Pike, a rice blast resistance allele consisting of two adjacent NBS-LRR genes, was identified as a novel allele at the Pik locus

Pike identified as a novel rice blast resistance allele at the Pik locus

Jing Chen1, Pei Peng1, Jinshan Tian2, Yonggang He1, Lipan Zhang1, Zixu Liu1, Dandan Yin1, Zhihong Zhang1*

1 State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, China

2 Yichang Academy of Agricultural Sciences, Yichang 443000, China.

* Corresponding author

Phone: +86-13871491559

Fax: +86-27-68752095

E-mail:

Supplemental Tables

Table S1 PCR primers used in the present study

Primer name / Forward primer (5’-3’) / Enzyme site / Reverse primer(5’-3’) / Enzyme site / Use of primers
Gene mapping
RM4112 / CACTGGCAAAGTCAGTAGTCCTTCC / GGAGGCTAGCTCCGTAAATCTGG
RM1233 / GTGTAAATCATGGGCACGTG / AGATTGGCTCCTGAAGAAGG
ID6946 / TCGGATGAGCCACAA / TCCACGGTCCACAGA
ID6816 / TCGCCGATGCGGTTGATTTACTC / CGTATTTTGTGTTGTTAGGAGATAAGG
RM224 / ATCGATCGATCTTCACGAGG / TGCTATAAAAGGCATTCGGG
ID1159 / CACAGCCTAAGTTACCAAATGG / GCTCAAATAGACCACGACTTAAC
ID7711 / ACATACGTAGTAGGTAGCTAGTCG / GACCTGGCTGGACACATACA
ID6697 / ATTGTGCAACTTCTGGGATG / AATTGGGAGTCAAGAAAGGTG
ID6726 / CAGACCTTACAAGAACTGGAAAC / GTCATATCCCCTGTTTGATCTG
RM7443 / TGCTGCGTGTTACTTTGGTG / AACCCTTCATCAGGCTACGC
Presence/absence analysis
E1a / AAGCAATCCACATGGTTGAACAC / CAGGCCAATTTGGTCCAATATATC
E1b / AAGATGTCTGAAGCGATAGCCC / CGGCCAACAGGAGTAGGTTATTA
E2a / GCGCTCCATCCTCCATTTACT / GAGAGACCGTCACGCTTCATG
E2b / GAGGACTGCATCGACGACTTC / AGTTTGACACCACGCTTACATCTG
E3a / CATGTCGGGTACTCCAGAGTTTACT / AGATGCACCTCTTTTAGGATTTCCA
E3b / CCAGATGGATCACACAGCTTGAA / TCCAAATCCAGTGAGACTTGAACC
E4a / TCTCTACAAACCCTCTCATGCTGCC / TCTCCAATCCGAAGCCAAAATCAG
E4b / AGGAGCATGATCCAGCCAGTAGA / ATATGGTAAACCTGTGTCGGCATTG
E5a / GAGGCAACAAAAATTAAGAGG / GTGGACTCATATCTTCGAAATT
E5b / AATTAGTGCTAACGGCATACCATA / CTCCCGTCCTATCGAAGCT
E6a / AACTCTTGTCTTGAGCTCATCGAA / CAGACCAAATATCATCAATCAAGAGG
E6b / CAGGCCAAGGAAACTCTCAGC / TGGAAATTGTGACACCATAAATATAGC
Gene cloning
KE1 / TTTGGCGCGCCGAACAGCCTGAGGAAGCAGAACATCG / AscI / TTTGGCGCGCCCCGAAGGCGTCTGCGAAGCTCT / AscI
KE2 / TTTGGCGCGCCAGTGGTCTTTGATCATCTTCGGGATACGC / AscI / TTTGGCGCGCCATAATTGAATCCATTGGGAGCCGTAGCA / AscI
KE3 / TTTGGCGCGCCGGGAAGCCGGATTAAGGACAAGCTAG / AscI / TTTGGCGCGCCATCCAGCACCTGTATTATCCCATAGTTGAA / AscI
KE4 / TTGGCGCGCCGGCGATGGATAGCTGAAGGTTTTG / AscI / TTGGCGCGCCAGGGACAGAATGACAGTGTAAGTGAGTTTG / AscI
KE1ORF / TTTGGCGCGCCGAGTAGCACTCAACGCAAAAGGGCATCGGC / AscI / TTTGGCGCGCCGAGCGGGGACTTAGAGTATTACATGAAGGA / AscI / Pike-1 ORF
KE2 ORF / TTTGGCGCGCCAGCTGAAAGTTGCAGTGAGAAGTACAGA / AscI / TTTGGCGCGCCTCAGATGATTGGTAAGTTCTCCGATTTG / AscI / Pike-2 ORF
Expression analysis
RT-1 / ACTTTCTGATGACTTCCCATTCTTC / GACATTTTCGCTCAAGCACAAC / Pike-1
RT-2 / CCCTGTCACTGCTCAGAGGTAAT / GCAGACCAAATGTCATCAATCAAG / Pike-2
Actin / GAGACATTCAGCGTTCCAGC / GCATAACCTTCGTAGATTGGGAC / Internal reference
PBZ1 / GCCGCAAGTCATGTCCTAAAG / GCCGTCCTCGAGCTCGTA / Internal reference
Detection of transgenic lines
TR-KE1 / GCATGGATTATCCTCTCCAGTTCCG / GGTTGCAATCGCCGGTGACC / Pike-1
TR-KE2 / AACAAAGTAGACGAGCAAGTCCCTG / CTTGGAGATGGAGTGATGAGGGTAG / Pike-2
SNP analysis
d-G1328C / CGATGACATTTGGCATTGGGAAGA / CTTTGTTGCTATCCCCCAAGACATC / TaqαI / Pike-specific SNP
d-A3017T / AAGTTCCTTGTAAATGATTTGAACG / AclI / TGCAAGGTGAGCAATAATAACAATA / Pike-specific SNP

Table S2 Genotypes of T0 transgenic rice plants assayed by Pike-specific PCR and their resistant/susceptible reactions to M. oryzae isolates

Total number of T0 transgenic plantsa / Genes detected by Pike-specific PCR / Isolates inoculatedb / Numbers of T0 plants resistant or susceptible to isolates
R / S
30 / Pike-1 / CB1.1-1 / 0 / 30
41 / Pike-2 / CB1.1-1 / 0 / 41
5 / Pike-1 + Pike-2 / CB1.1-1 / 5 / 0
9 / Pike-1 + Pike-2 / RB22 / 9 / 0

a T0 plants were obtained with cv. Nipponbare as recipient. b isolates inoculated were avirulent to Pike.

R, blast resistant; S, fully blast susceptible.

Table S3 Resistant/susceptible reactions of T1 transgenic plants carrying Pike (Pike-1 + Pike-2) to six M. oryzae isolates incompatible to the Pike carrier Xiangzao143 but compatible to at least one of the other six Pik allele carriers

R gene carriers / Blast R genes / M. oryzae isolatesa
TM2.1-1 / CB1.2-1 / TF2.2-1 / NC2.2-3 / FC1.1-2 / HLJ-856
Transgenetic plants
T1 plants / Pike (Pike-1 + Pike-2) / R / R / R / R / R / R
The recepient cv.
Nipponbare / — / S / S / S / S / S / S
The donor cv.
Xiangzao143 / Pike (Pike-1 + Pike-2) / R / R / R / R / R / R
Reference lines
IRBLks-S / Piks / S / S / S / S / S / S
IRBLk-Ka / Pik / R / R / S / R / S / S
IRBLkp-K60 / Pikp / R / R / S / S / S / S
IRBLkh-K3 / Pikh / R / R / R / S / S / S
IRBLkm-Ts / Pikm / R / R / R / S / S / S
C101LAC / Pi1 / R / S / R / S / S / S

a M. oryzae isolates TM2.1-1, CB1.2-1 and TF2.2-1 were collected from Tianmen, Chibi and Tuanfeng of Hubei province, China, respectively; NC2.2-3 and FC1.1-2 were collected from Nanchang and Fengcheng of Jiangxi province, China, respectively; HLJ-856 was collected from Heilongjiang province, China.

R, resistant; S, susceptible; —, none.