Improving Rice Blast Resistance by Mining Broad-Spectrum Resistance Genes at Pik Locus

Zhou Ying, Wan Tao, Yuan Bin, Lei Fang, Chen Meijuan, Wang Qiong, Huang Ping, Kou Shuyan, Qiu Wenxiu, Liu Li

Research Paper

Improving Rice Blast Resistance by Mining Broad-Spectrum Resistance Genes atLocus

Zhou Ying1, #, Wan Tao1, #, Yuan Bin3, Lei Fang4, Chen Meijuan5, Wang Qiong1, Huang Ping6, Kou Shuyan6, Qiu Wenxiu1, Liu Li2

(College of Life Science and Health, Wuhan University of Science and Technology, Wuhan 430065, China; College of Life Sciences, Hubei University, Wuhan 430062, China; Key Laboratory of Integrated Management of Crops of Central China, Ministry of Agriculture and Rural Affairs / Hubei Key Laboratory of Crop Disease, Insect Pests and Weeds Control, Wuhan 430064, China; College of Information, Huazhong Agricultural University, Wuhan 430070, China; College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Institute of Food Crops, Yunnan Academy of Agriculture Sciences, Kunming 650205, China; These authors contributed equally to this work)

is known for its genetic diversity and pathogenic variability, leading to rapid breakdown of resistance in rice. Incorporating multiple broad-spectrum blast resistance genes into rice cultivars would extend disease resistance longevity. Effective resistance breeding in rice therefore requires continual enrichment of the reservoir of resistance genes and alleles. We conducted a large-scale screen of rice blast resistance in about 2 000 rice accessions. Among them, 247 accessions showed at least medium resistance to the natural infection of rice blast and 7 novelalleles were identified from them. Variations in gene sequences were then correlated with the phenotypic trait to enable the identification of favorable alleles. Among the seven novelalleles, the resistant rate ofdonors was greater than 80%, and the disease score was less than 3. Through molecular marker-assisted backcross breeding, we successfully transferred the threealleles,, into an elite cultivated line Kongyu 131 to obtain BC3F2lines, which showed enhanced resistance to rice blast compared with the recurrent parent. Assessment of these near-isogenic lines in the greenhouse using 31 isolates offrom Heilongjiang Province of China revealed that the resistant levels of the BC3F2lines withwere significantly higher than those of the established cloned resistance genesand. Exploring such alleles will enrich our gene library for resistance to rice blast.

genetic diversity; near-isogenic line;gene; resistance gene allele; rice blast; Rgene

Rice (L.) is a staple food crop that is a major food for more than 4 billion people worldwide. However, rice growth and yield are seriously affected by rice blast. About 10%?30% of annual rice harvest is lost because of the infection of rice blast fungi (Skamnioti and Gurr, 2009; Deng et al, 2017; Xie et al, 2019). Therefore, improving the resistance of rice varieties to rice blast is considered a cost-effective and eco-friendly method to increase rice yield and quality (Ashkani et al, 2015; Yadav et al, 2019). Compared with traditional chemical pesticide methods, which cause additional costs during rice production and environmental pollution problems, the application of disease resistance (R) genes is considered to be the best strategy to minimize the yield loss and quality losscaused by the disease. More than 100 blast resistance genes and about 500 QTLs have been identified and mapped (Li et al, 2019). Resistant rice varieties can be generated by transferring broad-spectrum rice blast resistance genes, identified by molecular marker- assisted selection, into susceptible genotypes (Hittalmaniet al, 2000; Miah et al, 2013; Tian et al, 2016; Wu et al, 2016; Xiao et al, 2017; Mi et al, 2018).

Plants have evolved various mechanisms that protect them from pathogen invasion and colonization. Hence, incorporating multiple broad-spectrum blast resistance genes into rice cultivars would extend disease resistance longevity. Many resistance genes located in tandem repeat regions (such as,andon the long arm of chromosome 11 and,,andon the short arm of chromosome 6), have been proven to have broad-spectrum resistance (Sharma et al, 2005; Qu et al, 2006; Ashikawa et al, 2008; Dai et al, 2010; Hua et al, 2012; Wu et al, 2012). Among them, a series of broad-spectrum disease resistance genes cloned in the tandem repeat region on the short arm of rice chromosome 6 show better resistance levels in most rice-producing areas in China. However, the broad-spectrum resistance genes, such as,and, located on the tandem repeat region on the long arm of chromosome 11, show different levels of resistance in different rice-producing regions in China. These genes show excellent resistance in the southern rice-producing areas, but present susceptibility in the northern areas. Because disease resistance longevity would be largely extended by incorporating multiple broad-spectrum blast resistance genes into rice cultivars, mining broad- spectrum disease resistance genes at thelocus is important for the northern rice-producing areas.

Allele mining of thegene from rice cultivars, core genetic resources or core germplasms and overseas varieties would not only help identify genes that impart high resistance in plants growing in the northern rice-producing areas, but also serve to identify genetic variation and provide insight into the evolution of this locus. Variations in gene sequence were then correlated with the phenotypic trait to identify favourable alleles for future applications.

The objectives of this study were to (1) amplify and sequence theallele from screened resistant accessions, (2) analyze structural variation and understand the molecular evolution ofalleles, and (3) associate the resistance observed with the presence ofalleles using BC3F2near-isogenic lines (NILs). A total of 247 accessions were selected from a collection of about 2 000 accessions from throughout China, and they are resistant to rice blast in at least one rice-growing region. Seven novelalleles were obtained by sequencing thegene from these selected accessions. We established that thealleles of//had a good resistance ratio and disease score. Blast resistance to different.isolates from Heilongjiang Province in China was greatly enhanced in an elite rice cultivar Kongyu 131 introduced with//. This research demonstrated the potential for breeding new rice varieties with broad-spectrum blast resistance.

RESULTS

Screening resistant accessions for allele mining

More than 2 000 rice accessions were collected, including 1 214 core germplasm resources (mainly in landrace) provided by Huazhong Agricultural University, 514 varieties by Heilongjiang Institute of Agricultural Sciences, and more than 200 varieties by the United States Department of Agriculture, China National Rice Research Institute and other institutions. The selected accessions represent two major subspecies,(about 75%) and(about 25%). To evaluate the blast phenotype of these accessions, we grew the rice plants in high incidence areas of rice blast, such as Enshi and Yichang in Hubei Province and Jiamusi in Heilongjiang Province of China.

According to the International Rice Research Institute (IRRI) classification criteria for rice blast, disease resistance is divided into six categories: HR (high resistance), R (resistance), MR (medium resistance), MS (medium sensitivity), S (sensitivity) and HS (high sensitivity). We identified 247 accessions that displayed resistance phenotypes (HR, R or MR) in Enshi, Yichang or Jiamusi. A PCR-based screen for the presence of,,or(Tables S1 and S2) identified 119 accessions as candidates for allele mining.

Isolation of Pik alleles by sequencing

About 13 kb of genomic sequences were amplified from 119 resistant accessions. The 13-kb fragment included the full-length coding regions of(3 229 bp) and(6 319 bp), the sequence (2 542 bp) betweenandand the 5′ UTR regions ofandBy comparing all the obtained sequences with the reported genomic sequence of, we identified 7 novelalleles from these 119 accessions. These seven alleles were identified as novelalleles because they contained unique single nucleotide polymorphisms (SNPs), insertions and deletions (InDels), and changes in the Pik-1 or Pik-2 protein sequences. Fig. 1-A shows a comparison of the identified novelallele sequence with the reportedgenomic sequence.

By analyzing the sequences of the 119 accessions of this segment, we established that,,and, located in this segment, had very different distributions among the accessions. About 13 accessions carried, 15 accessions carried, 4 accessions carriedand none carried(Tables 1 and S2). Furthermore, theallele was the most widely distributedallele and appeared in 49 rice accessions.andwere also widely distributed, appeared in 19 and 10 rice accessions, respectively. The remaining alleles were relatively less abundant and were detected under five accessions. Among them,andwere the rarest and were only detected in one accession. There were few alleles in these 119 accessions, indicating that there is selective pressure at this locus. Mining novel alleles at this locus can enrich the source of resistance genes.

Sequence analysis of Pik alleles showed Pik-2 is conserved

Among the sevenalleles identified,had the lowest level of genomic sequence identity (97.16%) with the referencegene, andhad the highest level (99.85%) (Table 1). These seven alleles differed from,,,andby SNPs and InDels, either uniquely or shared among the different alleles (Figs. S1 and S2).

includesand, which are arranged back to back (Fig. 1-A). The novel alleles ofincluded one large InDel (about 36 bp) and three small InDels (one of 9 bp and two of 3 bp). The large insertion and two deletions of 3 bp were present in, and the insertion of 9 bp was present in. The novel alleles ofonly included SNPs and without InDels. The Pik-m protein is encoded by two genes,and. The protein encoded byharbors a typical coiled coil- nucleotide binding site-leucine rich repeat (CC-NBS- LRR) disease resistance domain, whereas the Pik-2 protein has a CC-NBS domain (Fig. 1-B and -C). All newly identified alleles had complete open reading frames (ORFs), similar toand. The predicted encoded proteins of the allele were highly similar to the controls Pik-1 (94.59%–99.91%) and Pik-2 (99.51%–100.00%) protein sequences (Table 2). Compared with the protein encoded by, the protein encoded byhad a higher level of conservation. Compared with the reference Pik-m protein, the amino acid changes of the allele proteins were mainly at thesite. At thesite, the clonedandgenes encode the same amino acids with the reference sequence. Sequence analysis of thealleles showed high levels of similarity at this locus, especially at thesite, implying that this site is important for the survival of plants and therefore is subjected to positive selection.

Phylogeny and distribution of novel Pik alleles

Four established broad-spectrumR genes,,,and, are located at the end of the long arm of rice chromosome 11 at about 27.5 Mb. To test the genetic correlation between thealleles, we performed a phylogenetic analysis of the seven novel alleles and the cloned reference genes. The tandem repeat region of thelocus contained five functional loci (,,,and), thelocus contained four functional loci (,,and), and these cloned genes were used as reference genes. Phylogenetic trees were constructed using genomic sequences (Fig. 2-A) and protein sequences (Fig. 2-B). The results obtained from genomic sequences were similar to proteins (Fig. 2). The alleles at theandloci were highly similar, implying that the two loci ofmight be related to the resistance function.

Table 1. Single nucleotide polymorphisms (SNPs) and different alleles of Pik gene in various rice species.

Representative donor accession containing the allele. SNP, Single nucleotide polymorphism; InDel, Insertion and deletion.

Fig. 1. Schematic maps for sequence alignment of novelalleles, Pik-1 and Pik-2 proteins.

A, Schematic diagram of gene structure was shown above, black boxes indicate exons and white boxes indicate introns, and the start codon and the termination codon are labeled with ATG and TGA, respectively. Seven alleles ofwere isolated from the studied rice accessions and were compared withgene as shown in the middle, the unit scale indicates the location of nucleotides. Sliding-window analysis of nucleotide diversity (π) about novelalleles was shown below.

B and C, Seven alleles of Pik-1 (B) and Pik-2 proteins (C) were compared with Pik-m protein. The unit scale indicates the location of amino acids. The black line on the bar indicates the amino acid polymorphism compared with the reference sequence. The gap between allele strips indicates deletion, and the size of gap indicates the length of deletion sequence. CC, Coiled coil; NBS, Nucleotide-binding site; LRR, Leucine rich repeat.

Table 2. Summary of difference in each allele of Pikprotein.

Representative donor accession containing the alleles.Number of amino acids in Pik allele proteins. InDel, Insertion and deletion.

Screening for Pik resistance alleles in field test

Accessions carrying the seven novelalleles or three cloned genes (,and) were selected to evaluate blast resistance in a field test. The resistance of the selected accessions to leaf blast was tested in Enshi, Yichang and Jiamusi. The accessions that carriedorin Enshi and Yichang showed resistant levels above R (resistance) (Table 3). The accessions that carried,,orshowed similar results, exhibiting a significant increase in resistance to leaf blast compared to the control, and at least with medium resistance. The accessions that carrieddid not show significantly enhanced resistance to leaf blast, but showed even high sensitivity in plants grown in Hubei Province (Enshi and Yichang), and only medium resistance in Heilongjiang Province (Jiamusi). The accessions that carriedandshowed different levels of resistance in different regions. Based on the field phenotype,allele (except) donor accessions generally showed better resistance than the donor accessions with cloned-gene at this locus.

Screening for high resistance alleles using M. oryzae isolates

Leaf blast resistance analysis of the accessions that carried the seven novelallelesor the three cloned genes (,and) were assessed in the greenhouse using 31isolates from Heilongjiang Province, China. The accessions that carried,orgenes are Tsuyuake, Jiahua 1 and Tetep, respectively, which show broad-spectrum resistance to rice blast (Ashikawa et al, 2008; Hua et al, 2012). In the inoculation experiment usingisolates, although the resistant rates of donors (,and) were 77.42% to 100.00%, the resistant rates of NILs (and) were only 0.00% to 16.13% (Table 4). The resistant rates ofallele donors ranged from 25.81% to 100.00%. The accession IR70175-22-1-1-2-2 (with theallele) was resistant to all 31 blast strains with the resistant rate of 100%. The accession R03138 (with theallele) and Meixiangzhan (with theallele)shared resistant rates greater than 80%. These threealleles with resistant rates greater than 80% and four cloned genes (,,and) were introduced into a recurrent parent Kongyu 131. The resistant rates of the corresponding NILs were 29.03% to 48.34%, and the disease scores were 4.16 to 5.26, significantly better than that of the back- crossing parent Kongyu 131, which had a resistant rate of 0% and a disease score of 7.45.

Fig. 2. Phylogenetic relationship amongallele (includingand) genomic sequences (A) and protein sequences (B).

Table 3. Disease responses of cloned genes and Pik allele honor plants in field text.

Representative donor accessions containing the alleles, which were used as parents. S, Sensitivity; MS, Medium sensitivity; HS, High sensitivity; R, Resistance; MR, Medium resistance; HR, High resistance.

DISCUSSION

Most blast R genes, except for a few genes such as,,,,and, contain the NLR (nucleotide-binding domain leucine-rich repeat containing) domain (Chen et al, 2006; Liu et al, 2007; Fukuoka et al, 2009; Hayashi et al, 2010; Xu et al, 2014; Inoue et al, 2017; Chen et al, 2018; Zhao et al, 2018). Analysis of the cloned blastR genes showed that most of the broad-spectrum R genes are located in tandem repeat regions of the genome. In addition, many R genes with highly similar structures replicate in tandem with pseudogenes (Li et al, 2019). Whole genome analysis of the R genes against rice blast showed that such tandem repeat regions with the CC-NBS domain exist on chromosomes 1, 6, 9, 11 and 12 (Lin et al, 2007; Ishihara et al, 2014). Among them, thelocus with cloned genes of,,,andlocated on the short arm of chromosome 6 (Liu et al, 2002), and thelocus with cloned genes of,,andlocated on the long arm of chromosome 11, confer broad-spectrum resistance to variousisolates (Zhou et al, 2006). Thelocus is generally composed of two copies connected in series. Like most rice blast resistance genes, the disease resistance clones located at thelocus also contain the CC-NBS-LRR disease resistance conserved domain.has the complete CC-NBS-LRR structure, whilehas only the CC-NBS conserved domain (Fig. 1-B and -C). According to our analysis (Fig. 1-A), this site was significantly positively selected, which implies that this site is related to important rice functions, such as rice blast resistance. These broad- spectrum resistance genes have been transferred into sensitive rice cultivars in several studies, and the genes located in the tandem repeat region of chromosomes 6 and 11 have the broadest spectrum of resistance to rice blast (Yadav et al, 2019). Therefore, we believe that mining alleles at thelocus would greatly enrich resistance genes (Zhu et al, 2000).

Table 4. Disease responses of cloned genes and Pik allele honor plants to Magnaporthe grisea isolates.

Representative donor accession containing the allele.The proportion below five inoculated with 31isolates.The average disease score inoculated with 31isolates.

China is one of the most important rice-producing regions in Asia, and Asian rice cultivars are mainly grown in China. Asian rice cultivars include two main subspecies,and. In China,rice is mostly grown in southern regions andrice in northern regions. Rice blast is a devastating disease in all rice cultivated areas, and the majorisolates of rice blast are distinctive in different rice- producing areas. Therefore, R gene resistance varies greatly in each rice-producing area. The results of the field test of rice blast in different rice-producing regions of China aligned with this observation (Table 3).at thelocus on the short arm of chromosome 6 shows at least medium resistance in major rice-producing regions of China (Zhou et al, 2020). However, seven genes of,,,,,and, at thelocus on the long arm of chromosome 11, only show good resistance in the southern rice area (Wang et al, 2017). Our test results (Table 3) were also consistent with existing reports (Wang and Valent, 2017). Furthermore,is known for its genetic diversity and pathogenic variability, leading to rapid breakdown of resistance in rice varieties (Jiang et al, 2012; Li et al, 2019). When only a single resistance gene is transferred, a rice variety often remains effective for only a few years before new dominant pathogenic races of the fungus emerge (Lee et al, 2009; Li et al, 2017). Durable resistance to rice blast depends on simultaneously stacking at least two resistance loci into rice cultivars. Therefore, it is urgent to mine genes on the tandem repeat region of chromosome 11 that can enhance resistance in northern rice-producing areas.

In this study, we investigated the allelic diversity of, a major blast resistance gene located in the tandem repeat region of chromosome 11, conferring resistance to rice blast in the southern rice-producing areas of China. Although the cloned genes in this region, such as,and, are associated with susceptibility to rice blast in the northern rice- producing areas of China, thealleles of//showed strong resistance to rice blast in this area. Although transgenic experiments are the best way to confirm the resistance phenotype associated with the alleles of thelocus, the complex structure of tandem repeat regions at thelocus makes it very difficult. Thelocus contains a dozen or so genes with similar structures, connected in series to form a region of several hundred kilobases. These tandem repeat genes have as many as 99% identity with each other, making the transgenic verification become challenging. However, as transgenic technology cannot be directly applied to rice cultivars, a possible route to address these issues would be to construct NILs instead (Jiang et al, 2015). The resistancealleles have been introduced into rice cultivars, such as Kongyu 131, to improve the rice blast resistance level (Zhou et al, 2018).

In the present study, through molecular marker- assisted backcross (MAB) breeding, we successfully transferred threealleles,//, into an elite cultivar, Kongyu 131, to obtain BC3F2lines, which showed enhanced resistance to rice blast compared with the controls. The resistant levels of BC3F2lines of//were significantly higher than those of lines harboringand,when these NILs were assessed in the greenhouse using 31isolates from Heilongjiang Province. The NILs containing theandalleles, with a resistant frequency of more than 40%, can be used for MAB breeding. Therefore, mining for alleles of(such asand) is an important strategy for controlling rice blast in rice-producing areas of northern China, and we believe that exploring such genotypes will help enrich our gene library for resistance to rice blast.

METHODS

Selection of rice genotypes for Pik allele mining

About 2 000 rice accessions were obtained from major rice- growing provinces of China and more than 200 rice accessions from abroad were stored at Huazhong Agricultural University for this study. These rice accessions were chosen to study the allelic variation of thegene at thelocus. In two uniform rice blast nurseries in Enshi and Yichang, Hubei Province, and a uniform rice blast nursery in Jiamusi, Heilongjiang Province, China, the rice blast resistant accessions were determined by the natural induction methods. Therice variety Lijiangxintuanheigu is susceptible to rice blast and was used as the susceptible control. The rice varieties Tsuyuake (containing), Jiahua 1 (containing) and Tetep (containing) were used as the resistant controls. Based on the standard scoring system for leaf blast, 247 accessions that were resistant with a phenotypic score of MR to HR against a field mix-inoculum were selected for molecular screening. These 247 accessions were further screened for the presence ofusing the primers shown in Table S1 (PikQ1P1-F/ PikQ1P1-R for, and PikQ2P1-F/PikQ2P1-R and PikQ2P2-F/ PikQ2P2-R for), which were the functional co-dominant markers for detecting the presence of theallele. From these 247 resistant accessions, only 119 accessions can be amplified. These 119 PCR products were selected as candidates for allele mining of(Table S2).

PCR for allele mining and blast resistance genes

PCR primers were designed using rice genomic sequences ofcvs. Nipponbare and 9311 (www.ncbi.nlm.nih.gov), Zhenshan 97 and Minghui 63 (http://rice.hzau.edu.cn/rice/), the sequence ofcv. Tsuyuake (AB462256.1, containing), the BAC clone sequence ofsubsp.(HQ662329.1, containing), the sequence ofcv. K60 (HM035360, containing) and the BAC clone sequence ofcv. C101LAC (HQ606329.1, containing), corresponding to the location of the,,orgenes, respectively. These sequences correspond to the LOC_Os11g46210 sequence on TIGR (http://rice.plantbiology. msu.edu/cgi-bin/gbrowse/rice/#search).

Three sets of primers flanking to the whole genomic DNA ofandgenes were designed using the Primer 5.0 software. Genomic DNA extracted from rice leaves using the CTAB method were used as templates for PCR (Murray and Thompson, 1980). The 50 μL PCR reaction included 100 ng of template DNA, 0.2 μmol/L of both forward and reverse primers, 5 μL of 10× LABuffer II, 8 μL dNTP Mixture (2.5 mmol/L each) and 2.5 U TaKaRa LA(RR02MQ, TaKaRa, China). The details of primers are provided in Table S1. Amplified PCR products were purified and sequenced using DNA Analyzer Sequencer, ABI 3730XL (ABI, Applied Biosystems/Amersham, USA). Three PCRs were performed for each allele, and each PCR product was sequenced using sequencing primers to ensure that sequencing errors were eliminated.

DNA and protein sequences analyses

All the sequence reads generated for each allele by sequencing primers were assembled separately using ChromasPro 2.4.1 (Technelysium Pty Ltd, Australia). The cloned genes at thelocus (including,,,and) andlocus (including,,and) were used as reference sequences. The high-quality sequence assembled from each allele was analyzed against the reference sequence of thegene, using the Blast Sequences analysis (https:// blast.ncbi.nlm.nih.gov/Blast), to assess its similarity. Insertions, deletions and SNPs were counted and mapped to the DNA sequences of all alleles. Multiple sequence alignments were performed using CLUSTALW (http://www.ebi.ac.uk/Tools/ msa/clustalw2/) and a phylogenetic tree was constructed using the MEGA6.0 software (http://www.megasoftware.net). Differences were shown on the DNA and protein sequences of all alleles using customized R scripts.

Crossing and selection scheme

Seven accessions containing novelalleles were crossed with Kongyu 131, and their F1offspring were backcrossed with Kongyu 131 to obtain BC1F1populations. Markers closely linked to thealleles were used to examine the correspondingalleles in the aforementioned BC1F1population. From each BC1F1population, 24 plants with the targetallele were selected, and by background marker detection, a plant with the targetallele and the closest background to Kongyu 131 was selected to backcross with Kongyu 131 until BC2F1. BC3F1with the targetallele was obtained by backcrossing in the same way. After selfing, the obtained BC1F2, BC2F2and BC3F2populations were tested by markers to obtain homozygous seeds at thelocus, which were then used to evaluate the role of a singleallele in the Kongyu 131 background.

Scoring leaf blast severity

The control variety and the accessions carryingalleles were randomly designed in Enshi and Yichang, Hubei Province, and Jiamusi, Heilongjiang Province, China. Enshi, Yichang and Jiamusi are all regions with high humidity, resulting in a high incidence of rice blast. To fully induce explosive disease infection, arice variety Lijiangxintuanheigu (susceptible control), was planted on both sides of each row and around the population. Except for the absence of bactericides, field management essentially followed normal agricultural practices.

All the plants were scored for leaf blast severity at the tillering stage using the scale rating system from IRRI (2002). Due to the inconsistent growing stage of the varieties, only the leaf blast severity was assessed, and the neck blast severity at maturity was not assessed.

Pathogen inoculation and disease scoring

For resistance spectrum analysis, we used 31 blast isolates of different races and virulence. These isolates, which were collected from Heilongjiang Province, China, are genetically distinct and belong to different blast lineages (Shen and Lin, 2004). We used these isolates to analyze the phenotype of parents with disease resistance genes. The 31isolates, which are highly virulent on most of rice lines, were also used for phenotypic analysis of theallele. Twelve-day-old seedlings were spray-inoculated with blast spore suspensions (approximately 1 × 105spores/mL), and were then grown in a dark chamber for 24 h (26 oC, 90% humidity). Then, the growth conditions were changed to 12 h light/12 h of darkness. At 7 d post inoculation, the disease reaction of each line was recorded (IRRI, 2002).

ACKNOWLEDGEMENTS

This study was supported by the National Natural Science Foundation of China (Grant No.62176192), Key Laboratory of Integrated Management of Crops of Central China and Hubei Key Laboratory of Crop Disease, Insect Pests and Weeds Control (Grant No. 2019ZTSJJ2), and China Scholarship Council (CSC No. 201908420374). We are very grateful to Professor Zhou Fasong in Greenfafa Company, Professors YuSibin and Xing Yongzhong in Huazhong Agricultural University, Professor Jiang Gonghao in Heilongjiang University for providing seeds for the donor parents of blast resistance.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

Fig. S1. Sequences of seven novel alleles.

Fig. S2. Alignments ofwith seven novel alleles.

Table S1. Primers used in this study.

Table S2. Gene type analysis oflocus.

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3 June 2021;

16 August 2021

Liu Li (liuli2020@hubu.edu.cn); Qiu Wenxiu (656601270@qq.com); Zhou Ying (18062565621@163.com)

Copyright ? 2022, China National Rice Research Institute. Hosting by Elsevier B V

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Peer review under responsibility of China National Rice Research Institute

http://dx.doi.org/10.1016/j.rsci.2022.01.002

(Managing Editor: Wu Yawen)