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Fine mapping and identification of the rice blast-resistance locus Pi-kf2(t) as a new member of the Pi2/Pi9 multigene family

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Abstract

The identification and utilization of broad-spectrum resistance genes is the most effective and economical strategy of controlling rice blast disease. Kangfeng B (KFB), an elite Chinese rice cultivar, has been shown to exhibit broad-spectrum resistance to 53 isolates of Magnaporthe oryzae, the rice blast causative agent, from different regions of China. In this study, we identified a dominant blast-resistance gene at the Pi2/Pi9 locus from cultivar KFB, through genetic analysis and physical mapping. Allele-specific marker-based assessment revealed that Pi2, Pi9, and Piz-t are not the blast-resistance genes in KFB. By combining bulked segregation analysis (BSA) and recessive class analysis (RCA), the blast-resistance gene was fine-mapped to an approximately 249-kb interval between markers InDel-22 and Rm7213 on chromosome 6. Three bacterial artificial chromosome (BAC) clones spanning the region were identified. This region contains 19 predicted genes, including 7 nucleotide binding site–leucine-rich repeat (NBS-LRR) genes at the Pi2/Pi9 locus in japonica cv. Nipponbare genome. Further sequence comparison of the four functional NBS-LRR genes revealed that NBS-LRR2 and NBS-LRR4, as evidenced by their allelic/orthologous to Pi9 or Pi2, had significant differences of 9 to 43 and 14 to 48 amino acids in KFB, respectively, unlike the other known Pi2/Pi9 alleles, suggesting that KFB carries a hitherto undocumented member of the Pi2/Pi9 multigene family. It was tentatively designated as Pi-kf2(t). Our results provide essential information for the isolation of the Pi-kf2(t) gene and will facilitate both map-based cloning and marker-assisted selection of this gene for rice blast-resistance breeding.

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Abbreviations

BSA :

bulked segregation analysis

CAPS :

cleaved amplified polymorphic sequence

CTAB :

cetyltrimethyl ammonium bromide

dCAPS :

derived cleaved amplified polymorphic sequence

InDel :

insertion–deletion

NBS-LRR :

nucleotide binding site–leucine-rich repeat

RCA :

recessive class analysis

SNP :

single nucleotide polymorphism

SSR :

simple sequence repeat

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Acknowledgments

We are grateful to Dr. Xiao-Yuan Zhu, Plant Protection Research Institute, the Guangdong Academy of Agricultural Sciences, for kindly providing the rice lines, IRBLzt-T (Piz-t), EBZ (Pi50), and Gumei 4 (Pigm); to Dr. Zong-Hua Wang, College of Plant Protection, the Fujian Agricultural and Forestry University, for kindly providing the M. oryzae isolate 12JL-907-1; and to Dr. Jian-Fu Zhang, Rice Research Institute, the Fujian Academy of Agricultural Sciences, for kindly providing the rice lines C101A51 (Pi2) and 75-1-127 (Pi9).

Funding

This work was partially supported by the Fujian Provincial Natural Science Foundation (2018J01141), the China Agriculture Research System (CARS-01-69), and the Sanming Municipal Science and Technology Project (2017-N-2).

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Authors and Affiliations

  1. Biotechnology Research Institute, Sanming Academy of Agricultural Sciences, Sanming, 365500, China

    Xinyu Wei, Yuehui Zeng, Rui Zhang & Jianhong Huang

  2. Rice Research Institute, Sanming Academy of Agricultural Sciences, Sanming, 365500, China

    Wangxing Yang, Wenguang Zou & Xuming Xu

Authors
  1. Xinyu Wei
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  2. Yuehui Zeng
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  3. Rui Zhang
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  4. Jianhong Huang
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  6. Wenguang Zou
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  7. Xuming Xu
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Contributions

XW and YZ contributed equally to this work. XW carried out molecular marker development, genetic analysis, molecular mapping, resistance spectrum analysis, and physical map and phylogenetic tree construction and wrote the manuscript. YZ carried out molecular marker development, genetic analysis, molecular mapping, and resistance spectrum analysis. RZ participated in genetic analysis, molecular mapping, and physical map construction. JH carried out mapping population construction and participated in resistance spectrum analysis and genetic analysis. WY participated in genetic analysis and molecular mapping. WZ participated in molecular mapping and resistance spectrum analysis. XX designed the research and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Xuming Xu.

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Online Resource 1

(PDF 97 kb)

Online Resource 2

The structure of NBS2-Pi2 and NBS4-Pi2 (Pi2) and the location of their corresponding specific primers PiNBS2 and PiNBS4 (PNG 1401 kb)

High resolution image (TIF 557 kb)

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(PDF 110 kb)

Online Resource 4

The NBS-LRR genes clusters present at the Pi9, Pi2, Piz-t, Pigm, and Pi50 loci. Data from the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/), corresponding GenBank IDs were: Pi2 (DQ352453), Pi9 (DQ285630), Piz-t (DQ352040), Pigm (KU904633), and Pi50_NBS4_1/3 (KP985761/KU999983). NBS-LRR genes with oblique lines are pseudogenes with no function. NBS-LRRs in yellow are highly conserved, showing little difference within the Pi2/Pi9 family. NBS-LRRs in red are a set of orthologous/allelic members of NBS-LRR2 or NBS-LRR4, the key components of this gene family (PNG 2013 kb)

High resolution image (TIF 672 kb)

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Wei, X., Zeng, Y., Zhang, R. et al. Fine mapping and identification of the rice blast-resistance locus Pi-kf2(t) as a new member of the Pi2/Pi9 multigene family. Mol Breeding 39, 108 (2019). https://doi.org/10.1007/s11032-019-1017-0

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