Identification of novel rice blast resistance alleles through sequence-based allele mining

DOI: https://doi.org/10.21203/rs.2.18859/v1

Abstract

Background: As rice ( Oryza sativa ) is the staple food of more than half the world’s population, rice production contributes greatly to global food security. Rice blast caused by the fungus M agnaporthe oryzae is a devastating fungal disease of rice, affecting yield and grain quality and resulting in substantial annual economic losses. Because the fungus evolves rapidly,, resistance conferred by most of the single blast race resistance genes is often broken after a few years of intensive agricultural use. Effective resistance breeding in rice therefore requires continual enrichment of the reservoir of resistance genes and alleles. Seed banks represent a rich source of genetic diversity; however, they have not been extensively used to identify novel genes and alleles.

Results: We carried out a large-scale screen for novel blast resistance alleles in 1883 rice varieties from major rice producing areas across China. Of these, 107 varieties showed at least moderate resistance to natural infection by rice blast at rice blast nurseries in Enshi and Yichang, Hubei Province. Using sequence-based allele mining to amplify and clone the allelic variants of major rice blast resistance genes at the Pi2/9/gm/zt locus of chromosome 6 from the 107 blast-resistant varieties, we identified 13 novel blast resistance alleles. We then used controlled infections to assess the resistance of rice varieties carrying the novel alleles to 34 single rice blast isolates from Hubei, Guangdong, Jiangsu, Hunan, Jangxi, Sichuan, Heilongjiang, and Fujin Provinces. The varieties identified as being resistant in the nursery trials showed varied disease responses when infected with the single blast isolates, suggesting that the novel Pi2/9/gm/zt alleles vary in their blast resistance spectra. Some of the newly identified alleles have unique single nucleotide polymorphisms (SNPs), insertions, or deletions, in addition to polymorphic residues that are shared between the different alleles.

Conclusions: These alleles expand the allelic series of blast resistance genes, enriching the genetic resource for rice blast resistance breeding programs and for studies aimed at deciphering rice–rice blast molecular interactions.

Key words : Pi9 , R-genes, Nucleotide diversity, Gene conversion, Resistance gene alleles, Rice blast

Introduction

Rice blast is an acute and destructive disease that can reduce yield or even wipe out an entire harvest. Grain blast also affects the quality of rice and is a serious food safety problem (Deng et al., 2017; Ishihara et al., 2014). In China, the disease affects more than 3.8 million hectares per year, reducing rice yield by 1 billion kg per year (Jiang et al., 2015; Tian et al., 2016). Rice blast, caused by the fungus Magnaporthe oryzae, is the most devastating disease affecting rice, under high temperatures and humidity conditions that favor its spread (Shen et al., 2004; Wang et al., 2017). Rice blast has been reported in almost all rice-producing areas in the world, including the main rice-producing areas of 85 countries and regions (Miah et al., 2013). Effective host resistance, conferred by resistance (R) genes, is considered to be the most economic approach to control plant diseases (Xiao et al., 2017). To date, 86 rice blast R genes have been isolated (Hua et al., 2012; Zhao et al., 2018), While these genes have advanced our understanding of the molecular mechanisms underlying disease resistance, maintaining genetic resistance in rice is challenging, because single varieties of rice are grown over large areas in monoculture and the pathogen evolves quickly. M. oryzae is known for its genetic instability and pathogenic variability, leading to rapid breakdown of resistance in rice varieties (Bryan et al., 2000; Jiang et al., 2012). Resistant rice varieties often remain effective for only a few years before new dominant pathogenic races of the fungus emerge (Lee et al., 2009; Li et al., 2017). Plants have evolved various mechanisms that protect them from pathogen invasion and colonization. Previous studies have shown that most R genes, which encode receptors containing a nucleotide-binding site and leucine-rich repeats (NBS-LRR), are organized into tight clusters containing multiple gene copies. Nine of the 13 major rice blast R genes are clustered (Qu et al., 2006; Wu et al., 2012) and most of them are broad-spectrum R genes with variable resistance. The Pi9 locus contains at least six known R genes (Pi2, Pi9, Piz-t, Piz, Pigm and Pi50) and is situated location, close to the centromere of chromosome 6. Four R genes atthis locus (Pi2, Pi9, Pigm and Piz-t) have been cloned (Dai et al. 2010; Zhou et al., 2006). Numerous studies have indicated that the clustered arrangement of R genes has contributed to the evolution of novel resistance specificities via gene conversion, recombination, or unequal crossing over (Ashikawa et al., 2008; Dai et al., 2010). Some NBS-LRR gene homologs at the same locus exhibit a different evolutionary pattern. Genomic analysis of the Pi9 locus in rice cultivars and wild rice lines has shown that the copy number and SNP genotypes of Pi9 homologs vary, indicating a complex evolutionary history for this R gene locus..

In order to gain insight into the origin and evolution of this locus, and to explore new alleles with broad-spectrum resistance for rice molecular breeding, among 1883 rice varieties from different rice regions, 107 varieties resistant to rice blast at least in one rice region were selected, and the genomic sequences of Pi9 homologs were analyzed. We identified 13 novel alleles in these 107 resistant rice varieties. Meanwhile, we inoculated these 13 alleles with Magnaporthe grisea single spore. By comparing with the resistance spectrum of the donor materials of Pi2, Pi9, Pigm, Piz-t, we proved that these 13 alleles are new alleles, Moreover, the resistance of Pi9-Type2, Pi9-Type3 and Pi9-Type5 were better than that of the cloned broad-spectrum rice blast resistance genes. These alleles extend the allele sequence and enrich the genetic resources of rice blast resistance breeding and rice blast interaction research at the molecular level. We plan to further evaluate the resistance level of new alleles by constructing near isogenic lines and transgenic verification. Finally, our goal is to introduce new alleles of broad-spectrum resistance into high-quality rice varieties by molecular breeding, improve the blast resistance of the original varieties, and cultivate new rice varieties resistant to blast.

Materials and Methods

Plant materials

About 2000 rice cultivars was obtained from major rice-growing provinces of China, including indica and Japonica, and then maintained at Huazhong Agricultural University for this study. Rice blast resistant varieties were measured through natural inducement in two rice uniform blast nursery, which are located at Enshi and Yichang in Hubei province. Based on the standard scoring system for leaf blast (scale HS-HR), choosing the varieties that were resistant with a phenotypic score of MR-HR against field mix-inoculum were selected for molecular screening. Lijiangxin Tuan Heigu (LTH), which is highly susceptible to rice blast, was used as a control for disease evaluation.

Pathogen collection, inoculation and disease evaluation

For resistance spectrum analysis, about 34 blast isolates of different races a virulent were used in this study. All these isolates were collected from whole China major rice-growing provinces and have genetic differentiation and belong to different blast lineages (Shen et al. 1998, 2004). We used these isolates to analyze phenotypic of parents with disease resistance genes. The 34 Magnaporthe oryzae isolates, which are highly virulent on most of the rice lines were also been used for phenotypic analysis of Pi9 allele. Twelve day-old seedlings were spray-inoculated with blast spore suspensions (approximately 1 × 105 spores/ml), and then grew in a dark chamber for 24 h (26℃, 90% humidity). After that, the growth conditions were changed to 12 hours of light and 12 hours of dark treatment every day. After 7 days post inoculation, disease reaction (0–5 disease rating scale) of each line was recorded (IRRI, 2002).

PCR for allele mining and blast resistance genes

The rice genomic sequence of the O. sativa cv. Nipponbare (www.ncbi.nlm.nih.gov), the BAC clone sequence (DQ454158.1, containing Pi2) of the O. sativa cv. C101A51, the sequence (DQ285630.1, containing Pi9) of the O. sativa cv. 75-1-127, the BAC clone sequence (KU904633.2, containing Pigm) of the O. sativa cv. GM4H and the sequence (DQ352040, containing Piz-t) of the O. sativa cv. ZY1H, corresponding to the location of Pi2/9/gm/z-t genes were used for designing PCR primers. This region corresponds to the sequence on TIGR coordinate LOC_Os06g17900. Two sets of primers flanking to the full length gene were designed using Primer 3 software (http://primer3.ut.ee/). PCR amplification of alleles was done using genomic DNA extracted from rice leaves using CTAB method (Murray and Thompson 1980). The PCR reaction of 50 µl was setup with 50 ng of template DNA, 50 ng both forward and reverse primers, 5 µl 10X LA Taq Buffer II, 8 µl dNTP Mixture (2.5 mM each) and 2.5 U TaKaRa LA Taq (RR02MQ, TAKARA). The details of various primers used in present study are given in Table S2.

Amplified PCR products were purified and sequenced using Sanger’s method based DNA Analyzer Sequencer, ABI 3730XL (ABI, Applied BiosystemsAmersham, USA). Each allele was sequenced three replications using sequencing primers.

DNA sequence analysis

All the sequence reads generated for each allele by sequencing primers were assembled separately for each allele by using Sequencing Analysis Software Version 5.1 (Applied Biosystems). The sequence of high quality was assembled and the assembled DNA sequence of each allele was used to do Blast2Sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=blastn&BLAST_SPEC=GlobalAln&LINK_LOC=BlastHomeLink) analysis against the Pi9 genes to check its similarity.

The Pi9 genes from the rice blast resistant lines were used as a reference for comparison studies. Structural analyses of Pi9 alleles were performed to predict gene structure and conserved domains using CDD software (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and the data were recorded as their position, such as CC, NBS and LRR domain. For Single Nucleotide Polymorphism (SNP) search, multiple sequence alignment was done for all the alleles along with Pi9 alleles from 75-1-127 as references by using Software of Sequencer (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=blastn&BLAST_SPEC=GlobalAln&LINK_LOC=BlastHomeLink). InDels and SNPs were identified, according to Pi9 sequence as reference sequence. Multiple sequence alignment of DNA sequences from amplified gene fragments were done by using CLUSTALW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and MEGA5.0 (http://www.megasoftware.net) softwares. The parameters used in MEGA 5.0 were bootstrap (1,000 replications) and neighbour joining with the p-distance model. A phylogenetic tree was constructed with MEGA to analysis the evolution of the Pi9 allele. The average nucleotide diversity (π), the average nucleotide polymorphism (θ) and the Tajima’s D significant test value were used for calculating variation among all the alleles isolated from different varieties by using DnaSP 5.0 software (Librado and Rozas 2009). Various domains, such as CC, NBS and LRR, which play vital role in disease resistance were shown on the DNA sequences of all alleles using the customized R scripts.

Crossing and selection scheme

Thirteen Pi9 allele genes were crossed with J23B, and then their F1 hybrids were backcrossed with J23B to obtain the BC1F1 populations. Markers closely linked with the Pi9 resistance genes were used to check the corresponding Pi9 allele genes among the above BC1F1 populations. Six plants with the target Pi9 allele gene from each BC1F1 populations were selected and genetic background of these BC1F1 were profiled by RICE6K (with 5102 SNP and InDel markers), a whole-genome single nucleotide polymorphism (SNP) array (Yu et al. 2014). Only one plant with the target Pi9 allele gene and background closest to J23 was selected to backcross with J23B up to BC2F1. Similarly, BC3F1 was obtained from BC2F1 populations by backcrossing the plant with the target gene and the background closest to J23B. After selfing, the BC1F2, BC2F2 and BC3F2 populations were obtained, which were then used to evaluate the effects of individual Pi9 alleles genes in J23B backgrounds.

Scoring rice blast

The BC3F2 families and control varieties were planted in a randomized complete block design in 2017 in Enshi and Yichang, Hubei Province, China. Enshi and Yichang are both mountainous areas, showing high humidity and heavy fog annually. The tests were performed in three replications. In each replication, each of the plots consisted of 4 rows with 6 plants per row at a planting density of 16 cm between plants and 16 cm between rows. To adequately induce blast disease infection, LTH was planted at both sides of each row and around the population. Field management essentially followed normal agricultural practices with the exception of not using bactericides.

All the plants were scored for leaf blast severity at the tillering stage and for neck blast severity at maturity stage using the HS-HR (HS, S, MS, MR, R and HR) scale rating system from IRRI (2002). The most seriously infected leaf among the top two or three new leaves was scored for each plant at the tillering stage as leaf blast rate. And the percentage of the infection on the neck was scored for each plant at physiological maturity stage as neck blast rate.

Results

Selection of varieties resistant to Magnaporthe oryzae

We collected 1883 rice varieties originating from regions across China, including 729 farm-cultivated varieties, 485 varieties with core germplasm resources, 514 japonica varieties from northern China, and 155 varieties from other sources (Fig. 1). Among these varieties, 72.7% are indica and 27.3% are japonica.

Evaluate the blast resistance of these varieties, plants were grown at Enshi and Yichang, Hubei Province, where test nurserie have been established to evaluate the blast resistance of rice varieties in regional trials. We classified the disease resistance of cultivar into six categories: HR, R, MR, MS, S, and HS, according to formula: disease resistance of cultivar = Leaf blast grade × 0.25 + Disease grade of ear blast × 0.25 + Loss rate of ear blast × 0.5. When the disease resistance rate of variety is less than 0.1, it is determined as HR; and when it is between 0.1 and 2.0, it is called R. We identified 361 varieties that displayed HR or R resistance phenotypes in Enshi or Yichang (Fig. 1). A PCR-based screen for the presence of Pi2, Pi9, Pigm, or Piz-t (Table S1; Table S2) identified 107 varieties as candidates for allele mining,, including 31 varieties for Pi2, 4 varieties for Pi9, 6 varieties for Pigm and 18 varieties for Piz-t (Table S1).

Isolation of Pi9 alleles

Pi9 genomic sequences of about 2 kb, including the promoters and full-length coding regions, were amplified from all 107 resistant varieties. Based on our analysis of these sequences, we identified 13 Pi9 alleles from these 107 varieties. The obtained sequences were compared with the reported Pi9 gene sequence specifically in the coding region. The 13 new alleles are novel and contain unique SNPs, insertions, and deletions. Figure 1 diagrams the sequence alignment of the identified Pi9 alleles. Among the 107 varieties, 4 varieties as honor of Pi9 gene (Table S2). The Pi9-Type5 allele was the most widespread Pi9 allele, appearing in 56 rice varieties. Moreover, the donors of Pi2 and Piz-t genes all had the Pi9-5 allele at this locus; therefore, Pi9-Type5 is considered to be the allele of Pi2 or Piz-t genes at this locus. Similarly, the donor of the Pigm gene had the Pi9-Type4 allele at this locus, which was detected in eight rice varieties (Table S2).

Sequence analysis of the Pi9 alleles

Among the 13 Pi9 alleles identified, Pi9-Type08 had the lowest level of amino acid sequence identity (92%) with the reference allele Pi9 and five alleles had more than 99% identity (Pi9-Type1, Pi9-Type5, Pi9-Type6, Pi9-Type8, Pi9-Type10, Pi9-Type12 and Pi9-Type13). These 13 alleles differed from Pi9 by many nucleotide polymorphisms, insertions and deletions, either uniquely or shared among the different alleles.

The alleles include several large insertions/deletions in the nucleotide sequence between − 728 and 2844, which encompasses the promoter region from about 728 bp upstream of the start codon, the first exon, and part of the first intron (Fig. 1).

Two alleles, Pi9-type10 and Pi9-type11, have insertions at different positions in the promoter region (Fig. 2, Table 1). In the first intron, we identified three large insertion regions located at 149–514 bp, 1099–1234 bp, and 2796–2844 bp. The insertions at 14–514 bp and 2796–2844 bp were present in only one allele each: Pi9-Type08 has the insertion at 149–514 bp and Pi9-Type09 has the insertion at 2796–2844 bp. The insertion at 1099–1234 bp was present in Pi9-Type02, Pi9-Type04, Pi9-Type05, Pi9-Type08, and Pi9-Type09. All Pi9 alleles have conserved CC and NBS domains, suggesting that these domains are important for Pi9 function. By contrast, the LRR domain is a highly polymorphic SNP-rich region. The allelic variation in the LRR domain indicates that this region is under less selective pressure than the other domains (Fig. 1; Table 2). We also performed nucleotide polymorphism analyses for all thirteen Pi9 alleles by DnaSP5.10. The average nucleotide diversity (π) of the alleles was 0.01674. Sliding window analysis of Pi9 allele nucleotide diversity showed that the diversity rate was higher in regions with abundant nucleotide polymorphisms and that there were more deletion/insertion in the first intron than elsewhere in the allele (Fig. 2; Table 2). The D test value of Tajima was less than 1 (-0.64099), indicating that the Pi9 locus was under positive selection, especially the conservative domains of CC and NBS (Fig. 2; Table 2).

Analysis of the derived Pi9 protein sequences

Pi9 proteins are composed of three conserved domains: CC, NBS and LRR. Eight of the new alleles (Pi9-Type1, Pi9-Type5, Pi9-Type6, Pi9-Type8, Pi9-Type10, Pi9-Type12 and Pi9-Type13) have complete open reading frames (ORFs) similar to that of Pi9. The remaining six alleles (Pi9-Type2, Pi9-Type3, Pi9-Type4, Pi9-Type7, Pi9-Type9 and Pi9-Type11) appear to have shorter ORFs due to sequence changes resulting in early termination codon. Among these six alleles, Pi9-Type2, Pi9-Type3, Pi9-Type4, and Pi9-Type7 have an SNP deletion in the CC domain that changes the reading frame, such that the translated protein has only 100 amino acids. Pi9-Type9 and Pi9-Type11 have some insertions/deletions in the LRR domain, which make the translated protein shorter than that of Pi9 (Table 1).

Phylogeny and distribution of the new Pi9 alleles in rice subspecies

In addition to the cloned Pi9 gene, we identified thirteen Pi9 alleles in this chromosomal region.. The reference allele Pi9 from the donor 75-1-127 is widespread among indica varieties and was also identified in somejaponica varieties through genomic breeding. The presence of other Pi9 alleles varied among rice subspecies. Three of the thirteen new Pi9 alleles (i.e., Pi9-Type01, Pi9-Type06 and Pi9-Type12), were found in only one rice line each. The donor of Pi9-Type06 is japonica subspecies and the other two alleles are derived from indica subspecies. Pi9-Type02, Pi9-Type09 and Pi9-Type11 are present only in the indica subspecies, whereas Pi9-Type0 and Pi9-Type13 exist only in the japonica subspecies. However, the remaining alleles are distributed in both indica and japonica (Fig. 3).

To establish the genetic relatedness among the Pi9 alleles, we analyzed the phylogeny of the thirteen new alleles and the cloned Pi9 reference gene. A phylogenetic tree was constructed using nucleotide sequences that includedthe complete ORF and 840 bp of the promoter sequence upstream of the start codon. Three major clusters were observed. Among them, cluster I and cluster III were composed of Pi9 alleles from indica rice, while cluster II was divided into three clear sub-clusters (Fig. 3). Only the Pi9 alleles in cluster II-subgroup III, including Pi9-Type06 and Pi9-Type13, were derived from japonica rice. The Pi9 alleles in the other two subgroups of cluster II are distributed between both indica and japonica.

Four established broad-spectrum R genes, Pi9, Pi2, Piz-t, and Pigm, are located in the same ~ 10.38-Mb region on the short arm of chromosome 6. The thirteen new alleles of Pi9 were sequenced using sequencing primers for Pi2, Piz-t and Pigm. The donor of the Pi2 gene is the Pi9-Type05 allele, the donor of the Pigm gene is the Pi9-Type04 allele, and the donor of the Piz-t is either the Pi9-type05 or Pi9-type08. Due to genomic breeding, these Pi9 alleles are widely present in both indica and japonica varieties.

Evaluation of blast resistance with Magnaporthe oryzae

Leaf blast resistance of the 13 allele of Pi9 donors and their Pi2, Pi9, Pigm and Piz-t donors were assessed in green house using 34 isolates of Magnaporthe oryzae from Hubei, Jiangxi, Hunan, Fujian and Guangdong provinces, China. The donors of Pigm, Pi2, Pi9 and Piz-t genes are Gumei4, C101A51, 75-1-127 and Dianyu1, which showed broad-spectrum resistance to rice blast with resistance frequencies ranging 58.8 to 94.1%, The resistance frequencies of donors of the Pi9 allele ranged from 23.5 to 100%. The donor GD-1S, (containing Pi9-Type5 allele) and the donor THAVALU (containing Pi9-Type9 allele) were resistant to all 34 blast isolates with a resistance frequency of 100%, an even higher resistance frequency than GM4H. The donors YD4038 and ZWH210, containing the Pi9-Type6 and Pi9-Type10 allele, were resistant to more than 30 of 34 blast isolates with a resistance frequency more than 91.2% (Table 3; Table S3).

Evaluation of blast resistance by field test

Six Pi9 allele genes with a resistance ratio greater than 85% (Pi9-Type3, Pi9-Type5, Pi9-Type6, Pi9-Type9, Pi9-Type10, Pi9-Type11) and three cloned genes (Pigm, Pi2 and Pi9) were introduced into recurrent parent J23B. The resistance to leaf blast and neck blast of Pi9 allele genes and three cloned genes introduced lines were tested in 2017 in Enshi and Yichang. Among the three cloned genes, Pigm showed greatest resistance to both leaf blast and neck blast in the background of J23B or donor parent, at Enshi or Yichang (Table 4). Like cloned genes, among six Pi9 allele genes, Pi9-Type6, Pi9-Type10 and Pi9-Type11 showed significantly enhanced resistance to leaf blast and neck blast in the background of J23B than the control, recurrent parent J23B, at Enshi and Yichang (Table 4). Pi9-Type3, Pi9-Type5 and Pi9-Type9 showed significantly enhanced resistance to leaf blast at Enshi and significantly enhanced resistance to neck blast at Yichang (Table 4). Pi9-Type3 showed significantly enhanced resistance to neck blast at Enshi. All of the Pi9 allele genes showed enhanced resistance to leaf or neck blast at least at one place. Moreover, the Pi9 allele genes of Pi9-Type6 and Pi9-Type11 were greater resistance to both leaf blast and neck blast than cloned gene Pi2 or Piz-t (Table 4).

Discussion

Current Status of Cloning and Evolutionary Analysis of Rice Blast R Genes

Identifying and cloning novel broad-spectrum blast-R genes is critical for breeding resistant rice varieties and has been a major focus of rice genome research. With the development of molecular marker technology, the construction of a high-density genetic linkage map and the improvement of related molecular techniques, great progress has been made in the cloning of rice blast R genes (Chen et al., 2006; Hayashi and Yoshida, 2009; Hittalmai et al., 2000; Lin et al, 2007; Sharma et al., 2005). Since Sasaki first presented the theory of rice blast resistance genes in 1922, initiating nearly 100 years of discovery and utilization of these genes in rice breeding. In 1966, Yamasaki cloned the rice blast R genes Pia, Pii and Pik (Yamasaki and Kiyosawa, 1966) from Aichi Asahi, Ishikari Shiroke and Kanto 51. In 1999, Wang cloned the R gene Pib (Wang et al., 1999) by map-based cloning. With improvements in technologies in the 21st century, the cloning and exploitation of rice blast R genes has seen explosive development To date, 119 blast R genes and more than 400 quantitative trait loci (QTLs) have been mapped, and more than 30 blast resistance genes have been mapped and cloned (Bryan et al., 2000; Chen et al., 2018; Chen et al., 2006; Chen et al., 2018; Chujo et al., 2014; Fukuoka et al., 2009; Inoue et al., 2010; Hayashi et al., 2010; Hayae et al., 2017; Ishihara et al., 2014; Jia et al., 2011; Li et al., 2017; Lin et al., 2007; Liu et al., 2002; Liu et al., 2007; Zhai et al., 2011; Zhao et al., 2018).

Most blast R genes, except for a few genes such as pi21, Pid2, Pid3, and Ptr, encode nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins (Chen et al., 2006; Fukuoka et al., 2009; Liu et al., 2007; Xu et al., 2014). Analysis of the cloned blast R genes revealed that most of the broad-spectrum R genes are located in tandemly repeated gene clusters on chromosomes 6, 9, 11 and 12. Generally, rice blast genes that have an NBS-LRR structure and occur in tandem repeat regions have broad-spectrum resistance. The structures of tandem repeat regions differ greatly in different rice varieties, harboring various inversions and deletions. Furthermore, many R genes with highly similar structures are tandemly duplicated with pseudogenes (Dai et al., 2010).

The short arm of chromosome 6 contains at least 10 blast R genes (Pigm, Pi2, Pi9, Piz-t, Piz, Pi22, Pi25, Pi26, Pi40, Pi42) in tandem repeats. Pi2 was cloned from the variety "C101 A51" and encodes an NBS-LRR protein composed of 1032 amino acids (Hittalmani et al., 2000; Zhou et al., 2006). Pi9 is highly similar to Pi2 but has a different resistance profile (Queet al., 2006). Puiz-t, derived from Xiushui 209, differs from Pi2 at eight amino acids (Fig. 1) due to differences at more than 20 bases in the coding regions of the two genes. Pigm is an unusual R gene formed by one copy each of PigmR and PigmS in series. PigmR imparts broad-spectrum disease resistance but reduces yield. PigmS does not confer resistance but improves seed setting rate. Because of its high concentration of R genes, we focused our analysis on this region of chromosome 6 and found 13 new alleles.

Current Situation of Molecular Breeding of Rice Blast Resistance Genes

Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most important rice diseases in the world (Ashkani et al., 2015; Shen et al., 2004). Rice blast has been reported in almost all rice-producing areas in the world, including the main rice-producing areas of 85 countries and regions (Mgonja et al., 2017; Miah et al., 2013; Zhu et al., 2000). High temperatures and humidity favor the development of rice blast. Rice blast is an acute and destructive disease that can reduce yield or even wipe out an entire harvest. Grain blast also affects the quality of rice and is a serious food safety problem (Miah et a., 2013).

In China, the disease affects more than 3.8 million hectares per year, reducing rice yield by 1 billion kg per year (Deng et al., 2017). At present, fungicides are the main method used to control rice diseases in rice production. From 1980 to 1990, the area requiring rice blast control in China increased from 2.4 million hectares to 8.9 million hectares (Shen et al., 2004). Over the past 20 years, the use of pesticides in China has increased each year. Although chemical control can reduce diseases and yield losses to a certain extent, the use of chemicals is a burden to farmers and pollutes the environment (Mi et al., 2018). Therefore, the best way to prevent rice blast is to breed blast-resistant rice varieties (Wu et al., 2016; Zhou et al., 2018).

Blast R genes (Mi et al., 2018; Zhou et al., 2018) can be introduced into high-quality susceptible rice varieties by molecular marker-assisted selection (MAS). Locating and cloning genes with broad-spectrum resistance to rice blast is of great significance for rice blast resistance breeding at this stage. In order to identify the resistance spectrum of the cloned rice blast resistance genes, we used the donor materials "Tetep", "TSUYUAKE", "Gumei No. 4", "C101 A51", "75-1-127", "Xiushui 209", "RIL260", "Jin23B", "Jin23B", "Tetep", "K59" corresponding to the genes Pi1, Pikm, Pigm, Pi2, Pi9, Piz-t, Pi5, Pid2, Pid3, Pikh, Pit, Pita, Pib and pi21. The donor varieties containing Pigm and Pi9 genes had the best resistance to 94.1% and 91.4% of 34 monospores. Donor varieties containing Pi2 and Piz-t genes could resist most of the monospores of rice blast in northern China (Jiang et al., 2015; Tian et al., 2016), while the donor varieties containing Pi1 and Pikm genes showed better resistance to monospores from southern China. Non-tandemly repeated blast R genes were not associated with broad-spectrum resistance.

Because the physiological races of rice blast pathogen are highly variable and change rapidly, any gene conferring resistance to a single race is easily overcome The breakdown of resistance can be avoided by developing rice varieties with a large number of broad-spectrum R genes associated with strong resistance. This is of great importancefor breeding disease-resistant rice varieties and preventing rice blast.

In this study, we examined more than 1883 rice germplasm resources in China. We screened 107 rice varieties with broad-spectrum resistance to rice blast and sequenced their Pi9 alleles. Seventeen alleles of Pi9 were obtained, including Pi9, Pigm, Piz-t, and Pi2. Although these alleles were isolated from rice germplasm resources that showed resistance to rice blast in our own tests, the resistance in these varieties could also be influenced by the presence of other major R genes and QTLs. To exclude this possibility, we used a BSA strategy to identify plants in the F2 populations and BC3F2 near-isogenic lines that were resistant to rice blast. After further functional testing, such as by complementation experiments or gene silencing, the new broad-spectrum resistance alleles and previously cloned rice blast R genes can be used in molecular marker-assisted breeding of different gene combinations to improve the durability of rice blast resistance.

Declarations

Acknowledgements

We are very grateful to Professor Fasong Zhou in Greenfafa company, Professor Sibin Yu and Professor Yongzhong Xing in Huazhong Agricultural University, Professor Gonghao Jiang in Heilongjiang University for providing seeds for the donor parents of the blast resistance cultivars.

Funding

This work was supported by grants from the National Nature Science Foundation of China (Grant No. 31772154 and 31872811).

Availability of data and materials

The data sets supporting the results of this article are included within the article and its supporting files.

Authors contributions

YZ and WY designed the experiments. YZ, FL, QW and WH performed the experiments. BY and YZ helped with field management. YZ and FL analyzed the data. YZ and WY wrote the manuscript. All authors approved the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests

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  7. Dai L, Wu J, Li X, Wang X, Liu X, Jantasuriyarat C, Kudrna D, Yu Y, Wing R, Han B, Zhou B, Wang G (2010) Genomic structure and evolution of the Pi2/9 locus in wild rice species. Theoretical and Applied Genetics 121:295-309 
  8. Deng Y, Zhai K, Xie Z, Yang D, Zhu X, Liu J, Wang X, Qin P, Yang Y, Zhang G, Li Q, Zhang J, Wu S, Milazzo J, Mao B, Wang E, Xie H, Tharreau D, He Z (2017) Epigenetic regulation of antagonistic receptors confers rice blast resistance with yield balance. Science 355:962-965
  9. Fukuoka S, Saka N, Koga H, Ono K, Shimizu T, Ebana K, Hayashi N, Takahashi A, Hirochika H, Okuno K, Yano M (2009) Loss of Function of a Proline-Containing Protein Confers Durable Disease Resistance in Rice. Science 325:998-1001
  10. Hayashi K, Yoshida H (2009) Refunctionalization of the ancient rice blast diseaseresistance gene Pit by the recruitment of aretrotransposon as a promoter. The Plant Journal 57:413-425
  11. Hayashi N, Inoue H, Kato T, Funao T, Shirota M, Shimizu T, Kanamori H, Yamane H, Hayano-Saito Y, Matsumoto T, Yano M, Takatsuji H (2010) Durable panicle blast-resistance gene Pb1 encodes an atypical CC-NBS-LRR protein and was generated by acquiring a promoter through local genome duplication. The Plant Journal 64:498-510
  12. Hittalmani S, Parco A, Mew T, Zeigler R, Huang N (2000) Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice. Theoretical and Applied Genetics 100:1121-1128
  13. Hua L, Wu J, Chen C, Wu W, He X, Lin F, Wang L, Ashikawa I, Matsumoto T, Wang L, Pan Q (2012) The isolation of Pi1, an allele at the Pik locus which confers broad spectrum resistance to rice blast. Theoretical and Applied Genetics 125:1047-1055
  14. IRRI (2002) Standard Evaluation System for Rice. IRRI, Manila
  15. Inoue H, Nakamura M, Mizubayashi T, Takahashi A, Sugano S, Fukuoka S, Hayashi N (2017) Panicle blast 1 (Pb1) resistance is dependent on at least four QTLs in the rice genome. Rice 10:36
  16. Ishihara T, Hayano-Saito Y, Oide S, Ebana K, La N, Hayashi K, Wang L, Ashikawa I, Matsumoto T, Wang L, Koizumi S (2014) Quantitative trait locus analysis of resistance to panicle blast in the rice cultivar Miyazakimochi. Rice 7:2
  17. Jiang J, Mou T, Yu H and Zhou F (2015) Molecular breeding of thermo-sensitive genic male sterile (TGMS) lines of rice for blast resistance using Pi2 Rice 8:11
  18. Jiang N, Li Z, Wu J, Wang Y, Wu L, Wang S, Wang D, Wen T, Liang Y, Sun P, Liu J, Dai L, Wang Z, Wang C, Luo M, Liu X, Wang G (2012) Molecular mapping of the Pi2/9 allelic gene Pi2-2 conferring broad-spectrum resistance to Magnaporthe oryzae in the rice cultivar Jefferson. Rice 5:29
  19. Lee S, Song M, Seo Y, Kim H, Ko S, Cao P, Suh J, Yi G, Roh J, Lee S, An G, Hahn T, Wang G, Ronald P, Jeon J (2009) Rice Pi5-mediated resistance to Magnaporthe oryzae requires the presence of two Coiled-Coil-Nucleotide-Binding-Leucine-Rich Repeat Genes. Genetics 181: 1627-1638
  20. Li W, Zhu Z, Chern M, Yin J, Yang C, Ran L, Cheng M, He M, Wang K, Wang J, Zhou X, Zhu X, Chen Z, Wang J, Zhao W, Ma B, Qin P, Chen W, Wang Y, Liu J, Wang W, Wu X, Li P, Wang J, Zhu L, Li S, Chen X (2017) A Natural Allele of a Transcription Factor in Rice Confers Broad-Spectrum Blast Resistance. Cell 170:114-126
  21. Librado P, Rozas J (2009) DnaSP: v5 A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451-1452
  22. Lin F, Chen S, Que Z, Wang L, Liu X, Pan Q (2007) The Blast Resistance Gene Pi37 Encodes a Nucleotide Binding Site-Leucine-Rich Repeat Protein and Is a Member of a Resistance Gene Cluster on Rice Chromosome 1. Genetics 177:1871-1880
  23. Liu G, Lu G, Zeng L, Wang G (2002) Two broad-spectrum blast resistance genes, Pi9(t) and Pi2(t), are physically linked on rice chromosome 6. Mol Genet Genomics 267: 472-480
  24. Liu X, Lin F, Wang L, Pan Q (2007) The in Silico Map-Based Cloning of Pi36, a Rice Coiled-Coil-Nucleotide-Binding Site-Leucine-Rich Repeat Gene That Confers Race-Specific Resistance to the Blast Fungus. Genetics 176:2541-2549
  25. Miah G, Rafii M, Ismail M, Puteh A, Rahim H, Asfaliza R, Latif M (2013) Blast resistance in rice: a review of conventional breeding to molecular approaches. Molecular biology reports 40:2369-2388
  26. Mi J, Yang D, Chen Y, Jiang J, Mou H, Huang J, Ouyang Y, Mou T (2018) Accelerated molecular breeding of a novel P/TGMS line with broad-spectrum resistance to rice blast and bacterial blight in two-line hybrid rice. Rice 11:11
  27. Qu S, Liu G, Zhou B, Bellizzi M, Zeng L, Dai L, Han B, Wang G (2006) The Broad-Spectrum Blast Resistance Gene Pi9 Encodes an NBS-LRR Protein and is a Member of a Multigene Family in Rice. Genetics 172:1901-1914
  28. Sharma T, Madhav M, Singh B, Shanker P, Jana T, Dalal V, Pandit A, Singh A, GaikwadK, Upreti H, Singh N (2005) High-resolution mapping, cloning and molecular characterization of the Pi-kh gene of rice, which confers resistance to Magnaporthe grisea. Molecular Genetics and Genomics 274: 569-578
  29. Shen M, Lin J (2004) The economic impact of rice blast disease in China. Pages 321-331 in: Rice Blast Disease. R. S. Zeigler, S. A. Leong, and P. S. Teng, eds. CAB International/IRRI, Wallingford, U.K.
  30. Tian D, Chen Z, Chen Z, Zhou Y, Wang Z, Wang F, Chen S (2016) Allele-specific marker-based assessment revealed that the rice blast resistance genes Pi2 and Pi9 have not been widely deployed in Chinese indica rice cultivars. Rice 9:19
  31. Wang G, Valent B (2017) Durable resistance to rice blast. Science 355:906-907
  32. Wang Z, Yano M, Yamanouchi U, Iwamoto M, Monna L, Hayasaka H, Katayose Y, Sasaki T (1999) The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes. The Plant Journal 19:55-64
  33. Wu K, Xu T, Guo C, Zhang X, Yang S (2012) Heterogeneous evolutionary rates of Pi2/9 homologs in rice. BMC Genet 13:73
  34. Wu Y, Yu L, Pan C, Dai Z, Li Y, Xiao N, Zhang X, Ji H, Huang N, Zhao B, Zhou C, Liu G, Liu X, Pan X, Liang C, Li A (2016) Development of near-isogenic lines with different alleles of Piz locus and analysis of their breeding effect under Yangdao 6 background. Molecular Breeding 36(2):12
  35. Xiao N, Wu YY, Pan CH, Yu L, Chen Y, Liu GQ, Li YH, Zhang XX, Wang ZP, Dai ZY, Liang CZ, Li AH (2017) Improving of rice blast resistances in japonica by pyramiding major R Genes. Front Plant Sci 7:1918
  36. Xu X, Lv Q, Shang J, Pang Z, Zhou Z, Wang J, Jiang G, Tao Y, Xu Q, Li X, Zhao Z, Li S, Xu J, Zhu L (2014) Excavation of Pid3 Orthologs with Differential Resistance Spectra to Magnaporthe oryzae in Rice Resource. Plos One 9:3(e93275)
  1. Yamasaki Y, Kiyosawa S (1966) Studies on inheritance of resistance of rice varieties to blast I. Inheritance of resistance of Japanese varieties to several strains of the fungus (in Jappanese). Bull Natl Inst Agric Sci D 14:39-69
  2. Zhao H, Wang X, Jia Y, Minkenberg B, Wheatley M, Fan J, Jia M, Famoso A, Edwards J, Yeshi W, Valent B, Wang G, Yang Y (2018) The rice blast resistance gene Ptrencodes an atypical protein required for broad-spectrum disease resistance. Nature Communications 9: 2039 
  3. Zhou B, Qu S, Liu G, Dolan M, Sakai H, Lu G, Bellizzi M, Wang G (2006) The eight amino-acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea. Molecular Plant-Microbe Interactions 19:1216-1228
  4. Zhou X, Jiang G, Yang L, Qiu L, He P, Nong C, Wang Y, He Y, Xing Y (2018) Gene diagnosis and targeted breeding for blast-resistant Kongyu 131 without changing regional adaptability. Journal of Genetics and Genomics 45:539-547
  5. Zhu YY, Chen HR, Fan JH, Wang YY, Li Y, Chen JB, Fan JX, Yang SS, Hu LP, Leung H, Mew TW, Teng PS, Wang ZH, Mundt CC (2000) Genetic diversity and disease control in rice. Nature 406(6797):718-722
  6. Wu S, Milazzo J, Mao B, Wang E, Xie H, Tharreau D, He Z (2017) Epigenetic regulation of antagonistic receptors confers rice blast resistance with yield balance. Science 355:962-965
  7. Fukuoka S, Saka N, Koga H, Ono K, Shimizu T, Ebana K, Hayashi N, Takahashi A, Hirochika H, Okuno K, Yano M (2009) Loss of Function of a Proline-Containing Protein Confers Durable Disease Resistance in Rice. Science 325:998-1001
  8. Hayashi K, Yoshida H (2009) Refunctionalization of the ancient rice blast diseaseresistance gene Pit by the recruitment of aretrotransposon as a promoter. The Plant Journal 57:413-425
  9. Hayashi N, Inoue H, Kato T, Funao T, Shirota M, Shimizu T, Kanamori H, Yamane H, Hayano-Saito Y, Matsumoto T, Yano M, Takatsuji H (2010) Durable panicle blast-resistance gene Pb1 encodes an atypical CC-NBS-LRR protein and was generated by acquiring a promoter through local genome duplication. The Plant Journal 64:498-510
  10. Hittalmani S, Parco A, Mew T, Zeigler R, Huang N (2000) Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in rice. Theoretical and Applied Genetics 100:1121-1128
  11. Hua L, Wu J, Chen C, Wu W, He X, Lin F, Wang L, Ashikawa I, Matsumoto T, Wang L, Pan Q (2002) The isolation of Pi1, an allele at the Pik locus which confers broad spectrum resistance to rice blast. Theoretical and Applied Genetics 125:1047-1055
  12. IRRI (2002) Standard Evaluation System for Rice. IRRI, Manila
  13. Inoue H, Nakamura M, Mizubayashi T, Takahashi A, Sugano S, Fukuoka S, Hayashi N (2017) Panicle blast 1 (Pb1) resistance is dependent on at least four QTLs in the rice genome. Rice 10:36
  14. Ishihara T, Hayano-Saito Y, Oide S, Ebana K, La N, Hayashi K, Wang L, Ashikawa I, Matsumoto T, Wang L, Koizumi S (2014) Quantitative trait locus analysis of resistance to panicle blast in the rice cultivar Miyazakimochi. Rice 7:2
  15. Jiang J, Mou T, Yu H, Zhou F (2015)Molecular breeding of thermo-sensitive genic male sterile (TGMS) lines of rice for blast resistance using Pi2 Rice 8:11
  16. Jiang N, Li Z, Wu J, Wang Y, Wu L, Wang S, Wang D, Wen T, Liang Y, Sun P, Liu J, Dai L, Wang Z, Wang C, Luo M, Liu X, Wang G (2012) Molecular mapping of the Pi2/9 allelic gene Pi2-2 conferring broad-spectrum resistance to Magnaporthe oryzae in the rice cultivar Jefferson. Rice 5:29
  17. Lee S, Song M, Seo Y, Kim H, Ko S, Cao P, Suh J, Yi G, Roh J, Lee S, An G, Hahn T, Wang G, Ronald P, Jeon J (2009) Rice Pi5-mediated resistance to Magnaporthe oryzae requires the presence of two Coiled-Coil-Nucleotide- Binding-Leucine-Rich Repeat Genes. Genetics 181: 1627-1638
  18. Li W, Zhu Z, Chern M, Yin J, Yang C, Ran L, Cheng M, He M, Wang K, Wang J, Zhou X, Zhu X, Chen Z, Wang J, Zhao W, Ma B, Qin P, Chen W, Wang Y, Liu J, Wang W, Wu X, Li P, Wang J, Zhu L, Li S, Chen X (2017) A Natural Allele of a Transcription Factor in Rice Confers Broad-Spectrum Blast Resistance. Cell 170:114-126
  19. Librado P, Rozas J (2009) DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451-1452
  20. Lin F, Chen S, Que Z, Wang L, Liu X, Pan Q (2007) The Blast Resistance Gene Pi37 Encodes a Nucleotide Binding Site-Leucine-Rich Repeat Protein and Is a Member of a Resistance Gene Cluster on Rice Chromosome 1. Genetics 177:1871-1880
  21. Liu G, Lu G, Zeng L, Wang G (2002) Two broad-spectrum blast resistance genes, Pi9(t) and Pi2(t), are physically linked on rice chromosome 6. Mol Genet Genomics 267: 472-480
  22. Liu X, Lin F, Wang L, Pan Q (2007) The in Silico Map-Based Cloning of Pi36, a Rice Coiled-Coil-Nucleotide-Binding Site-Leucine-Rich Repeat Gene That Confers Race-Specific Resistance to the Blast Fungus. Genetics 176:2541-2549
  23. Miah G, Rafii M, Ismail M, Puteh A, Rahim H, Asfaliza R, Latif M (2013) Blast resistance in rice: a review of conventional breeding to molecular approaches. Molecular biology reports 40:2369-2388
  24. Mi J, Yang D, Chen Y, Jiang J, Mou H, Huang J, Ouyang Y, Mou T (2018) Accelerated molecular breeding of a novel P/TGMS line with broad-spectrum resistance to rice blast and bacterial blight in two-line hybrid rice. Rice 11:11
  25. Qu S, Liu G, Zhou B, Bellizzi M, Zeng L, Dai L, Han B, Wang G (2006) The Broad-Spectrum Blast Resistance Gene Pi9 Encodes an NBS-LRR Protein and is a Member of a Multigene Family in Rice. Genetics 172:1901-1914
  26. Sharma T, Madhav M, Singh B, Shanker P, Jana T, Dalal V, Pandit A, Singh A, GaikwadK, Upreti H, Singh N (2005) High-resolution mapping, cloning and molecular characterization of the Pi-kh gene of rice, which confers resistance to Magnaporthe grisea. Molecular Genetics and Genomics 274: 569-578
  27. Shen M, Lin J (2004) The economic impact of rice blast disease in China. Pages 321-331 in: Rice Blast Disease. R. S. Zeigler, S. A. Leong, and P. S. Teng, eds. CAB International/IRRI, Wallingford, U.K.
  28. Tian D, Chen Z, Chen Z, Zhou Y, Wang Z, Wang F, Chen S (2016) Allele-specific marker-based assessment revealed that the rice blast resistance genes Pi2 and Pi9 have not been widely deployed in Chinese indica rice cultivars. Rice 9:19
  29. Wang G, Valent B (2017) Durable resistance to rice blast. Science 355:906-907
  30. Wang Z, Yano M, Yamanouchi U, Iwamoto M, Monna L, Hayasaka H, Katayose Y, Sasaki T (1999) The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes. The Plant Journal 19:55-64
  31. Wu K, Xu T, Guo C, Zhang X, Yang S (2012) Heterogeneous evolutionary rates of Pi2/9 homologs in rice. BMC Genet 13:73
  32. Wu Y, Yu L, Pan C, Dai Z, Li Y, Xiao N, Zhang X, Ji H, Huang N, Zhao B, Zhou C, Liu G, Liu X, Pan X, Liang C, Li A (2016) Development of near-isogenic lines with different alleles of Piz locus and analysis of their breeding effect under Yangdao 6 background. Molecular Breeding 36(2):12
  33. Xiao N, Wu Y, Pan C, Yu L, Chen Y, Liu G, Li Y, Zhang X, Wang Z, Dai Z, Liang C, Li A (2017) Improving of rice blast resistances in japonica by pyramiding major R Genes. Front Plant Sci 7:1918
  34. Xu X, Lv Q, Shang J, Pang Z, Zhou Z, Wang J, Jiang G, Tao Y, Xu Q, Li X, Zhao Z, Li S, Xu J, Zhu L (2014) Excavation of Pid3 Orthologs with Differential Resistance Spectra to Magnaporthe oryzae in Rice Resource. Plos One 9:3(e93275)
  35. Yamasaki Y, Kiyosawa S (1966) Studies on inheritance of resistance of rice varieties to blast I. Inheritance of resistance of Japanese varieties to several strains of the fungus (in Jappanese). Bull Natl Inst Agric Sci D 14:39-69
  36. Zhao H, Wang X, Jia Y, Minkenberg B, Wheatley M, Fan J, Jia M, Famoso A, Edwards J, Yeshi W, Valent B, Wang G, Yang Y (2018) The rice blast resistance gene Ptrencodes an atypical protein required for broad-spectrum disease resistance. Nature Communications 9: 2039 
  37. Zhou B, Qu S, Liu G, Dolan M, Sakai H, Lu G, Bellizzi M, Wang G (2006) The eight amino-acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea. Molecular Plant-Microbe Interactions 19:1216-1228
  38. Zhou X, Jiang G, Yang L, Qiu L, He P, Nong C, Wang Y, He Y, Xing Y (2018) Gene diagnosis and targeted breeding for blast-resistant Kongyu 131 without changing regional adaptability. Journal of Genetics and Genomics 45:539-547
  39. Zhu Y, Chen H, Fan J, Wang Y, Li Y, Chen J, Fan J, Yang S, Hu L, Leung H, Mew T, Teng P, Wang Z, Mundt C (2000) Genetic diversity and disease control in rice. Nature 406(6797):718-722

Tables

Table 1 Summary of SNP and different alleles of the Pi9 gene in different species

Pi9 allele

No.

Varieties

Haplotype

Identity to Pi9

Number of SNP sites

Number of SNP sites

Number of inserts/deletions

Number of inserts/deletions

Pt

Ex

In

Pt

Ex

In

Pi9

 

75-1-127

2

-

-

-

-

-

-

-

-

-

Pi9-Type01

GRP00494

Heo Trang

1

99%

79

29

47

3

17

9

0

8

Pi9-Type02

GRP00617

IR64

2

95%

443

246

94

103

259

10

0

249

Pi9-Type03

GRP01331

HC1H

13

98%

134

33

61

40

26

20

3

3

Pi9-Type04*

GRP00328

PⅡB

11

96%

410

253

56

101

266

10

0

256

Pi9-Type05*

GRP00574

XS209

56

95%

451

259

57

135

320

9

0

311

Pi9-Type06

GRP00234

YD4038

1

99%

104

25

31

48

241

9

3

12

Pi9-Type07

GRP00537

KAUKKYI ANI

2

99%

105

30

36

39

23

20

0

3

Pi9-Type08*

GRP00713

DY1H

6

92%

710

519

56

135

581

9

0

572

Pi9-Type09

GRP00499

THAVALU

1

93%

608

312

103

193

342

109

3

230

Pi9-Type10

GRP00249

ZWH210

6

97%

316

282

16

18

269

259

0

10

Pi9-Type11

GRP00077

R03138

3

96%

387

339

22

26

339

234

0

105

Pi9-Type12

GRP01188

JP-5

1

99%

32

22

4

6

9

7

0

2

Pi9-Type13

GRP01106

ZD5H

2

99%

105

26

30

49

26

9

3

14

* Pigm is contained in the donor of Pi9-Type04；Pizt is contained in the donor of Pi9-Type08 and a part of Pi9-Type05; Pi2 is contained in the donor of a part of Pi9-Type05.

Table 2 Summary of the natural variation of different Pi9 allele genes in different species

Allele

π 

θ 

Tajima’s D

CC

0.00670

0.01475

-2.23881

NBS

0.01035

0.01574

-1.49472

LRR

0.03311

0.03529

-0.27483

Promoter

0.01401

0.01823

-0.94844

Exson1

0

0

-

Intron1

0.01499

0.01665

-0.45014

Exson2

0.02137

0.02604

-0.80967

Intron2

0

0

-

Part of Exson3

0

0

-

Total

0.01674

0.01950

-0.64099


 

 

Table 3 Disease reactions of Pigm, Pi2, Pizt, Pi9 and Pi9 alleles honor plants to Magnaporthe grisea isolates.

Gene

No.

Varieties

Resistance ratio

Resistance

CK1

GRP00704

LTH

0.00%

4.82±0.39

CK2

GRP02083

J23B

0.00%

3.76±0.70

Pigm

GRP02064

GM4H

91.20%

1.50±1.02

Pi2

GRP02074

C101 A51

67.60%

2.29±1.12

Pi9

GRP02075

75-1-127

94.10%

1.62±0.60

Pizt

GRP00713

DY1H

58.80%

2.53±1.58

Pi9-Type1

GRP00494

Heo Trang

26.50%

3.09±1.26

Pi9-Type2

GRP00617

IR64

23.50%

3.38±1.04

Pi9-Type3*

GRP01331

HC1H

85.30%

1.76±1.21

Pi9-Type4-Pigm*

GRP00328

PⅡB

88.20%

1.71±1.03

Pi9-Type5*

GRP00727

GD-1S

100.00%

1.15±0.36

Pi9-Type5-Pi2

GRP00884

CT 18664-9-18-1-3-2

70.60%

1.91±1.14

Pi9-Type5-Pizt

GRP01232

ASD 18

67.60%

2.15±1.05

Pi9-Type6*

GRP00234

YD4038

91.20%

1.29±0.72

Pi9-Type7

GRP00537

KAUKKYI ANI

61.80%

2.44±1.44

Pi9-Type8-Pizt

GRP00713

DY1H

58.80%

2.53±1.58

Pi9-Type9*

GRP00499

THAVALU

100.00%

1.00±0.00

Pi9-Type10*

GRP00249

ZWH210

94.10%

1.32±0.91

Pi9-Type11*

GRP00077

R03138

88.20%

1.68±0.91

Pi9-Type12

GRP01188

JP-5

79.40%

1.68±0.94

Pi9-Type13

GRP01106

ZD5H

55.90%

2.68±1.41

*When Resistance Ratio is greater than 85% and Resistance value is less than 2, NIL of allele material is done. 

Table 4 Disease reactions of Pigm, Pi2, Pizt, Pi9 and Pi9 alleles honor plants to Magnaporthe grisea isolates.

Pi9 allele

No.

Varieties

Generation

Enshi

Yichang

Lr

Nr

Lr

Nr

CK1

GRP00704

LTH

F0

S

S

HS

S

CK2

GRP01884

J23B

F0

MS

S

HS

MS

Pigm

GRP02064

GM4H

F0

MR

R

HR

HR

Pi2

GRP02074

C101 A51

F0

R

MR

MR

MR

Pi9

GRP02075

75-1-127

F0

MR

R

HR

R

Pizt

GRP00713

DY1H

F0

MR

MR

R

MR

Pi9-Type1

GRP00494

Heo Trang

F0

HR 

R

HR

R

Pi9-Type2

GRP00617

IR64

F0

MS

MS

R

R

Pi9-Type3*

GRP01331

HC1H

F0

R

MR

R

R

Pi9-Type4-Pigm*

GRP00328

PⅡB

F0

R

R

HR

HR

Pi9-Type5*

GRP00727

GD-1S

F0

HR

HR

HR

HR

Pi9-Type5-Pi2

GRP00884

CT 18664-9-18-1-3-2

F0

MR

MR

MR

MR

Pi9-Type5-Pizt

GRP01232

ASD 18

F0

MR

MS

MR

MR

Pi9-Type6*

GRP00234

YD4038

F0

R

HR

HR

HR

Pi9-Type7

GRP00537

KAUKKYI ANI

F0

MR

HR

HR

R

Pi9-Type8-Pizt

GRP00713

DY1H

F0

S

HS

HR

HR

Pi9-Type9*

GRP00499

THAVALU

F0

HR

R

HR

R

Pi9-Type10*

GRP00249

ZWH210

F0

R

HR

HR

MR

Pi9-Type11*

GRP00077

R03138

F0

HR

HR

HR

HR

Pi9-Type12

GRP01188

JP-5

F0

R

HR

HR

MS

Pi9-Type13

GRP01106

ZD5H

F0

MR

MR

R

HS

Pigm

GRP02064

GM4H

BC3F2

MR

R

R

HR

Pi2

GRP02074

C101 A51

BC2F2

MR

MS

MR

R

Pi9

GRP02075

75-1-127

BC3F2

MR

MR

R

R

Pi9-Type3

GRP01331

HC1H

BC3F2

MR

MR

MS

R

Pi9-Type5

GRP00727

GD-1S

BC3F2

MR

MS

R

R

Pi9-Type6

GRP00234

YD4038

BC3F2

R

MR

R

R

Pi9-Type9

GRP00499

THAVALU

BC3F2

MR

MS

R

MR

Pi9-Type10

GRP00249

ZWH210

BC2F2

R

MR

R

MR

Pi9-Type11

GRP00077

R03138

BC3F2

R

MR

R

R

Additional Files

Additional file 1 :

Table S1. List of 107 rice varieties as candidates for allele mining

About 2000 varieties were grown at test nurserie of Enshi and Yichang, Hubei Province. We identified 361 varieties that displayed HR or R resistance phenotypes in Enshi or Yichang. A PCR-based screen for the presence of Pi2, Pi9, Pigm, or Piz-t identified 107 varieties as candidates for allele mining. The sequencing results of all 107 materials are shown in this table.

Table S2. Primers for sequence analysis of Pi9, Pi2, Pigm and Pizt allele genes in rice.

Among them, the alleles of Pi2, Pigm and Piz-t share a set of primers for amplification and sequencing; the alleles of Pi9 use a unique set of primers for amplification and sequencing. The amplified primers are labeled amplification in the table, and the sequenced primers are labeled sequence in the table.

Table S3. Disease reactions of Pigm, Pi2, Pizt, Pi9 and Pi9 alleles honor plants to Magnaporthe grisea isolates.

The sheet of resistance ratio and resistance shown the identification results of all materials to Magnaporthe grisea isolates. The information about of Magnaporthe grisea isolates, collected from all provinces of China, is also shown in the table. The resistance value of HR, R, MR, MS, S, showed in sheet of resistance ratio, is corresponding as 0-5 showed in sheet of resistance.