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.