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

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.


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 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  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 broadspectrum 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.

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(Shen et al. , 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 × 10 5 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).
P C R f o r a l l e l e m i n i n g a n d b l a s t r e s i s t a n c e g e n e s  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 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 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) (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).
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  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 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) 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. 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). 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

Discussion
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.

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.   *When Resistance Ratio is greater than 85% and Resistance value is less than 2, NIL of allele material is done. 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 33 amplification and sequencing. The amplified primers are labeled amplification in the