Fine Mapping of qBlsr5a, a QTL Controlling Resistance to Bacterial Leaf Streak in Rice

ACTA AGRONOMICA SINICA Volume 34, Issue 4, April 2008 Online English edition of the Chinese language journal Cite this article as: Acta Agron Sin, 2008, 34(4): 587–590.

RESEARCH PAPER

Fine Mapping of qBlsr5a, a QTL Controlling Resistance to Bacterial Leaf Streak in Rice HAN Qing-Dian, CHEN Zhi-Wei, DENG Yun, LAN Tao, GUAN Hua-Zhong, DUAN Yuan-Lin, ZHOU Yuan-Chang, LIN Min-Chuan, and WU Wei-Ren* Institute of Crop Genetic Improvement, Fujian Agriculture & Forestry University, Fuzhou 350002, China

Abstract: The quantitative trait locus (QTL) qBlsr5a on the short arm of rice (Oryza sativa L.) chromosome 5 has been proved to have the largest effect on the resistance to rice bacterial leaf streak (Xanthomonas oryzae pv. oryzicola, BLS). Using Acc8558 (highly resistant to BLS) as the donor and H359 (highly susceptible to BLS) as the recipient, a near-isogenic line (NIL) H359-BLSR5a was developed through backcross and only qBlsr5a was introgressed from the donor parent. A big F2 population (2,265 individuals) was constructed by hybridizing the NILs with H359 and 120 individuals with extreme phenotypes (lesion length < 2 cm) were selected. Eighty-five out of the 120 individuals were identified as homozygous resistant genotypes at the target QTL after examining their progeny lines (F2:3). By genotyping these homozygous individuals with SSR markers and performing linkage analysis, qBlsr5a was mapped to an interval between SSR markers RM153 and RM159, which covered a range of 2.4 cM or 290 kb. Keywords:

rice bacterial leaf streak; QTL; fine mapping

Bacterial leaf streak (BLS) is a major rice (Oryza sativa L.) disease subject to quarantine regulations in China [1]. The disease was first observed in Guangdong, China [2] and has proved to be a new bacterial disease in rice caused by Xanthomonas oryzae pv. oryzicola [3]. When rice is infected by the BLS pathogen, leaves become yellow or even blasted, the rate of unfilled grains will increase, and the grain weight will decrease. Bacterial leaf streak may cause 10–20% and up to 40% grain yield reduction, when the disease is serious [4–6]. This disease is widely distributed in rice production areas in southern China including Guangdong, Fujian, Hunan, and Jiangxi Provinces, and the Guangxi Autonomous Region. The resistant variety is the most effective solution to BLS. The BLS resistance in rice has proved to be a quantitative trait controlled by polygenes [7–10], but some major genes may be involved [11–14]. In recent years, some QTLs for BLS resistance have been mapped on rice genome using molecular markers [9, 10, 15]. Using a recombinant inbred line population derived from the cross between the highly resistant variety, Acc8558, and the highly susceptible variety H359, Tang et al. [9] mapped 11 QTLs for resistance to BLS in rice including qBlsr5a, on the

short arm of chromosome 5, with the largest contribution to phenotypic variations. This QTL was further verified and more precisely mapped by Chen et al. [16]. In this article, we aimed at fine mapping qBlsr5a, by developing a nearisogenic line (NIL) that only carries the target QTL, accordingly, to facilitate the marker-assisted selection and positional cloning of gene(s) resistant to BLS in rice.

1 1.1

Materials and methods Construction and plantation of mapping population

The BLS highly resistant variety Acc8558 as the donor and the highly susceptible variety H359 as the recipient were crossed to develop a near-isogenic line H359-BLSR5a. After several rounds of backcrossing with marker-assisted selection, only the QTL qBlsr5a was introgressed from the donor parent. A large F2 population consisting of 2,265 individuals was constructed by hybridizing H359-BLSR5a and H359 for the fine mapping of qBlsr5a. The individuals of the F2 population with the highest resistance to BLS were then identified, for selecting a homozygous genotype at the target QTL, by examining their progeny (F2:3) lines. The F2 population was

Received: 6 September 2007; Accepted: 30 October 2007. * Corresponding author. E-mail: [email protected] Copyright © 2008, Crop Science Society of China and Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Published by Elsevier BV. All rights reserved. Chinese edition available online at http://www.chinacrops.org/zwxb/

HAN Qing-Dian et al. / Acta Agronomica Sinica, 2008, 34(4): 587–590

planted with a spacing regime of 20 cm × 20 cm between seedlings and rows, in the experimental field of Fujian Agriculture & Forestry University, in the late cropping season of 2006. Parents Acc8558 and H359 and line H359-BLSR5a were used as the controls with 84 seedlings each. The F2:3 lines (14 seedlings per line) and the 3 controls (28 seedlings each) were planted in the early cropping season of 2007, with the same spacing regime used for the F2 population. 1.2

Evaluation of BLS resistance

The strain of X. oryzae pv. oryzicola was kindly provided by professor CHENG Guo-Ying of the Huazhong Agricultural University. Rice plants at the active tillering stage were inoculated by an artificial pricking inoculation method [9]. The concentration of the pathogen strain used for inoculation was 9 × 108 colony forming units per milliliter. Eight complete leaves of similar age from each plant were inoculated with 5 pricking points per leaf. Lesion lengths on 5 leaves, randomly selected from the 8 inoculated leaves of each plant, were measured 20 d after inoculation. The resistance of each plant was indicated by the mean lesion length of these 5 leaves. 1.3 Analysis of SSR markers and construction of genetic linkage map The total genomic DNA was extracted from fresh leaves using the SDS method. SSR analysis was performed according to the method described by Duan et al. [17]. PCR amplification was conducted in a 20 µL reaction system containing 2 µL 10× PCR buffer (with Mg2+), 0.4 µL dNTPs (2.5 µmol mL−1), 2 µL primers (5 µmol mL−1), 1 U Taq enzyme, 30 ng DNA template, and 13.45 µL sterile H2O. DNA amplification was programmed at 94ºC for 4 min; followed by 35 cycles of denaturing at 94ºC for 30 s, annealing at 57ºC for 1 min, and extension at 72ºC for 50 s; and final extension at 72ºC for 10 min. PCR products were separated by nondenaturing polyacylamide gel electrophoresis and visualized by silver-staining. Bands were recorded as 1 for the susceptible parent type, 2 for the heterozygous type, 3 for the resistant parent type, and 0 for missing. A genetic linkage map was constructed using the software Mapmaker/Exp 3.0 and a recombination fraction was converted into genetic distance using Kosambi’s mapping function.

2 2.1

Results Identification of homozygous resistant genotype

In 2006, the mean lesion lengths of Acc8558, H359, and H359-BLSR5a were 0.82, 4.30, and 2.41 cm, respectively, and the lesion lengths of the F2 individuals ranged from 0.6 cm to 7.0 cm, approximately, following a normal distribution. To finely map qBlsr5a as a major gene, we tried to identify individuals with a homozygous resistant genotype or a

homozygous susceptible genotype in the F2 population. Considering no apparent dominance between the resistant and susceptible alleles and the longer lesions probably with larger variations (variances), to increase difficulties in distinguishing the heterozygous genotype and the homozygous susceptible genotype at the target QTL qBlsr5a, we only selected individuals with homozygous resistant genotype for fine mapping. Therefore, 120 individuals with extremely resistant phenotypes (lesion length < 2 cm) were selected as candidates of homozygous resistant genotype in the F2 population. In 2007, the F2:3 lines of these candidates were further verified by inoculation, taking Acc8558, H359-BLSR5a, and H359 as the controls. The average lesion lengths of Acc8558, H359-BLSR5a, and H359 were 0.35, 1.79, and 3.98 cm in 2007, respectively. Finally, on the basis of a conservative criterion, to avoid involving the heterozygous genotype and the homozygous susceptible genotype, we only identified 85 lines as homozygous resistant genotypes (average lesion length ranged from 1.65 to 2.13 cm). 2.2

Fine mapping of target QTL

The target chromosomal segment in H359-BLSR5a that introgressed from the donor parent was known to be located between SSR markers RM153 and RM413 [16]. By referring to the Gramene website (http://www.gramene.org/), 22 SSR markers in the target segment were selected. Eleven of the markers were polymorphic between H359-BLSR5a and H359. These primers included RM153, RM5816, RM122, RM17746, RM159, RM5361, RM7029, RM17768, RM17777, RM6317, and RM413, which were listed in the order of chromosomal positions. Five of the polymorphic markers, RM153, RM7029, RM17777, RM6317, and RM413, were used to analyze the 85 homozygous resistant genotype lines. Linkage analysis showed that qBlsr5a was located between the markers RM153 and RM7029 (Fig. 1-A). Thus, we further used markers RM5816, RM122, RM17746, RM159, and RM5361 to analyze the lines, with recombination that occurred in the interval. There were no recombinant lines for markers RM5816, RM122, and RM17746, hence their distances to qBlsr5a could not be determined. Finally, qBlsr5a was mapped to an interval of 2.4 cM or 290 kb between RM153 and RM159 with an equal distance of 1.2 cM to the 2 flanking markers (Fig. 1-B). 2.3 Construction of physical map covering qBlsr5a region The contig and sequence of the region where qBlsr5a was obtained, was from the IRGSP website (http://rgp.dna.affrc.go. jp/IRGSP/pdf/Chr05.pdf). The whole segment from RM153 to RM413 covered 9 BAC/PAC clones, and the interval between the closest flanking markers RM153 and RM159 contained 3 clones, covering a range of 290 kb (Fig. 1-C). By searching for the sequence between RM153 and RM159 on TIGR’s Rice

HAN Qing-Dian et al. / Acta Agronomica Sinica, 2008, 34(4): 587–590

Fig. 1

Results of fine mapping of qBlsr5a

A: primary genetic map; B: fine genetic map; C: contig.

Genome Annotation website (http://www.tigr.org/tdb/e2k1/ osa1/index.shtml), we found that the region contained a total of 51 predicted open reading frames, of which 27 had annotated functions and 24 were unknown proteins.

3

Discussion

In the fine mapping of major genes, it is usually to genotype the homozygous recessive individuals in F2 populations using molecular markers [18, 19]. The merit of this method is that it can greatly reduce the workload. A QTL with a large effect can also be finely mapped with the help of this method, by constructing a secondary mapping population. In the secondary mapping population, only the target QTL is segregated along with the phenotype of the target quantitative trait, therefore, it can segregate in a qualitative way. Many major QTLs have been precisely mapped or even cloned by this method [20, 21]. However, with respect to QTLs with small effects, especially those that have interactions with the environment, it is very difficult to make the phenotype segregate qualitatively, even in a secondary mapping population. In this case, the QTL mapping method is usually applied instead of the major gene mapping method. However, QTLs with small effects can be mapped using a method similar to that used for major genes, if individuals with homozygous genotype corresponding to a certain phenotype (e.g., recessive trait) are identified. Following this principle, in this study, we identified individuals with a homozygous resistant genotype at the target QTL, by selecting individuals with extreme phenotypes and verifying their progeny lines. The results validated the feasibility of this approach. In previous studies, Chen et al. [16] mapped qBlsr5a to an interval covering a range of 1,600 kb between the SSR

markers RM7028 and RM413 using the method of composite interval mapping (CIM) [22, 23]. In this article, we not only amend their result but also greatly narrow down the range of the target QTL. This also indicates the advantage of the major gene-based mapping method. In view of the principle, CIM infers a QTL’s position based on the statistical result rather than directly on the genotypes of individuals. Hence, it can hardly achieve the precision that is obtained by the fine mapping of major genes. For a QTL with a small effect, however, the phenotypes of different genotypes usually overlap a lot on account of large environmental errors. In this regard, the efficiency of using the major gene-based mapping method is very low because only selecting individuals with extreme phenotypes will ensure the acquiring of the target homozygous genotype. In this study, theoretically, the large F2 population (2,265 individuals) had approximately 560 homozygous individuals resistant to BLS, but only 85 individuals fitting the required genotype were actually identified. This is a limitation to the mapping precision of the target QTL. Therefore, an even larger segregating population is needed for obtaining sufficient individuals with the homozygous genotype, at the target QTL. Currently, we are trying to identify the candidate gene qBlsr5a by enlarging the mapping population and using other methods to facilitate the positional cloning and functional study of the QTL.

4

Conclusions

The major points of the method in this study are, constructing a large F2 population with only a single QTL segregation, selecting individuals with extremely resistant phenotypes as candidates, and verifying the progeny lines of the candidates to confirm the homozygous genotype. The BLS

HAN Qing-Dian et al. / Acta Agronomica Sinica, 2008, 34(4): 587–590

resistant QTL qBlsr5a of rice is finely mapped to an interval between SSR markers RM153 and RM159, covering a range of 2.4 cM or 290 kb.

Acknowledgments This study was partially supported by the National Natural Science Foundation of China (30170503), the Natural Science Foundation of Fujian Province (B0610012), and the Fujian Provincial Young Talent Innovation Foundation (2005J019).

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