Genetic Analysis and Gene Detection of Bacterial Blight Resistance in New Released Varieties Lvzhen8072 and Baixiangzhan  

Shen Chen1 , Jie Zhong1,2 , Xiaoyuan Zhu1 , Jianyuan Yang1 , Shengyuan Wu1 , Liangying Dai2 , Liexian Zeng1
1 Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, P.R. China
2 College of Biosafety Science and Technology, Hunan Agricultural University, Changsha, 410128, P.R. China
Author    Correspondence author
Rice Genomics and Genetics, 2012, Vol. 3, No. 9   doi: 10.5376/rgg.2012.03.0009
Received: 07 May, 2012    Accepted: 31 Aug., 2012    Published: 15 Oct., 2012
© 2012 BioPublisher Publishing Platform
This article was first published in Molecular Plant Breeding (2012, Vol.10, No.3, 357-362) in Chinese, and here was authorized to translate and publish the paper in English under the terms of Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Chen et al., 2012, Genetic Analysis and Gene Detection of Bacterial Blight Resistance in New Released Varieties Lvzhen8072 and Baixiangzhan, Vol.3, No.9 55-60 (doi: 10.5376/rgg.2012.03.0009)

Abstract

Bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) is one of the most serious bacterial diseases for rice all over the world. Breeding resistant varieties is one of the most economical and effective measures to control rice bacterial blight. In this research, gene identification and genetic analysis were carried out on the newly released varieties, Lvzhen8072 and Baixiangzhan, in order to provide references in rice production. The cross between Lvzhen8072indica rice cultivar Jingang30 (highly susceptible to Xoo strains) was made to generate F1 and F2 population, and then the parents and their F1 and F2 individuals were inoculated highly pathogenic GD6027, a V-type strain of bacterial blight in Guangdong Province to identify resistant phenotype. While Xa7-linked microsatellite markers were used to detect the polymorphism of the parents and F2 individuals in order to confirm the linkage between Xa7 gene and its makers among F2 individuals. The result confirmed that Lvzhen8072 harbored the resistance gene that isallelic to Xa7, which is independently inherited. In the same way, the bacterial blight resistance gene of Baixiangzhan and its genetic pattern were identified. The result showed that the resistant gene of Baixiangzhan was related to xa5 gene, but the segregation ratio of resistant to susceptible in F2 individuals didn’t match theoretical segregation radio (1:3) according to the alw of the Mendelian recessive single gene. Obviously, there was segregation distortion obviously existing in the F2 population.

 
Keywords
Rice bacterial blight; Resistance genes; Genetic analysis; Genes detection

Bacterial blight (Xanthomonas oryzae pv. Oryzae, Xoo) is the most serious bacterial diseases of rice, generally 20% to 30% yield loss, the incidence of serious plots up to 80% above, even completely lost. The disease is widely distributed in the north and south regions of rice in China, Japan and Southeast Asian countries, which is a major disaster of the diseases in rice production (Xu et al., 1994, Plant Protection, 20 (4): 7-9; Zeng et al., 1997; Pei et al., 2011), the application of resistant varieties is the most cost-effective measures to control bacterial blight (Khush et al., 1989).

Genetic studies on rice resistance have shown that the resistance to bacterial blight is controlled by major independent genes and modified by the effect of minor genes (Pei et al., 2011). Resistance of rice varieties to bacterial blight includes the quality resistance and quantitative resistance. Quality resistance generally is controlled by the major gene, so far, there are at least 34 resistance genes to bacterial blight identified at home and abroad (Chen et al., 2008; 2011), of which Xal, Xa5, Xal3, Xa21, Xa23, Xa26 and Xa27 have been cloned (Song et al., 1995; Yoshimura et al., 1998;Lyer and of McCouch, 2004; Sun et al., 2004; Gu et al., 2005); whereas quantitative resistance is controlled by tiny effective genes that may have significant gene effects and non-race specification.
Choosing the genotype that meets the requirements to detect the disease resistance of new varieties is an important step in the resistance breeding program, in the past the artificial inoculation was classic phenotypic identification methods. In order to avoid the mistakes of the phenotype judgment and to exclude the influence of environmental factors, molecular biology techniques were employed to determine the resistance gene that might be more accurate analysis on the resistant inheritance of new varieties.
In this study, genetic analysis and gene detection on new bacterial blight resistant varieties Lvzhen8072 and Baixiangzhan were carried out in order to provide a scientific basis for applying in rice production.
1 Results and analysis
1.1 Genetic analysis on the resistance in varieties of Lvzhen8072 and Baixiangzhan
Choosing GD6027 strain, the representative strain of rice bacterial blight â…¤ type in Guangdong province to be inoculated by leaf-cutting, the results showed (Table 1) that F1 derived from the cross of the resistant parent Lvzhen8072 and susceptible parent Jingang30 exhibited disease-resistant, the separation ratio of disease resistance to susceptible in the F2 generation was 3:1, Chi-square (χ2 ) test showed that there was no not significant difference between the actual value and the theoretical value, indicating that Lvzhen8072 should have a single dominant gene to control the resistance to rice bacterial blight â…¤ type strain. Whereas the F1 derived from the cross of the Baixiangzhan and susceptible parent of Jingang30 exhibited disease-susceptible; the separation ratio of disease resistance to susceptible in the F2 generation was 1: 1.85, and the value of χ2 test was 22.16 with extremely small p-value, indicating the difference should be extremely significant, the result showed that the separation of genetic resistance in Baixiangzhan is not in line with controlling by a pair of recessive gene.


Table 1 Resistance reaction of Lvzhen8072/Jingang30, Baixiangzhan/Jingang30 and their F1, F2 progenies against strain GD6027

 
1.2 Detecting resistant genotypes of Lvzhen8072 to bacterial blight
Four pair primers of SSR markers, RM20580, RM20590, RM20591 and RM20595, tightly linked to Xa7 genes were chosen to detect Xa7 gene in parents of Lvzhen8072 and Jingang30 and their offspring of F2 generation including 11 resistant and 37 susceptible individuals (Table 2). The results showed the four selected SSR markers had excellent polymorphism between the parents. Specific band genotype of resistant donor was treated as AA, assigning "1"; while the genotypes of susceptible parent Jingang30 was treated as aa, assigning "2", and heterozygous genotype with two specific bands was treated as Aa, assigning "3". The detecting results found that the ratio of resistant individuals (AA and Aa) to susceptible individuals (aa) was in line with the theoretical ratio of 3:1, which meets the Mendelian genetic law of single dominant gene segregation. Figure 1 presented the genetic testing result of SSR markers RM20580, the result indicated that Lvzhen- 8072 should have the target gene Xa7.


Table 2 Linkage analysis of bacterial blight resistance gene Xa7 in Lvzhen8072



Figure 1 Linkage analysis of bacterial blight resistance gene Xa7 in Lvzhen8072 by SSR marker RM20580


1.3 Detecting resistant genotypes of Baixianzhan to bacterial blight
Six pairs of primers RM17743, RM6320, RM17756, RM17757, RM17763 and RM17768, tightly linked to xa5 gene were chosen to detect xa5 gene in parents of Baixiangzhan and Jingang30 and their offspring of F2 generation including 67 resistant and 20 susceptible individuals (Table3). Specific band genotype of Baixiangzhan was treated as aa, assigning "1"; while the genotypes of susceptible parent Jingang30 was treated as AA, assigning "2", and heterozygous genotype with two specific bands was treated as Aa, assigning "3". The detecting results showed that the recombination rates of the five makers in disease-resistant individuals were larger than 60% except RM 17743 was less than 30%. Detecting the resistant individual indicated that the bacterial blight resistance gene in Baixiangzhan should not conferred to xa5 gene. Figure 2 presented the genetic testing result of SSR markers RM17743. However, in term of the test results of the F2 susceptible individuals with less than 30% recombinant rate indicating that the resistant gene to bacterial blight in Baixiangzhan should confer to xa5 gene. There would be two possibilities appearing these different results, the Baixiangzhan might possess xa5 gene that might be interfered by other linked resistance genes, or the Baixiangzhan may possess some tiny resistant genes to bacterial blight.


Table 3Linkage analysis of bacterial blight resistance gene xa5 in Baixiangzhan

 


Figure 2 Linkage analysis of bacterial blight resistance gene xa5 in Baixiangzhan by SSR marker RM17743


2 Discussions
Xa7 is a broad spectrum and high resistant gene to bacterial blight that can be resistant to all strains of bacterial blight in Guangdong. Although the gene has important value in use, poor agronomic traits in the germplasm of gene source, 8072-2, does not directly apply in production yet. Lvzhen8072 was developed from the hybrid cross of resistant variety 8072-2 and susceptible cultivar Lvzhan. The Lvzhen8072 was confirmed to contain Xa7 gene or to be allelic to Xa7 in this research, and also Lvzhen8072 planted in rice region of Guangdong exhibited effective against bacterial blight infection, which proved the new released cultivar, Lvzhen8072, can apply in hybrid rice production
Allele segregation distortion in hereditymay adversely affect breeding resistant variety and maintaining resistance. The character of resistance in Baixiangzhan derived from IRBB5 that carries xa5 gene (Zeng et al., 2009, Guangdong Agricultural Sciences, 5: 19-28). However, Baixiangzhan seems to contain xa5 recessive resistance gene, resulting in separation distortion in F2 offspring. Definitely, the reason for separation distortion need to be further studied.
3 materials and methods
3.1 Materials used in this research
Lvzhen8072, a new variety with high resistance to bacterial blight, and a highly susceptible Jingang30 were used as parents to make F1, and then develop F2 population including 278 individuals derived by F1 selfing. Sampling parents and 11 individuals of resistance phenotypes, 37 individuals of susceptible phenotype were used for molecular detection.
Baixiangzhan, a new variety with high resistance to bacterial blight, and a highly susceptible Jingang30 were used as parents to make F1, and then develop F2 population including 416 individuals derived by F1 selfing. Sampling parents and 67 individuals of resistance phenotypes, 20 individuals of susceptible phenotype were used for molecular detection.
The strain GD6027, the representative strain of rice bacterial blight â…¤ type strains in Guangdong Province was used to be inoculated with the culture age 72 hours and bacterial concentration 6×108/mL.
3.2 Resistance detection
The Inoculated strain was stored in glycerol nutrient solution at -20 degrees and rejuvenated in PSA medium (agar 17 g, glucose 20 g, potato 22 g, add water to 1 000 mL), and then test its pathogenicity in the standard test varieties, finally developed several slopping pipes of inoculating seeds stored at 4℃refrigerator. The tested varieties were planted individually in the test field, artificial leaf-cutting inoculation was carried out in the growing stage with inoculum concentration 6×108/mL. After 21days inoculated ion-by trees, the relative average lesion length of the diseased leaves were measured leaf by leaf each plant to figure out the average. The resistant and susceptible plants were classified based on the peak slot value of the relative lesion length distribution in the segregating populations. Due to slightly difference in annual climatic conditions, from 20% to 25% of the relative average lesion length of the diseased leaves were adopted as the bounders between resistance and susceptive (Zeng et al., 2002, Guangdong Agricultural Sciences (1): 35-37).
3.3 Molecular detection of resistant gene
Chemincals, dNTPs and Taq enzyme etc. used for molecular detection and reagents were purchased from TAKARA (Takara Biotechnology Co., Ltd.). The modified CTAB method for extraction of plant genomic DNA was followed the reference of Murray and Thompson (1980). Quality of the extracted DNA was detected with 1.0% agarose gel containing DNA GREEN (FMC Corporation, USA). The amplification of the microsatellite markers were detected by the modified Temnykh’s method (2000) .
Total 20 μL of PCR reaction volume included 7.0 μL of double distilled water, 10.0 μL of 10×PCR Mixture (Guangzhou BioTek Biological Technology Co., Ltd.), 1.0 μL of forward primer (10 μmol/L), 1.0 μL of reverse primer (10 μmol/L), and 1 μL of DNA template (concentration approximately 50 ng/μL). PCR reaction was carried out in the iCycler Gradient Temperature Cycler (Bio-Rad) or GeneAmpTM PCR System 9700 (PE Company) after PCR reaction system prepared well. SSR reaction program was as following: pre- denaturation at 94℃ for 4 min, then 35 cycles for denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, 72℃ extension of 1 min, final extension at 72℃ for 5 min.
3 μL of sample buffer (containing 0.25% bromophenol blue, 0.25% xylene cyanol FF) were added to amplified product with instant centrifugation. 2.0 μL of PCR amplification products were loaded by 5.0 μL range of trace sample injector (Shanghai Anting) into a 6% polyacrylamide gel in 1.0×TBE electrophoresis buffer for electrophoresis, and electrophoresis conditions were set up under 120 V voltage and 2 hours. After electrophoresis ended, taking vertical electrophoresis gel was stained by the silver staining method based on the reference of Ouyang et al (2009).
Primer sequences of SSR marker came from GRAMENE database (http://www.gramene.org/), and synthesized by Shanghai Biological Engineering Technology Co., Ltd.. Four SSR markers, RM20580, RM20590, RM20591, and RM20595 were selected by identifying the SSR makers tightly linked to Xa7 gene through GRAMENE public database, thereinto, RM20580 marker was located at 0.3cM of the left side of Xa7 gene, and RM20591 maker co-separated with Xa7 (Chen et al., 2008). Polymorphism between the Lvzhen8072 and Jingang30 was analyzed by using SSR markers firstly, and then using parental polymorphic markers determine whether Lvzhen8072 contained target resistance gene. Information about maker primers was shown in Table 4.
Likewise, searching SSR marker primers, RM17743 RM6320, RM17756, RM17757, RM17763, and RM17768, analyzed the polymorphism between Baixiangzhan and Jingang30, then detected F2 population by using the parental polymorphic makers to determine whether Baixiangzhan whether contained the target gene xa5. Information about maker primers was shown in Table 4.


Table 4 Markers’ primers for gene detecting

 
Author contributions
ZLX, CS and ZJ were the executor of the experimental design and experimental operation in this study; CS and ZJ also completed the data analysis, draft writing of the manuscript; ZXY and DLY participated in experimental design and result analysis; ZLX conceived the project and was the person in charge to guide experimental design, data analysis, manuscript writing as well as modification. All authors read and approved the final manuscript.
Acknowledgement
This project was jointly supported by the special project of modern agricultural technology system construction (Yuecaijiao [2009] No. 356: CARS-01-24), the program of Guangdong Provincial Science and Technology (Yuecaijiao [2011] No. 511), the Guangdong Provincial Natural Science Foundation (10151064001000029) and the Guangzhou Science and Technology Project (2010Z1-E041).
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