes at Pik locus"

Table 8

Resistance spectrum analysis of Pi47 and the nearby genes"

稻瘟菌小种
Isolate		 来源
Origin		 水稻品种 Rice cultivar
CO39 CX18
(Pi47)		 IRBLk-Ka
(Pik)		 IRBLkm-Ts
(Pikm)		 IRBLkh-K3
(Pikh)		 IRBLkp-K60
(Pikp)
318-2 中国湖南 Hunan, China S R R R R R
CHL473 中国湖南 Hunan, China S R R R R R
X2007A-3 中国湖南 Hunan, China S R R R R S
CHL440 中国湖南 Hunan, China S R R R R S
CHL438 中国湖南 Hunan, China S R R R R R
X2007A-7 中国湖南 Hunan, China S R R R R R
195-2-2 中国湖南 Hunan, China S S R R R S
87-4 中国湖南 Hunan, China S R R R R R
193-1-1 中国湖南 Hunan, China S R R R R R
110-2 中国湖南 Hunan, China S R R R R R
236-1 中国湖南 Hunan, China S R R R R R
RB4 中国广东 Guangdong, China S R R R R S
RB6 中国广东 Guangdong, China S R R R R S
RB18 中国福建 Fujian, China S R S R R S
RB19 中国福建 Fujian, China S R R R R R
RB12 中国 China MR MR MR MR MR MR
KOH 日本 Japan S S S S S S
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