Rice (Oryza sativa L.) belongs to the genus Oryza, and is one of the three major food crops (wheat, rice and corn) in the world. In recent decades, the continuous expansion and frequent outbreaks of various rice diseases have seriously affected the rice yield and quality. Bacterial leaf streak (BLS) of rice, referred to as streak disease or leaf streak, is a pathogen that usually penetrates leaves through stomata or wounds and propagates in the outer body of mesophyll tissue, eventually leading to pathological symptoms of water immersion. It has the characteristics of early occurrence, rapid spread, large occurrence area, long onset time and serious damage (Liang, 2016). According to research and investigation, under natural conditions, the bacterial leaf streak will cause 15%~25% yield reduction of susceptible varieties, and in case of serious disease, the yield will be reduced by 40%~60% (Tang et al., 2018). At present, the control of rice bacterial leaf streak mainly depends on spraying chemical drugs for 1 to 2 times, which often fails to achieve ideal results (Wang et al., 2018), and the abuse of pesticides will cause great harm to the ecological environment (Zhang et al., 2015). It is an important means of modern crop breeding to cultivate pollution-free and pollution-free disease resistant varieties by means of molecular biology and improve plant disease resistance to achieve the purpose of control. Therefore, it is of great significance to excavate rice bacterial leaf streak resistance gene/QTL for breeding rice resistant varieties.

The lesion length of rice bacterial leaf streak showed continuous variation, and its resistance was mostly controlled by QTL, which was a typical quantitative trait. In recent years, there have been many studies on the mapping of rice bacterial leaf streak resistance gene/QTL. Tang et al. (2000) constructed F₂ mapping population for susceptible parent H359 and resistant parent Acc8558, Using composite interval mapping, a total of 11 QTLs with resistance to bacterial leaf streak on chromosome 6 were detected, Chen (2002) further constructed backcross population with materials from Tang et al. (2000) as parents, In addition to the validation of 3 major QTLs, 6 molecular markers linked to the QTL of resistance to bacterial leaf streak were found; Jing et al. (2004) identified QTL qBlsr5b sperm in the long arm region of chromosome 5, and the explanation rate of phenotypic variation was 4.64%; He et al. (2010) crossed the wild rice material with high and stable resistance with the susceptible variety '9311' to construct a high generation population, and detected a major gene on chromosome 6; Xie et al. (2014) used a set of secondary chromosome segment replacement lines to reduce the qBlsr5a with a large phenotypic contribution rate (14%) to the range of 30 kb, confirming its candidate gene LOC_Os05g01710 is the same gene as rice bacterial blight resistance gene xa5. Ju et al. (2017) overexpression of OsHsp18.0-CI improved the resistance of rice to XooC; Triplett et al. (2016) identified a dominant resistance gene Xo1 located within 1.09 Mb of the long arm of chromosome 4 from the American genetic rice variety ‘Carolina Gold Select’, and expressed complete resistance to the physiological races isolated from Africa; Wu et al. (2020) studied the OsDRxoc1 gene located on qBlsr5b, which has a strong effect on resistance to bacterial leaf streak, and found that OsDRxoc1 interacted with OsbHLH81 to jointly regulate rice resistance to BLS. Although many rice bacterial leaf streak resistance loci have been obtained, only a few resistant QTLs have been reported to be cloned (Zhang et al., 2019), mainly because the identified QTLs are basically small in effect, and there are few major QTLs, which makes the cloning of resistance genes and breeding applications face greater difficulties.

In this study, we used the multi growth period bacterial leaf streak resistant material ‘HD10’ (Hong et al., 2017) obtained on the basis of early broad-spectrum resistance resource screening to cross with the whole growth period sensitive material ‘93111’ to construct a F₂ mapping population. We found the molecular markers closely linked to the resistance site through the bulk segregation analysis (BSA), and located the bacterial leaf streak resistant site on chromosome 2. It provides reference for further fine mapping, cloning and functional verification, and provides excellent gene resources for resistance breeding.

1 Results and Analysis

1.1 Phenotype identification of parents and F₁ and F₂ generations

The resistance of parent ‘HD10’, parent ‘9311’ and hybrid F₁ were identified by strain GX01 at seedling stage and tillering stage respectively. The results showed (Table 1) that the resistance level of the parent 'HD10' was anti immune; The resistance level of the parent ‘9311’ is from sense to high sense (Figure 1); All F₁ generations had no disease resistant plants. According to the analysis of resistance performance of F₂ generation plants to bacterial leaf streak at seedling stage and tillering stage, the overall lesion length is continuously distributed (Figure 2A; Figure 2B), indicating that the resistance is a quantitative trait controlled by QTL. Among them, 154 plants were resistant (high resistance, low resistance and medium resistance) at seedling stage, accounting for 50.16% of the total; There were 61 resistant plants at tillering stage, accounting for 25.0% of the total.

Table 1 Resistance reaction of parents, F1 and F2 to bacterial leaf streak Note: The number of resistance identification plants at seedling and tillering stage is shown inside and outside brackets respectively

Figure 2  Lesion length statistics of F2 population Note: A: seedling stage; B: Tillering stage

1.2 Molecular marker screening of polymorphism

The location of the main resistance QTL was determined by referring to the classical map based cloning method. 440 pairs of polymorphic markers distributed on 12 chromosomes were screened among the parents by using 2 244 pairs of SSR and InDel markers (Table 2). The average polymorphisms percentage between the two parents was 19.6%, of which the highest percentage was chromosome 6, reaching 40.3%, and the lowest percentage was chromosome 10, only 7.4%.

Table 2  Polymorphism and average distance of molecular markers between 'HD10' and '9311'

 1.3 Screening of resistance locus linked markers

440 pairs of molecular markers with polymorphism between parents, as well as non denatured PAGE electrophoresis resistant and susceptible gene pools were used for scanning detection. Among them, B03021 and B03045, which are close to each other, also have polymorphism between resistant and susceptible gene pools and are consistent with the two parents' bands (Figure 3; Figure 4), indicating that B03021 and B03045 are closely linked to the resistant sites. According to the published rice genome database, B03021 and B03045 are located on chromosome 2. Five polymorphic markers (RM6, B03059, B03029, B0301, B02065) were further found by expanding the range and increasing the molecular marker density near these two markers (Table 3).

Figure 3 Amplification of the marker B03021 linked to the resistance locus on the resistant and susceptible pool and individual plants Note: M: DL2000 DNA marker; P1: resistant parent 'HD10’; P2: Susceptible parent '9311'; RB: Resistant pool; SB: Susceptible pool; 1~10: Individual plants from resistant pool; 11~20: Individual plants from susceptible pool

Figure 4  Amplification of the marker B03045 linked to the resistance locus on the resistant and susceptible pool and individual plants Note: M: DL2000 DNA marker; P1: resistant parent HD10; P2: Susceptible parent 9311; RB: Resistant pool; SB: Susceptible pool; 1~10: Individual plants from resistant pool; 11~20: Individual plants from susceptible pool

Table 3   Primer information for localization

1.4 There is a peak value of LOD greater than 3.0 between B03021 and B03029

Use the software JoinMap 3.0 to construct the genetic linkage map (Figure 5A; Figure 6A). The sequence of molecular markers on the linkage map is consistent with the physical sequence on the chromosome. Scanning with MapQTL 5.0 software, it was found that there was a peak value of LOD greater than 3.0 between B03021 and B03029 markers at seedling and tillering stages (the curve was the trend of LOD value change), indicating that there was a major QTL between the two markers. That is, the resistance loci are located in the region with a genetic distance of 7 cM between the markers B03021 and B03029 at both seedling and tillering stages (Figure 5B; Figure 6B). Further analysis showed that the LOD values of the main QTL at seedling stage and tillering stage were 8.68 and 11.80, respectively, which could explain the phenotypic variation rates of 13.1% and 17.5%, respectively.

Figure 5  QTL for resistance to BLS in seedling stage Note: A: Linkage map of the markers; B: Multiple OTLs model

Figure 6 QTL for resistance to BLS in tillering stage Note: A: Linkage map of the markers; B: Multiple QTLs model

2 Discussion

In this study, the high quality resistance source 'HD10' with broad-spectrum resistance in multiple growth stages was crossed with the susceptible material '9311'. From the resistance performance of parents, F₁ and F₂ generations to the disease at seedling and tillering stages, it was known that 'HD10' was a stable resistant material with resistance grades of HR and R. Li et al. (2017) found that the disease resistance of Dongxiang wild rice at seedling stage is generally lower than that at booting stage, so 'HD10' is also a resistant material with high research value. At seedling stage and tillering stage, the resistance loci were initially located in the 7 cM region between chromosome 2 B03021 and B03029, and the contribution rates of phenotypic variation were 13.1% and 17.5%, respectively, indicating that there was a major resistance QTL in this region. Zheng et al. (2005) used Jiafuzhan and indica rice variety 'Minghui 86' to construct F₂ population, and detected a QTL (marker interval RM279~RM154) with a phenotype contribution rate of 13.7% on chromosome 2. Ma et al. Their localization results are located in the same chromosome region, which is far from the physical distance of this study. However, the QTL mapped in this study and the resistance gene bls2 mapped in BC₃F₂ population constructed by Shi et al.

According to the genetic analysis of resistance in F₂ population, the resistance of 'HD10' to bacterial leaf streak was controlled by QTL, but only one major QTL locus was obtained in this study, and the resistant parent DH10 may also contain other QTL loci. The reasons for the loss of other resistance QTLs may be: (1) BSA method is generally used to locate genes controlling quality traits, while for genes controlling quantitative traits, only major QTLs with large contribution can be screened. In this study, BSA method was used to rapidly screen major resistance QTLs, which may lead to the omission of some minor resistance QTLs. (2) Both parents are conventional cultivated rice, with similar genetic background, uneven distribution of polymorphic primers, and excessive spacing of some adjacent polymorphic molecular markers, leading to non linkage with resistance sites.

At present, most of the international researches on the gene location and pathogenesis of rice bacterial leaf streak are based on the tillering stage or adult plant stage, and there is little research on multiple growth stages of the same strain. The resistance of a certain growth period to the strain does not mean that the whole growth period has resistance, so it is of great significance to dig out the genes showing resistance to streak disease in multiple growth periods. Therefore, it is necessary to verify whether the QTL locus has the same resistance in other growth stages except seedling stage and tillering stage.

The resistance ability of the same resistance gene may be different under different genetic backgrounds (Dixon et al., 2000). Although many rice resources resistant to bacterial leaf streak have been found, most of them come from tropical regions, with poor agronomic characteristics, and are difficult to be directly used in breeding. In this study, the tested parents of resistance are all conventional rice varieties (lines), and conventional rice is formed through long-term natural selection and artificial cultivation, which has high production and application value. Therefore, compared with foreign varieties or wild rice resistance sources, the main resistance QTL loci contained in conventional rice resistance source 'HD10' appear particularly important. Quantitative resistance controlled by QTL is usually non race specific, which can slow the development of pathogens at the infection site of plants. Therefore, quantitative resistance is more durable and more popular in production. The main resistance QTL identified in this study provides an excellent gene resource for the cloning and breeding application of the resistance gene to bacterial leaf streak.

3 Materials and Methods

3.1 Test materials and pathogen sources

Resistant parents: the cultivated rice line 'HD10' with high resistance to bacterial leaf streak and stable resistance in multiple growth stages identified by our laboratory in the early stage; Susceptible parent: Indica rice variety '9311', provided by China Rice Research Institute, is highly susceptible to the disease and has strong disease susceptibility stability. Test strain: GX01, a highly pathogenic strains of rice bacterial leaf streak pathogen in Guangxi, was provided by Professor He Yongqiang, the State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources.

3.2 Group construction

Hybrid F1 was obtained by crossing 'HD10' (male parent) and '9311' (female parent), and multiple F₂ populations were obtained by F₁ self crossing for genetic analysis of resistance. The seedling identification was carried out in the greenhouse of the College of Agriculture of Guangxi University, and the tillering stage was carried out in the net room field. The field management was carried out according to the conventional field management methods.

3.3 Strain inoculation and resistance identification

Inoculate the test strain coating method on the NA medium at 28℃ for 48 h, select a single colony with a full and smooth surface, and cultivate it to the logarithmic phase at the shaking table at 28℃ at 220 r/min, then prepare the collected bacteria with sterile water and dilute it to 3×10⁸ CFU/mL suspension was immediately used for inoculation. The method of strain inoculation refers to the method of Zhao et al. (2018). According to the width of the leaves, two needles are fixed on rubber blocks at appropriate intervals (the needles are exposed about 0.5 cm). The leaves are placed flat on a sponge with a thickness of about 2 cm in the culture dish and sufficient bacterial fluid is absorbed. After the needle tip pierces both sides of the middle vein of the leaves, the sponge is squeezed by the rubber to make the bacterial fluid fully penetrate into the wound hole. Each material is inoculated on 2 healthy fully developed leaves, with 4 needle points per leaf. The inoculation period of bacteria should avoid high temperature and dry weather, and should pay attention to selecting cloudy days or cover film for inoculation, so as to avoid failure of bacterial infection.

Refer to the method of Nong et al. (1991). When the lesion length of '9311' in the susceptible control group is more than 3 cm (about 15 days later), select 4 lesions with the same length at the inoculation point, measure their length, that is, investigate 4 data for each plant, and take the average value as the resistance data for each plant. The identification levels of disease resistance were: immunity (I), no symptoms or only brown spots in the wound; High resistance (HR), lesion length 0.1~0.5 cm; Resistance (R), lesion length 0.6~1.0 cm; Moderate resistance (MR), lesion length 1.1~1.5 cm; Susceptibility (S), lesion length 1.6~2.5 cm; High sensitivity (HS): the lesion length is more than 2.5 cm.

3.4 Construction of anti susceptibility pool

With reference to Michelmore et al. (1991)'s BSA (Bulked aggregation analysis) method, 10 individual plants of extreme disease resistance (HR) and extreme disease susceptibility (HS) types were selected from F₂ population, and DNA was extracted. After the concentration was diluted to the same value, DNA of equal volume of liquid was mixed into a pool of disease resistance genes and a pool of disease susceptibility genes.

3.5 DNA extraction and polymorphism detection

CTAB (cetyltrimethylammonium bromide) method was used to extract DNA from plants (Murray and Thompson, 1980); PCR reaction system 10 μL. DNA Template 1 μL. 0.4 μL for upstream and downstream primers respectively，2×Rapid Taq Master Mix 5 μL, ddH₂O 3.2 μL. The PCR reaction program is set as follows: 95℃ pre denaturation for 5 min, 95℃ denaturation for 30 s, 54℃~63℃ (adjusted according to the actual Tm value of the primer) annealing for 30 s, 72℃ extension for 45 s, 72℃ extension for 5 min, 34 cycles. After PCR amplification, put it in a 4 ℃ refrigerator. DNA amplification products were detected by polyacrylamide gel electrophoresis and silver staining. The results showed that the same electrophoretic pattern with the female parent '93111' was marked as "A", the same electrophoretic pattern with the male parent 'HD10' was marked as "B", and the double band pattern was marked as "H".

3.6 Genetic map construction and QTL mapping

The 2244 pairs of primer markers (SSR and Indel markers) in our laboratory were used to detect the polymorphism of the two parents, and then the primers showing good polymorphism between the two parents were used to mark the polymorphism between the pool of resistant genes and the pool of susceptible genes, and then the single plant genotype of the targeted population was detected with the primers that may have linkage with the QTL of resistance to bacterial leaf streak. According to the genotype of the population, use JoinMap 3.0 software to draw the linkage map of these markers, combined with the phenotypic data of resistance identification, and then use MapQTL 5.0 software to map the QTL compound interval (MQM) of this area, and determine the area where the peak value of LOD curve is greater than the threshold value 3.0, which is regarded as the area where QTL is initially located.

Authors’ contributions

WY was the experimental designer and executor of this research; LDJ participated in data collation and the writing of the first draft of the paper; HSX and QXM participated in some experiments; LF was the designer and principal of the project; LRB and LF jointly guided experimental design, data analysis, thesis writing and revision. All authors read and approved the final manuscript.

Acknowledgements

This research was jointly funded by the National Natural Science Foundation of China (31460341), Guangxi Natural Science Foundation of China (2019GXNSFAA185043) and Guangxi Innovation Driven Development Project (Guangxi AA17204070).

References

Chen S., 2002, Mapping resistance QTLs to rice bacterial leaf streak (Xanthomonas campestris pv. Oryzicola Dye) by SSR, Thesis for M.S., Fujian Agriculture and Forestry University, Supervisor: Li W.M., and Wu W.R., pp.23-28.

Dixon M.S., Golstein C., Thomas C.M., Biezen E.A., and Jones J.D., 2000, Genetic complexity of pathogen perception by plants: The example of Rcr3, a tomato gene required specifically by Cf2, Proc. Natl. Acad. Sci. USA, 97(16): 8807-8814.

https://doi.org/10.1073/pnas.97.16.8807

He W.A., Huang D.H., Liu C., Cen Z.L., Zhang Y.H., Ma Z.F., Chen Y.Z., Lu S.N., Liu H.Y.,Wu B., and Li Y.B., 2010, Inheritance of resistance to bacterial leaf streak in four accessions of common wild rice (Oryzarufipogon Griff.), Zhiwu Bingli Xuebao (Acta Phytopathologica Sinica), 40(2): 180-185.

Hong D.W., Zhao Y., Luo D.J., Shi L.J., Li Y.B., and Liu F., 2017, Screening of broad-spectrum resistant resources against rice bacterial leaf streak, Nanfang Nongye Xuebao (Journal of Southern Agriculture), 48(2): 272-276.

Jing Y.J., 2004, Fine localization of bacterial leaf streak resistance QTLqBlsr5b in rice, Thesis for M.S., Fujian Agriculture and Forestry University, Supervisor: Wu W.R., pp.23-25.

Ju Y.H., Tian H.J.,Z hang R.H., Zuo L.P., Jin G.X., Xu Q., Ding X.H., Li X.K.,and Chun Z.H., 2017, Overexpression of OsHSP18.0-CI enhances resistance to bacterial leaf streak in rice, Rice, 10(1): 12

https://doi.org/10.1186/s12284-017-0153-6

Li X.H., 2005, Fine mapping of QTL, resistant to bacterial leaf streak in rice (Oryza sativa L.), Thesis for M.S., Fujian Agriculture and Forestry University, Supervisor: Wu W.R., pp.27-30.

Li X.S., Zhou L.F., Cai Y.H., Huang R.H., and Hua J.L., 2017, Pathotype monitoring of Xanthomonas oryzae pv. oryzicola and resistance identification of rice varieties to bacterial leaf streak in Jiangxi Province, Zhiwu Binglixue (Acta Phytopathologica Sinica), 47(6): 808-815.

Ma L., Fang Y., Xiao S.Q., Zhou C., Jing Z.L., Ye W.L., and Rao Y.C., 2018, QTL exploration of bacterial leaf streak and their gene expression in rice, Zhiwu Xuebao (Bulletin of Botany), 53(4): 468-476.

Michelmore R.W., Paran I., and Kesseli R.V., 1991, Identification of markers linked to disease resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating segregation populations, Proc. Natl. Acad. Sci. USA, 88(21): 9828-9832.

https://doi.org/10.1073/pnas.88.21.9828

Murray M.G., and Thompson W.F., 1980, Rapid isolation of high molecular weight plant DNA, Nucleic Acids Research, 8(19): 4321-4325.

https://doi.org/10.1093/nar/8.19.4321

Shi L.J., Luo D.J., Zhao Y., Cen Z.L., Liu F., and Li Y.B, 2019, Genetic analysis and mapping of bacterial leaf streak resistance genes in Oryzae rufipogon Griff, Huanan Nongye Daxue Xuebao (Journal of South China Agricultural University), 40(2): 1-5.

Tang D.Z., Wu W.R., Li W.M., Lu H., and Worland A.J., 2000, Mapping of QTLs conferring resistance to bacterial leaf streak in rice, Theor. Appl. Genet., 101(1): 286-291.

https://doi.org/10.1007/s001220051481

Tang S., Yang Y.H., Pan S.J., Lin M.Q., Dai L.Y., and Li W., 2018, Control of rice bacterial leaf streak: research progress, Nongxue Xuebao (Journal of Agriculture), 8(11): 16-20.

Triplett L.R., Cohen S.P., Heffelfinger C, Schmidt C.L., Huerta A., Tekete C., Verdier C., Bogdanove A.J., and Leach J.E., 2016, A resistance locus in the American heirloom rice variety carolina gold select is triggered by TAL effectors with diverse predicted targets and is effective against African strains of Xanthomonas oryzae pv. oryzicola. Plant J., 87(5): 472-483.

https://doi.org/10.1111/tpj.13212

Wang X.D., Zhang Y., Mao J.H., and Jiang L., 2018, Control effect of three fungicides on bacterial leaf streak of late rice, Zhejiang Nongye Kexue (Journal of Zhejiang Agricultural Sciences), 59(12): 2200-2201.

Wu W., 2020, The molecular mechanism analysis of OsDRxoc1-mediated resistance to bacterial leaf streak, Thesis for M.S., Shandong Agricultural University, Supervisor: Ding X H., pp.29-32.

Xie X.F., Chen Z.W., Cao J.L., Guan H.Z., Lin D.G., Li C.L.,Lan T., Duan Y.L., Mao D.M., and Wu W.R., 2014, Toward the positional cloning of qBlsr5a, a QTL underlying resistance to bacterial leaf streak, using overlapping Sub-CSSLs in rice, PLoS One, 9(4): e95751.

https://doi.org/10.1371/journal.pone.0095751

Zhang M.J., Wang S.P., and Yuan M , 2019, An update on molecular mechanism of disease resistance genes and their application for genetic improvement of rice, Mol. Breeding, 39(10-11):154.

https://doi.org/10.1007/s11032-019-1056-6

Zhang R.S., Chen Z.Y., and Liu Y.F., 2015, Advance in rice bacterial leaf streak researches, Agr. Sci. Tech., 16(2): 298-305, 310.

Zhao Y., Luo D.J., He S.X., and Liu F., 2018, Comparative study on four inoculation methods of rice bacterial leaf streak, Yaredai Nongye Yanjiu (Subtropical Agriculture Research), 14(4):242-246.

Zheng J.S., Li Y.Z., Fang X.J., 2005, Detection of QTL conferring resistance to bacterial leaf streak in rice chromosome 2 (O. sativa L. spp. indica), Zhongguo Nongye Daxue (Scientia Agricultura Sinic), 38(9): 1923-1925.