Juan YANG, Lijuan WANG, Shengdong HUANG, Yusheng LI*

1. Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;

2. Agriculture Commission of Ganyu County, Ganyu 222100, China

Rice sheath blight is a fungal disease caused by Rhizoctonia solani Kühn. Since the mid-1990s, the surveys have shown that rice sheath blight has caused enormous yield losses and declined quality, and it has become the first major disease in some rice-growing areas in Southern China[1-4].The characteristics of rice sheath blight make it difficult to find high-level resistance sources. Even worse, the incidence of sheath blight is greatly influenced by the environment. All the factors above affect the studies on genetic breeding of sheath blight resistance, which explains why sheath blight-resistant rice varieties have not been promoted in large-scale cultivation. In current production, the prevention and control of rice sheath blight mainly relies on chemicals[5-9].

In recent years,with the development of molecular biological technology,genetic researches on rice sheath blight have made great progresses.Studies have shown that the resistance to sheath blight in rice is controlled by multiple genes, in which main-effect QTLs conferring sheath blight resistance exist.Currently,a total of 80 sheath blight resistance QTLs have been detected using F2, double haploid (DH)lines,backcross populations(BC2F2),recombinant inbred line(RILs) and other different genetic groups, and they are distributed in total 12 chromosomes of rice[10-13]. In order to further understand genetic characteristics of rice sheath blight resistance and find new sheath blightresistant QTLs, the RILs derived fromthe hybrid of Daguandao/IR28 were used as materials, and the rice sheath blight resistance-related QTLs were studied, thereby providing new information for promoting the development of sheath blight resistance breeding.

Materials and Methods

Materials

The recombinant inbred line population (F15) composed of 157 lines and the parents were used as materials. The susceptible rice variety was Daguandao, a local japonica rice variety in Jiangsu Province, and the disease-resistant rice variety was indica IR28 bred by the International Rice Research Institute. The parents and RILs were all provided by the Rice Research Institute of Nanjing Agricultural University.

Methods

Material cultivation The 157 inbred lines and their parents were all cultivated in the seedling pool of Jiangsu Academy of Agricultural Sciences in summer, 2013. Each line was cultivated in a row, and there were 15 plants in each row.There were two replicates for each material. The plant and row spacing was 12 cm×15 cm.The single grain cultivation was adopted,and during the entire growth period,fungicides were not used. The other cultivation and management measures were the same with those in conventional cultivation.

Rice sheath blight resistance screening The highly pathogenic Rhizoctonia solani Kühn strain RH-9 was provided by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences. The inoculation identification was carried out with the toothpick inoculation method at the tillering stage of rice[14]. The thin and flat wooden toothpicks were cut into segments in the length of 0.8-1.0 cm,and then the segments were split longitudinally into two parts.After soaked in PDB medium and sterilized, the toothpicks were inoculated with Rhizoctonia solani Kühn.After cultured for 3-5 d, the toothpicks were used for field inoculation. The toothpicks were embedded in the inside of the top third sheathes using tweezers. For each line,total 10 rice plants were inoculated;and for each rice plant, total 3 stems were inoculated, and the corresponding leaves were labeled. After the inoculation, the filed water level was controlled in order to maintain a high humidity in fields. The incidence was investigated 25 d after the inoculation.A total of 5 plants with relatively uniform performance in the middle of each row were selected. The lesion lengths and plant heights were first measured, and then the ratios between lesion length and plant height were calculated. According to the grading standards of Chen et al[15],the resistance or sensitivity of each material was determined.When the ratio ≤0.10, the material was treated as resistant; when the ratio ranged from 0.10 to 0.30, the material was treated as moderately sensitive; when the ratio ≥0.30,the material was treated as highly sensitive.

Data analysis and QTL analysis

The genotypes of the 157 RILs were detected using 167 SSR markers. The linkage map was constructed using Mapmarker/Exp 3.0, and the map distances were calculated using Kosambi function. The LOD threshold was 3.0, and the largest map distance was 50 cM. The linkage map was drawn using Map Draw. Finally, a molecular marker linkage map of rice with 167 markers was obtained. The total length was 15.4 cM, and the average distance between two adjacent markers was 15.4 cM. The order of all the markers was consistent with published results (http://www.gramene.org).

Combining the molecular marker detection results and ratios between lesion lengths and plant heights (disease index was treated as phenotypic value,and analysis results were treated as sine transformed average values between replicates), QTL detection was performed using interval mapping method[16]. The possible presence of QTLs was detected using QTL Cartographer 2.5 (LOD value of 2.5).The naming of QTLs was in line with the rules proposed by McCouch et al[17].

Results and Analysis

Performance of rice sheath blight resistance in parents Daguandao and IR28 and recombinant inbred line population

The rice sheath blight resistance of the parents and RILs was analyzed.The results showed that the resistance differed significantly between the 2 parents.The parent IR28,with disease index of 0.098, was resistant to rice sheath blight, while the parent Daguandao, with disease index of 0.224,was moderately sensitive to rice sheath blight. The distribution of disease indexes among the RILs was analyzed. The results showed that the resistance and susceptibility were distributed continuously among the 157 RILs; and the disease indexes ranged from 0.033 and 0.675,and were mainly concentrated in the range of 0.11-0.20 (Fig.1). It indicates that the rice sheath blight resistance in this population is a more complex quantitative trait controlled by multiple genes, and it is suitable for QTL mapping.

Detection of QTLs conferring rice sheath blight resistance

The molecular marker detection results and disease indexes of sheath blight were combined with QTL Cartographer software. A total of 4 rice sheath blight-resistant QTLs were detected, including qsb1, qsb2, qsb5-1 and qsb5-2, of which the LOD values were 2.74, 2.58, 9.028 and 5.80, respectively. The variance explained ranged from 10.41% and 36.92%(Table 1).The 4 QTLs were distributedon chromosomes 1, 2 and 5 (Fig.2).The additive effects of qsb1, qsb2 and qsb5-2 were positive, indicating that the QTLs derived from the donor parent Daguandao enlarged the disease index;while the additive effect of qsb5-1 was negative, indicating that the QTLs derived from the donor parent IR28 narrowed the disease index.

Table 1 Chromosomal location and characteristics of QTLs for rice sheath blight resistance

Conclusions and Discussion

In this study, the recombinant inbred line population composed of 157 lines derived from the hybrid of Indica IR28 and Japonica Daguandao, between which the rice sheath blight resistance differed significantly, was used as test materials, and the sheath blight resistance-related QTLs were detected with the toothpick inoculation method at the tillering stage. The incidence of sheath blight in rice was greatly influenced by the environment.The tillering stages of the RILs were almost simultaneous, thus the errors caused by different field microclimates due to asynchronous tillering stages which were optimum growth period for incidence of sheath blight were effectively reduced. In addition, replicates were arranged. There were total 10 rice plants for each line, and total 5 plants with relatively uniform incidence were selected from each replicate,improving the accuracy of phenotypic identification.

In this study, total 4 rice sheath blight resistance-related QTLs were detected,including qsb1,qsb2,qsb5-1 and qsb5-2,and the additive effects of single QTLs on phenotype ranged from 10.41% to 36.92%. The relative location on chromosome was compared between detected QTLs in this study and published rice sheath blightresistant QTLs. The results showed that among the 4 QTLs, there were 3 QTLs of which the locations were overlapped or close to those of previously located QTLs. The qsb1 was found to be located in the range of RM5410-RM3602 on chromosome 1,and the sheath blight-resistant QTL QRh1 (RM1361-RM104) located by Sharma et al.[18]was found to be located on the same chromosome region.The qsb2 was found to be located in the range of RM6122-RM13489 on chromosome 2, and the QTL QDs2a(RM341-RM5427)located by Li et al.[19]was found to be located on the same chromosome region. The qsb5-1 was found to be located in the range of RM5796-RM267 on chromosome 5,and the locations of resistant QTLs by Han et al[9],Li et al[19],Liu et al[20],Zheng et al[21]and Pan et al.[22]were overlapped completely or partially. Due to different mapping populations and molecular markers, the QTL intervals were relatively large. Whether the close-distance QTLs were the same QTLs or adjacent QTLs was still needed to be studied further. The qsb5-2 detected in this study had not been detected in previous studies,which might be due to great effect of genetic background on the expression of rice sheath blight resistant QTLs.

The effect size of QTL is usually referred to as phenotypic variance percentage explained by QTL. Generally,in primary populations,the QTL of which the variance explained is higher than 10% is treated as main-effect QTL. The rice sheath blight resistant QTLs with variance explained higher than 10% have been found in many researches, and they are distributed on the 16 segments of chromosomes 1,2, 3, 5, 7, 8, 9 and 11. In this study,on chromosome 5, the qsb5-1 was detected, and the variance explained was 30.68% . The additive effect of qsb5-1 reduced the disease index by 5.05, enhancing the resistance to sheath blight in rice. However, although some QTLs are located on the same chromosome segments, their variances explained are greatly different among different studies, which may be due to different donor parents,different population type, identification methods, experimental environments,etc.

There have been currently onlythree sheath blight resistant QTLs that have been mapped finely, including qSB-11Le,qSB-9Tqand qSBR11-1. Zuo et al.[23]located the qSB-11Lewithin a interval of 74 kb using chromosome segment substitution lines.There were 11 open reading frames(ORFs)in this interval,and one out the 11 open reading frames was considered to be a candidate gene. Yin et al.[24]finely located the qSB-9Tqusing chromosome fragments iteration lines. They found that qSB-9Tqwas made up of 2 QTLs,including L-qSB-9Tqand R-Qsb-9Tq, of which the molecular weights were 180.9 and 207.7 kb, respectively. The QTL qsb5-2 detected in this study has not been reported in previous studies.The genotypic variance explained by qsb5-2 was 18.28%.The QTLs can be mapped more finely using a secondary segregation population constructed with recombinant inbred lines.

[1]MENG QZ (孟庆忠),LIU ZH (刘志恒),WANG HY (王鹤影), et al. Research progress in rice sheath blight (水稻纹枯病研究进展) [J]. Journal of Shenyang Agricultural University(沈阳农业大学学报),2001,32(5):376-381.

[2]CHEN W(陈伟),QIAN J(钱建),CHENG FY (程枫叶). Incidence characteristics and comprehensive control technology of rice sheath blight in Nantong (南通地区水稻纹枯病的发生危害特点与综防技术)[J].Jiangsu Agricultural Sciences(江苏农业科学),2012,40(9):133-134.

[3]ZHANG GL (张国良). Occurrence reasons and control strategies of rice sheath blight in Jiangsu Province (江苏省近几年水稻纹枯病重发生原因及防治策略) [J]. Jiangsu Agricultural Sciences(江苏农业科学),2012,40(9):116-118.

[4]JI MX(吉沐祥), ZHUANG YQ(庄义庆),MIU K(缪康),et al.Antimicrobial activity of difenoconazole·hexaconazole against Rhizoctonia solani in laboratory and control effect on Thanatephorus cumumeris (苯醚·己唑醇对水稻纹枯病菌室内抑菌活性及田间防治效果)[J].Jiangsu Journal of Agricultural Sciences(江苏农业学报),2011,27(1):219-220.

[5]ZHU XD(朱锡德),LI J(李健),LI PG(李品刚), et al. Control effect of different agent against sheath blight in directlysowed rice(不同药剂防治直播稻纹枯病药效试验)[J]. Jiangsu Agricultural Sciences (江苏农业科学), 2012, 40(6):103-104.

[6]SUN YW(孙以文),MING L(明亮),CHU XP(储西平).Control effect of 24%hexaconazole·azoxystrobin suspoemulsion against rice sheath blight (24%唑醇·嘧菌酯悬乳剂防治水稻纹枯病效果) [J].Jiangsu Agricultural Sciences(江苏农业科学),2012,40(6):131-132.

[7]WEI SJ (魏赛金), CHENG X (程新),ZHOU Y (周云), et al. Control effect evaluation of 0.3% Nongkang 702 against Rhizoctonia solani Kühn, pathogen of rice sheath blight(0.3%农抗702水剂防治水稻病原真菌的药效评价)[J].Jiangsu Agricultural Sciences(江苏农业科学),2012,40(4):118-120.

[8]KUNIHIRO Y(国广泰史),QIAN Q(钱前),SATO H(佐藤宏之),et al.QTL analysis of sheath blight resistance in rice(Oryza sativa L.)(水稻纹枯病抗性QTL 分析)[J]. Journal of Genetics and Genomics(遗传学报),2002,29(1):50-55.

[9]HAN YP (韩月澎), XING YZ (邢永忠),CHEN ZX(陈宗祥),et al.Mapping QTLs for horizontal resistance to sheath blight in an elite rice restorer line, Minghui 63(杂交水稻亲本明恢63 对纹枯病水平抗性的QTL 定位)[J].Journal of Genetics and Genomics (遗传学报),2002,29(7):565-570.

[10]CHANNAMALLIKARJUNA V, SONAH H, PRASAD M, et al. Identification of major quantitative trait loci qSB11-1 for sheath blight resistance in rice [J].Molecular Breeding,2010,25(1):155-166.

[11]PINSON SRM, CAPDEVIELLE FM,OARD JH. Confirming QTL S and finding additional loci conditioning sheath blight resistance in rice using recombinant inbred lines[J].Crop Science,2005,45:503-510.

[12]XIE XW (谢学文), XU MR (许美容),ZANG JP (藏金萍), et al. Genetic background and environmental effects on expression of QTL for sheath blight resistance in reciprocal introgression lines of rice(水稻抗纹枯病QTL 表达的遗传背景及环境效应) [J].Acta Agronomica Sinica (作物学报), 2008, 34(11):1885-1893.

[13]WANG JK,WAN XY,LI HH,et al.Application of identified QTL marker associations in rice quality improvement through a design-breeding approach[J]. Theor Appl Genet, 2007, 115: 87-100.

[14]PAN XB(潘学彪), CHEN ZX(陈宗祥),XU JY (徐敬友), et al. The effects of different methods of inoculation and investigation on genetic research of resistance to rice sheath blight(不同接种调查方法对抗水稻纹枯病遗传研究的影响)[J]. Jiangsu Agricultural Research (江苏农学院学报), 1997, 18:27-32.

[15]CHEN ZY(陈志谊),LU F(陆凡),LIU SF(刘水峰),et al.Assessment and evaluation of disease resistance of main rice varieties in Jiangsu(江苏省水稻主栽及区试品种抗病性鉴定和评价) [J].Jiangsu Journal of Agricultural Sciences (江苏农业学报), 2001, 17(2):82-86.

[16]COLLARD BCY, JAHUFER MZZ,BROUWER JB, et al. An introduction to markers, quantitative trait loci(QTL)mapping and marker-assisted selection for crop improvement: The basic concepts [J]. Euphytica, 2005, 142:169-196.

[17]MCCOUCH SR,CHO YG, PANL E, et al. Report on QTL nomenclature [J].Rice Genet Newsl,1997,14:11-13.

[18]SHARMA A,MCCLUNG AM,PINSON SRM,et al.Genetic mapping of sheath blight resistance QTLs within tropical japonica rice cultivars [J]. Crop Science,2009,49:256-264.

[19]LI F (李芳),CHENG LR (程立锐),XU MR (许美容), et al. QTL mining for sheath blight resistance using the backcross selected introgression lines for grain quality in rice (利用品质性状的回交选择导入系挖掘水稻抗纹枯病QTL)[J].Acta Agronomica Sinica(作物学报),2009,35:1729-l737.

[20]LIU G,JIA Y,CORREA-VICTORIA FJ,et al. Mapping quantitative trait loci responsible for resistance to sheath blight in rice[J]. Phytopathology, 2009,99(9):1078-1084.

[21]ZHENG TQ (郑天清),XU JL (徐建龙),FU BY(傅彬英),et al.Preliminary identification of genetic overlaps between sheath blight resistance and drought tolerance in the introgression lines from directional selection (回交高代选择导入系的纹枯病抗性与抗旱性的遗传重叠研究)[J].Acta Agronomica Sinica(作物学报), 2007, 33 (8): 1380-1384.

[22]PAN XB(潘学彪), CHEN ZX(陈宗祥),ZHANG YF(张亚芳),et al.Preliminary evaluation for breeding advancement of resistance to rice sheath blight(水稻抗纹枯病育种成效的初步评价) [J].Chinese Journal of Rice Science(中国水稻科学),2001,15:2l8-220.

[23]ZUO SM(左示敏).Researching on effects of a resistance QTL(qSB-11Le)against rice sheath blight and its fine mapping and candidate gene analysis(水稻抗纹枯病数量基因qSB-11 的精细定位、 效应研究及其候选基因分析)[D]. Yangzhou: Yangzhou University(扬州: 扬州大学),2006.

[24]YIN YJ (殷跃军).Genetic analysis and fine mapping of a QTL qSB-9Tqconferring resistance to sheath blight in rice(水稻抗纹枯病QTL qSB-9 的遗传分析和精细定位研究) [D]. Yangzhou:Yangzhou University(扬州: 扬州大学),2008.