Tibet Development and Breeding Application of the Fluorescence Molecular Marker of Rice Bacterial Blight Resistance Gene Xa7

In this study, the closely linked fluorescence molecular marker PM-Xa7 of  Xa7  gene was developed on the basis of sequence difference between the resistant variety ‘IRBB7’ containing  Xa7  and other nine bacterial blight susceptible rice varieties, and combine with PARMS technology (Penta-primer amplification refractory mutation system). Genotypes of  Xa7  were identified in 72 rice germplasm and the bacterial blight resistance level of ‘Mei B’ was improved by backcross and screening with this marker. The results showed that two rice germplasms containing  Xa7  were screened out from 72 rice germplasm; By introducing the resistant  Xa7  gene from ‘IRBB7’ into ‘Mei B’ with PM-Xa7, a stable new maintainer with resistance to bacterial blight was successfully obtained. The bacterial blight resistant fluorescence signal was detected in 289 plants and the bacterial blight susceptible fluorescence signal was detected in 93 plants of BC 2 F 2 population generated from ‘IRBB7’ and ‘Mei B’, and the genotypes were consistent with the phenotypes of  Xoo  inoculation. These research results indicated that PM-Xa7 can be effectively used for the marker-assisted breeding of bacterial blight resistant rice variety with the resistant Xa7  gene.

Rice bacterial blight is a bacterial disease caused by Xanthomonas oryzae pv. oryzae (Xoo). The disease was first detected in Fukuoka, Japan in 1884 (Li et al., 2017). Subsequently, the disease was found in every major rice-growing area in the world. In China, rice bacterial blight was first discovered in the suburbs of Nanjing in 1950 (Shen, 2001). At present, the disease has occurred in all rice areas in China, especially in the southern coastal rice areas (Yu et al., 2012). The outbreak of bacterial blight not only resulted in severe rice yield, but also reduced rice quality. Practice shows that breeding resistant varieties is the most economical and effective way to control bacterial blight. However, many resistant varieties in production lose their resistance after 3~5 years (Shi et al., 2008). Therefore, it is of great significance to breed rice varieties with long-lasting and broad-spectrum resistance to bacterial blight.
In order to develop rice varieties with long-lasting and broad-spectrum resistance to bacterial blight, the key lies in the discovery and utilization of genes with long-lasting, broad spectrum and high resistance to bacterial blight. Among the resistance genes to bacterial blight, Xa7 gene from the rice variety 'DV85' from Bangladesh is a gene with high resistance, broad-spectrum and persistent resistance to bacterial blight (Ma et al., 2013), which has not been cloned so far. Studies have shown that Xa7 gene shows high resistance to 52 different bacterial blight strains from China and Japan (Liu et al., 2016), and Xa7 is relatively stronger in high temperature environment (Webb et al., 2009), which makes it suitable for utilization in rice areas of South China. At present, some scholars have carried out molecular marker-assisted breeding for Xa7 gene. The 'DV85' Xa7 genes were incorporated into the 'Minghui63' and selected by markers STSP3 and M5 to improve the resistance of 'Minghui63' to bacterial blight (http://www.cropscience.org.au/icsc2004/poster/3/4/4/1041_heyuqing.htm). Lan et al. (2011) improved the resistance of 'Hua 201S' to bacterial blight by selecting the linkage marker STSP3 of Xa7 and combining with conventional breeding methods. Yan et al. (2013) used linkage markers to polymerize the anti-bacterial blight genes Xa7 and Xa21 and anti-borer gene cry1C* into 'Xianhui 207', and improved the resistance of 'Xianhui 207' to the blight and the borers. In the above studies, the markers selected for Xa7 gene were all linkage markers that were detected by electrophoresis after PCR amplification, and the detection process was relatively complicated, which was not conducive to the detection of large-scale samples. However, the fluorescent molecular markers developed by PARMS technology do not need electrophoresis detection, but can read the genotype signal directly by fluorescence scanning, and then the genotype can be obtained by combining with software data analysis, which can perform Xa7 genotyping on a large number of rice samples at the same time.

A fluorescent molecular marker for Xa7 gene selection
Referring to the locus of co-dominant marker M5 of Xa7 gene developed by Porter et al. (2003), the sequence was compared with that of 9 susceptible cultivars (Ce253, Gui582, Guanghui998, R128, Ce138, Guanghui308, Shen08S, 186B, Ⅱ-32B). It was found that there were differences in A/C and T/A between 'IRBB7' and the 9 susceptible cultivars at the location of marker M5 ( Figure 1). Based on this, primers Xa7-FaT and Xa7-FCA were designed to match the fluorescence primers of FAM and HEX in PARMS master mix, respectively. Primers Xa7-FaT, Xa7-FCA and general primers Xa7-R jointly constituted the fluorescent molecular marker PM-Xa7 of Xa7 gene (Table 1). The genotype of the Xa7 gene in the sample can be determined by analyzing the different signals obtained from the PCR amplification product scan of the marker PM-Xa7, so the marker can be used for selection of the Xa7 gene.  1.2 Two germplasms containing Xa7 gene were identified by PM-XA7 marker from 72 rice germplasms 72 rice germplasms were inoculated with the predominant species Ⅳ of Blight from Guangxi, and many of them showed resistance to bacterial blight. Furthermore, the Xa7 gene in 72 rice germplasm was identified by fluorescence molecular marker PM-Xa7. The results showed that two rice cultivars, 'IRBB7' and '82B', contained Xa7 allele. Other rice varieties, such as' IRBB5 'containing resistance gene Xa5 and' CBB23 'containing resistance gene Xa23, were all susceptible to Xa7 alleles ( Figure 2). The results of inoculation identification and marker detection indicated that the fluorescent molecular marker PM-Xa7 could specifically and effectively detect Xa7 gene in the 72 rice germplasm resources, which would be helpful to explore new germplasm carrying Xa7 gene and improve the utilization rate of Xa7 gene in breeding work.

A new maintainer material against bacterial blight was created by labeling PM-Xa7
By using the marker PM-Xa7 for selection and phenotypic identification, a new mainstay material resistant to bacterial blight was created ( Figure 3). In this process, the hybrid rice breeding parent 'MeiB' was cross-bred with the resistant parent 'IRBB7' and backcrossed in succession. 382 single plants of its BC2F2 generation were detected by using the marker PM-Xa7. The detection results showed (Figure 4) Figure 5) showed that 289 single plants were resistant, while the remaining 93 were all susceptible, which was consistent with the results of marker detection genotype. The separation ratio of resistant and susceptible single plants was close to 3:1 (chi-square 0.09). These results indicate that PM-Xa7 can accurately track Xa7 gene and can be used for molecular marker-assisted breeding of Xa7 gene.

Discussion
Molecular marker-assisted selection is not limited by environmental conditions or affected by physiological species of pathogenic bacteria, and can accelerate the selection of rice varieties resistant to bacterial blight (Li et al., 2011). At present, most of the molecular markers used in molecular marker-assisted breeding are those detected by polyacrylamide gel or agarose gel electrophoresis after PCR amplification. However, with the development of molecular marker technology, a fluorescent molecular marker technology based on PARMS established by PCR amplification of blocked mutation system has been applied in rice molecular marker-assisted breeding (Qing et al., 2018). The molecular markers developed by this technology do not need electrophoresis detection, and the genotype signal can be read directly by fluorescence scanning. Combined with software analysis, the genotype can be quickly realized. Compared with the traditional molecular markers, it has the advantages of convenience, fast, pollution-free, high detection efficiency and low detection cost, and is more suitable for large-scale genotyping. Therefore, the development of fluorescent molecular markers of rice resistance genes and their application in production practice will further promote the effective utilization of rice resistance genes and accelerate the breeding of resistant varieties. Porter et al. (2003) developed a linkage marker of Xa7 gene, M5, which was used to screen bacterial blight resistant varieties. The marker was only 0.5 cM away from Xa7 gene, and AFLP31-10 (3 cM) was closer to Xa7 gene than the marker G1091 (6.0 cM). In this study, a fluorescent marker PM-Xa7 was designed based on the sequence contrast between the resistant variety 'IRBB7' and 9 susceptible varieties by referring to the locus of marker M5. This marker is closely linked to Xa7 gene and has good stability. Compared with previous M5 markers, it does not need to detect PCR products by electrophoresis. The operation is simpler and faster, which is more suitable for the detection of large-scale samples. In this study, the PM-Xa7 marker was further used to identify the Xa7 gene in the rice germplasm resources, and the Xa7 gene was selected from the hybrid offspring of 'IRBB7' and the susceptible variety 'Mei B', and the combination inoculation identification confirmed the effectiveness and stability of the marker. It is believed that the further utilization of PM-Xa7 will improve the utilization of Xa7 gene in rice breeding against bacterial blight.
Xa7 is a highly resistant, broad-spectrum and persistent bacterial blight resistant gene. Many researchers have used different linkage markers to identify this gene in rice germplasm resources and carry out molecular marker breeding application. Wu et al. (2019) identified one disease-resistant material containing Xa7 gene from 148 weedy rice samples using RM20582 marker. Liu et al. (2016) analyzed some rice germplasm resources in Guangdong Province with linkage markers and identified two germplasm containing Xa7 gene. He (2012) detected Xa7 gene in 72 rice parental materials through inoculation identification and M5 labeling, and found several rice parents containing Xa7 gene. In this study, fluorescence marker PM-Xa7 was used to detect 72 rice germplasm resources and inoculation identification. Only two varieties' IRBB7 'and' 82B 'were found to contain Xa7 gene, which indicated that Xa7 gene had low utilization rate in rice breeding for resistance to bacterial blight, which may be related to the easier expression of resistance under high temperature conditions and the expression of non-whole growth period. In order to take advantage of the stronger resistance of Xa7 gene under high temperature conditions, this study used 'IRBB7' to improve the resistance of susceptible parent 'Mei B' to bacterial blight, and combined with the marker PM-Xa7 for selection, a new mainstay material with stable resistance was obtained. Predecessors' (http://www.cropscience.org.au/icsc2004/poster/3/4/4/1041_heyuqing.htm) using Xa7 gene to rice bacterial leaf blight resistant effect of improvement is consistent. It was confirmed that this gene is an excellent disease resistance gene which can play a role in production. In this study, only Xa7 resistance genes were polymerized into the varieties to be improved, but studies have shown that rice varieties with multiple resistance genes to bacterial blight can show more durable, stronger and broader resistance (Huang et al., 2012). The next step is to combine other resistance genes with existing materials to develop rice varieties with long duration, broad-spectrum and high resistance to bacterial blight.

Experimental materials
The experimental materials were 72 rice parents including 'IRBB7' containing Xa7 gene, 'IRBB5' containing Xa5 and 'CBB23' containing Xa23 collected by Guangxi Crop Genetic Improvement and Biotechnology Laboratory (Table 2), and 382 single plants of BC2F2 generation of 'IRBB7' and susceptible maintainer line 'Mei B'.

Development of fluorescent molecular marker for Xa7 gene
According to the locus of co-dominant marker M5 of Xa7 gene in rice variety 'IRBB7', the 200 kb sequence near was selected. According to the difference between 'IRBB7' and 9 susceptible cultivars, the sequence was compared with 9 susceptible cultivars (Ce253, Gui582, Guanghui998, R128, R138, Guanghui308, Shen08S, 186B, Ⅱ-32B). Primers Xa7-FaT and Xa7-FCA, which can match with the fluorescence primers of FAM and HEX in PARMS Master Mix, were designed and combined with the universal primer Xa7-R to form the fluorescence molecular marker PM-Xa7 of Xa7 gene.

Phenotypic identification of the tested material
The tested pathogen was the bacterial blight (Ⅳ) strain of rice in Guangxi, which had the highest occurrence frequency and the most extensive damage distribution in Guangxi, and was considered as the dominant pathogenic bacteria in Guangxi (Sun et al., 1993). According to the bacterial culture and inoculation methods of Wu et al. (2019), the disease was investigated about 20 days after inoculation. The disease was graded according to the percentage (%) of disordered spots in the total leaf length (Qin et al., 2014): 0~3.0% of high resistance (HR), 4%~12.0% of resistance (R) and 13%~25.0% of moderate resistance (MR), 26%~50.0% of moderate susceptible (MS), 51%~85.0% of susceptible (S), 86%~100.0% of high susceptible (HS).

Xa7 allele type
The fluorescence signal intensity of PCR amplification products was detected by using the enzyme label analyzer of FAM, HEX and ROX fluorescence detection channels. By SNP decoder (http://www.snpway.com/snpdecoder01/) software analysis each sample PCR amplification of FAM and HEX fluorescent signal values, and the corresponding scatter plot format output, genotyping of amplification products. If the fluorescence signal of FAM is obtained by scanning, it is the Xa7 allele containing A and T bases, which is divided into green dots on the scatter map. If the HEX fluorescence signal is obtained by scanning, it is the Xa7 allele containing C and A bases, which is the blue dot on the typing map. If the fluorescence signals of both FAM and HEX are obtained by scanning, the genotype contains heterozygous A/C and T/A base alleles, which are represented by red dots on the typing map.

Breeding application
Using Xa7 gene 'IRBB7' as the donor, cross with Guangxi high quality hybrid rice susceptible parent 'Mei B' and backcross to BC2F1 generation. After three successive self-crossing generations, each generation was selected by using the marker PM-Xa7 and combined with phenotypic identification to create a new maintenance line material resistant to bacterial blight. In this process, in order to verify the accuracy of Xa7 gene selection by marker PM-Xa7, 382 BC2F2 generation single strains were detected by marker PM-Xa7, and 2 negative controls were added.