Development of Specific DNA Markers for Detecting the Rice Blast Resistance Gene Alleles Pi2/9/z-t

Jing SU Wenjuan WANG Shen CHEN Congying WANG Jianyuan YANG Aiqing Feng Xiaoyuan ZHU

Abstract Specific molecular markers for detecting the alleles of Pi2/Pi9/Piz-t， designated as Pi2SNP， Pi9SNP and Pizt-PA， respectively， have been successfully developed through sequence comparison method. These three specific markers can distinctly distinguish target genes from others mapped on Pi2/9 locus， which facilitates the molecular marker-assistant selection and the development of pyramiding cultivars combined with other allele R genes. Additionally， 101 main cultivars and breeding parents collected from different rice-planting areas have been diagnosed with these three specific markers， and the results showed that only 2 out of the 101 cultivars carried Piz-t， but neither Pi2 nor Pi9 band had been detected. It indicates that most of the main cultivars and breeding parents in China do not carry Pi2/Pi9/Piz-t. These results provide important information for the application of Pi2/Pi9/Piz-t in the blast resistance breeding programs.

Key words Rice; Blast; Resistance gene; Molecular marker; MAS; Pi2/9 locus

Rice is one of the most important food crops in the world， and more than half of the population feed on rice as the staple food[1]. The rice blast caused by Magnapothe oryzae is one of the most serious diseases to rice production， causing serious food loss every year. In consideration of environmental protection and sustainable agricultural development， breeding and utilization of disease-resistant varieties is the safest and most effective method to control rice blast. In addition， due to the frequent pathogenic variation of rice blast physiological races， the resistance of a single resistant variety will be gradually lost after 3 to 5 years of planting[2-3]. Therefore， exploring and using broad-spectrum resistance genes is an important way to obtain durable disease-resistant varieties.

Traditional rice resistance breeding relies on the identification of resistance phenotypes， and the results of phenotypic identification are easily affected by environmental and human factors， reducing the efficiency of selection and breeding of disease-resistant varieties. The production of DNA molecular marker technology and the completion of rice whole genome sequencing work have greatly promoted the development of molecular markers based on PCR （polymerase chain reaction） technology. Its advantages such as convenience， directness， and freedom from environmental influences have attracted more and more attention to its application value and prospects. It is also used in R genes mapping and resistance breeding based on molecular marker-assistant selection （MAS） technology， thus playing an increasingly important role in work. In order to better carry out rice blast resistance breeding and improve the efficiency of resistance breeding， researchers have developed and obtained a batch of DNA molecular markers closely linked to the major resistance genes of rice blast， and introduced the target resistance genes into improved varieties combining with MAS technology[4-7]. However， molecular markers closely linked to target genes may be restricted by genetic background and genetic recombination in the application process. On the one hand， this type of molecular marker needs to detect the polymorphism of the parent in different populations. Once the resistant parent has no polymorphism， this type of marker cannot be applied. On the other hand， in the process of meiosis， there is a certain genetic distance between this type of molecular marker and the target gene. Therefore， there is still the possibility of losing the close linkage relationship with the target gene due to the exchange of chromosomes， which makes the wrong or missed selection during the target gene selection process[8-9]. As more and more rice blast resistance genes are cloned， it becomes possible to develop gene-specific molecular markers based on the internal sequences of genes. Such molecular markers are completely co-segregated with target genes， and the screening accuracy can theoretically reach 100%. Related studies have shown that there are multiple functional alleles with different resistance profiles at the same resistance locus， which are highly similar in sequence[10-14]. In gene aggregation or transgenic work， it is difficult for molecular markers linked to genes to accurately identify and screen functional alleles， while specific markers within genes can effectively solve the above problems， and improve the selection efficiency of resistance genes and the accuracy and efficiency of rice blast resistance breeding.

So far， at least 9 rice blast resistance genes （Pi2， Piz， Piz-t， Pi40， Pigm， Pi9， Pi26， Pi50， Pi2-2） have been located in the Pi2/9 gene cluster at the end of the short arm of chromosome 6 in rice. The genes present at this locus are considered to be broad-spectrum resistance genes for rice blast and have important application value in rice blast resistance breeding[15]. Among them， Pi9， Pi2 and Piz-t have been successfully cloned[10， 16]. Although some breeders have used molecular marker-assisted selection breeding to introduce resistance genes at this locus into varieties to be improved[3，5]， the molecular markers used are all developed based on gene flanking sequences， and there is a certain genetic distance from the target genes. In the work of resistance breeding， wrong selection or omission is likely to occur. CAPS （cleaved amplified polymorphic sequences） and dCAPS （derived cleaved amplified polymorphic sequences） method can simplify the detection of SNP signal to the polymorphism of specific PCR product restriction fragment length， which can be detected by agarose or polyacrylamide gel electrophoresis， thereby greatly improving the detection rate of SNP loci[17]. The purpose of this study was to find gene-specific SNPs based on the published complete gene sequences of Pi9， Pi2 and Piz-t， and convert them into gene-specific molecular markers detected by PCR technology， so as to improve the screening efficiency of Pi9， Pi2 and Piz-t in resistance breeding， allele aggregation and transgenic breeding based on MAS technology.

Materials and Methods

Rice materials

The rice materials used in this study included the genotype lines Pi9， Pi2 and Piz-t donor varieties IRBL9-W， C101A51， IRBLzt-T， and IRBLz-Fu （Piz）， rice varieties carrying other rice blast resistance genes Gumei 2 （Pi26）， Gumei 4 （Pigm） and 28 Zhan （Pi50）， hybrid rice maintainer lines and restorer lines and conventional rice varieties， as well as susceptible controls Nipponbare and Lijiang Xintuan Heigu （LTH）. The genotype lines were provided by the International Rice Research Institute， and other rice seed materials were collected by our unit.

Primer design

The complete gene sequences of Pi9， Pi2 and Piz-t were from the Gen-Bank database under the accession numbers DQ285630， DQ352453 and DQ352040， respectively. The allelic susceptibility gene sequences of Pi9， Pi2， and Piz-t on the Nipponbare genome were from NCBI （http：//www.ncbi.nlm.nih.gov/）. The sequence comparison was performed with the sub-software Seqman of DNAStar （http：//www.dnastar.com/）. The CAPS/dCAPS markers were developed through the online software dCAPS finder 2.0 （http：//helix.wustl.edu/dcaps/dcaps.html）[17]. The relevant information of each marker is shown in Table 1.

Molecular marker detection method

The total DNA of rice leaves was extracted with a new rapid plant genomic DNA extraction kit （BioTeke， cat#DP3112）. The primer sequence was synthesized by Sangon Biotech （Shanghai） Co.， Ltd. The total volume of the PCR system was 20 μl， including 2 μl of 10×PCR buffer Solution （Mg2+ Plus）， 1.6 μl of dNTPs （2.5 mmol/L， TaKaRa）， 1 μl of forward and reverse primers （10 μmol/L） each， 0.1 μl of TaKaRa TaqTM （5 U/μl）， and rice genomic DNA 50 ng/μl. The reaction procedure was as follows： 3 min at 94 ℃; 35 cycles of 30 s at 94 ℃， 30 s at 60 ℃ and 1 min at 72 ℃; and 5 min at 72 ℃. The PCR products obtained by amplification of Pi9 and Pi2 gene specific molecular markers Pi9SNP and Pi2SNP were detected by electrophoresis in 6%-8% denaturing polyacrylamide electrophoresis gel （90 V， 120 min）. The PCR products obtained by Piz-t PA were detected by 2% agarose gel electrophoresis.

Results and Analysis

Development of Pi9 gene-specific molecular markers

The complete gene sequence of Pi9 was compared with the gene sequences pi9-C101A51 and pi9-Nip from the corresponding genomic regions of rice varieties C101A51 and Nipponbare. The results of sequence alignment showed that there was a specific SNP in the first intron of Pi9 （Fig. 1）. In order to convert the SNP into a dCAPS molecular marker that could be detected by PCR technology， 23 bp upstream of the SNP was used as the forward primer Pi9SNP-F， and a reverse primer Pi9SNP-R was designed at 126 bp downstream of the SNP. A mismatched base G was introduced at the 3′ end of the forward primer （Table 1， underlined）， which constituted with the Pi9-specific SNP， a site that could be recognized by the restriction enzyme Hind III （Fig. 1）.

The primer pair Pi9SNP-F/R was used to amplify the total DNA of the donor varieties carrying Pi9 and other resistance genes identified at Pi2/9， and the amplified products were digested with restriction enzyme Hind III， while rice varieties Nipponbare and LTH were used as susceptible controls. Because the PCR products amplified from the Pi9 donor varieties could be recognized by Hind III and digested with restriction enzymes， the electrophoresis detection showed that the band was small （108 bp， Fig. 2， the second lane）. The PCR products obtained from other rice varieties could not be digested by Hind III， and the bands detected by electrophoresis were still the size of the PCR products， which was larger （126 bp， Fig. 2， lanes 3-8）. Therefore， the Pi9-specific molecular marker Pi9SNP developed in this study could clearly distinguish Pi9 from Pi2， Piz-t and other resistance genes located at this locus.

Development of Pi2 gene-specific molecular markers

The results of previous studies have shown that Pi2 and Piz-t are alleles， and there are only 8 amino acid residue differences between the two at the amino acid level， which are concentrated in the LRR region. In order to quickly find Pi2-specific SNPs， we compared the DNA sequences of Pi2， Piz-t， Pi9 and the LRR region of the susceptible alleles on Nipponbare， and found 4 Pi2-specific SNPs， creating a Hinf I recognition site （Fig. 3）.

Primer pair Pi2SNP-F/R （Table 1） was designed on the flanks of these SNPs， and the total DNA of donor varieties carrying Pi2 and other resistance genes identified at Pi2/9 locus were amplified， while using rice varieties Nipponbare and LTH as susceptible controls. The PCR products were digested with the restriction enzyme Hinf I and then detected by polyacrylamide gel electrophoresis. The PCR products amplified from the Pi2 donor varieties and could be cleaved by Hinf I because they had 3 Hinf I restriction sites， producing 4 nucleotide fragments with molecular weights of 22， 32， 173 and 235 bp， respectively. Polyacrylamide gel could clearly detect the 173 and 235 bp nucleic acid fragments （Fig. 4， lane 1）， while the PCR products obtained from the allelic donor varieties and susceptible varieties lacked a Hinf I restriction site and could only be digested to produce 3 nucleic acid fragments with molecular weights of 22， 173 and 267 bp， respectively （Fig. 4， lanes 2-9）.

Development of specific molecular markers for Piz-t gene

Through sequence alignment， no specific single SNP difference was found in Piz-t. In this study， the LRR region with a relatively concentrated SNP was selected to design the primers and these SNPs were placed at the 3′ end of the primers （Fig. 5）. By adjusting the PCR amplification conditions， a Piz-t-specific dominant molecular marker Pizt-PA was developed. This marker could specifically amplify the target fragment of Piz-t with a size of 176 bp （Fig. 6， lane 1）， but for other alleles or susceptible genes， no amplified bands could be obtained （Fig. 6， lanes 2-9）.

Verification of Pi9/Pi2/Piz-t gene-specific molecular markers

Understanding the resistance genetic background of different rice varieties can reduce the blindness of resistance breeding and improve the efficiency of resistance breeding. Therefore， it is very important to use gene-specific molecular markers to screen rice varieties for resistance genes. Pi9/Pi2/Piz-t is a rice blast resistance gene with a broad spectrum of resistance， which is suitable for introduction into resistance breeding in various rice areas. To understand the distribution of these 3 genes in the main varieties n South China， the Pi9/Pi2/Piz-t gene-specific molecular markers developed in this study were used to detect 101 rice varieties and their parents from different rice regions， including 31 restorer lines， 42 maintainer lines and 28 other main cultivated varieties. The detection results showed （shown in online auxiliary Table S1，http：//www.ricesci.cn/CN/ article/showSupportInfo.do？id=2524） that among these 101 varieties， except the donor varieties of Pi2 and Pi9， no varieties carrying Pi2 and Pi9 genes were detected. When detecting with the Piz-t dominant marker Pizt-PA， 2 restorer lines， namely Minghui 86 and Duoxi 1， could produce the specific band pattern of Piz-t by amplification. This result showed that most of the restorer lines， maintainer lines and tested main varieties did not carry the pest resistance genes Pi9， Pi2 and Piz-t （online auxiliary Table S1）.

Discussion

With the emergence of MAS technology， some breeders have used molecular markers linked to a target resistance gene to assist in the selection of rice varieties carrying the target resistance gene to achieve the resistance improvement of varieties[18-20]， which greatly improves the utilization efficiency of resistance genes in resistance breeding. However， the efficiency and accuracy of target gene screening largely depend on the closeness of linkage between the selected molecular marker and the target gene. So far， 23 rice blast resistance genes have been cloned[21-22]， providing sequence information for the development of gene-specific molecular markers. Based on these sequence information， some genes have been successfully developed， such as gene-specific molecular markers of Pit and Pi25[23-24].

In resistance breeding， accurate selection of broad-spectrum disease resistance genes can significantly improve the disease resistance of varieties[25]. However， currently reported broad-spectrum resistance gene loci have multiple alleles with different resistance spectra， and these multiple alleles are highly similar in sequence[13-14， 26-27]. Therefore， in the process of resistance breeding， accurate selection of multiple alleles with different broad-spectrum resistance genes requires the development of accurate and effective gene-specific molecular markers. For example， there are multiple functional alleles with different resistance profiles at the Pik locus， and gene-specific molecular markers that can distinguish each allele have been successfully developed at this locus[12-14]. These markers can be used not only to identify different resistance genes in rice seed resources， but also to screen different multiple alleles in the process of gene aggregation.

Pi2/Pi9 is a locus with broad-spectrum resistance to rice blast. Except for the cloned Pi9/Pi2/Piz-t， genes with broad-spectrum resistance to rice blast， which have important application value in rice resistance breeding， have been successively identified at this locus[15， 27]. The genomic region where the Pi2/Pi9 locus is located has multiple candidate genes that are highly similar in sequence， and their sequence identity at the nucleotide level ranges from 71.8% to 98.6%[16]. This study successfully developed Pi9/Pi2/Piz-t gene-specific molecular markers that could distinguish Pi9/Pi2/Piz-t from other alleles identified at Pi2/9 locus， which provides an important basis for the selection of alleles in the region. These gene-specific molecular markers were developed based on the cloned Pi9/Pi2/Piz-t gene sequence and were completely co-segregated with the target gene， which could more effectively improve the screening accuracy of Pi9/Pi2/Piz-t in the process of resistance breeding.

This study used Pi9/Pi2/Piz-t gene-specific molecular markers to carry out genetic screening of different rice resource varieties， and found that the tested maintainer， restorer and conventional rice varieties did not contain Pi2 and Pi9 blast resistance genes， and except the two restorer lines Minghui 86 and Duoxi 1 which contained the Piz-t gene， other tested parents and varieties did not carry the gene， indicating that Pi9/Pi2/Piz-t has great application potential in resistance breeding in China. Therefore， in the breeding of hybrid rice in China and conventional rice in South China， the above three disease resistance genes can be introduced to enrich the blast resistance genotypes of the selected varieties. In addition， the 101 rice varieties tested carry known or unknown rice blast resistance genes， and the Pi9/Pi2/Piz-t gene-specific molecular markers developed in this study showed clear and identifiable polymorphisms in these rice varieties with different resistance genetic backgrounds， which fully demonstrated the wide applicability of these markers， which meant that they are suitable for the detection of resistance breeding under different genetic backgrounds.

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