QTL Detection for Rice Grain Shape Using Chromosome Single Segment Substitution Lines

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1 Rice Science, 2011, 18(4): Copyright 2011, China National Rice Research Institute Published by Elsevier BV. All rights reserved QTL Detection for Rice Grain Shape Using Chromosome Single Segment Substitution Lines LI Sheng-qiang, CUI Guo-kun, GUAN Cheng-ran, WANG Jun, LIANG Guo-hua (Jiangsu Key Laboratory of Crop Genetics and Physiology / Key Laboratory of the Ministry of Education for Plant Functional Genomics, Yangzhou University, Yangzhou , China) Abstract: Rice grain shape is one of the important factors affecting grain quality and yield, but it is liable to be influenced by genetic backgrounds and environments. The chromosome single segment substitution lines (SSSLs) in rice have been considered as ideal populations to identify the quantitative trait loci (QTLs). In this study, 22 QTLs affecting rice grain shape were detected to be distributed on eight chromosomes except chromosomes 6, 9, 11 and 12 by using SSSLs. Among them, seven QTLs conditioned grain length, six conditioned grain width, five affected grain length-width ratio and four controlled grain thickness. Key words: rice; single segment substitution line; grain shape; quantitative trait locus Rice is one of the most important food crops in the world, and more than half of the world s population depend on rice as the main source of nutrition. Food security for an ever-increasing world population largely depends on increasing the grain yield of cereal crops, including rice, which in turn largely depends on several yield components, especially grain size. Moreover, rice grain appearance quality is mainly specified by grain shape including grain length, grain width and length-width ratio (LWR). Many independent studies showed that grain shape of rice is a typical quantitative trait controlled by several genes. Using two RFLP maps from two F 2 populations, QTLs for grain shape of rice were identified by Lin et al (1995). In the Tesan ai 2 / CB1128 population, five QTLs for grain length were located on chromosomes 1, 1, 7, 8 and 10, respectively, whereas in the Waiyin 2 / CB1128 population, five QTLs for grain length were located on the chromosomes 2, 3, 6, 7 and 10, respectively (Lin et al, 1995). Huang et al (1997) detected 12 QTLs for grain length, grain width and length-width ratio using 146 RFLP markers in a doubled haploid (DH) population. Lin and Wu (2003) identified 15 QTLs for grain length and 17 QTLs for grain width using a recombinant inbred line (RIL) population derived Received: 29 December 2010; Accepted: 20 March 2011 Corresponding author: LIANG Guo-hua (ricegb@yzu.edu.cn) from a cross between two indica rice varieties, H359 and Acc Li et al (2009) detected three QTLs for grain length, two QTLs for grain width, four QTLs for grain thickness and four QTLs for length-width ratio using an F 2 population derived from a cross between japonica rice varieties DL115 and XL005. Most of QTL analyses in plants used traditionally primary populations such as F 2 /F 3 and BC 1, however, it is difficult to replicate accurate phenotypic values in these populations for precise QTL mapping, which allows us to proceed with further analysis, such as fine mapping and characterization of the target QTLs. Even though QTLs of interest can be identified using primary mapping populations, further development of mapping population is required for fine mapping and cloning of QTLs. Furthermore, some QTLs with minor effects and those with epistatic interactions with other loci might not be detected in primary QTL analysis. Genome-wide chromosome single segment substitution lines (SSSLs) consist of the lines carrying a single fragment from donor parent into a genetic background. As a result, each of the QTLs can be considered as a single Mendelian factor because of the minimized genetic background noise. A set of SSSLs composed of 110 lines had been developed from a backcross between a japonica recipient Nipponbare and an indica donor Guanglu ai 4. In this study, QTLs controlling grain length, grain

2 274 Rice Science, Vol. 18, No. 4, 2011 width, grain thickness and length-width ratio were detected and mapped using 22 SSSLs. The results supplied useful information for QTL cloning and molecular breeding of rice grain shape. MATERIALS AND METHODS Experimental population The population including 22 SSSLs was used to detect and map QTLs for grain shape (Table 1). In 2009, two parents (Nipponbare and Guanglu ai 4) and the 22 SSSLs were grown in the experimental plot of Yangzhou University, Jiangsu Province, China. The parents and each SSSL were grown four rows under natural conditions with single plant per hill and ten plants per row spaced at 15 cm 20 cm. Phenotypic evaluation Ten seeds from each SSSL and parent were sampled and investigated for grain length, grain width, grain thickness and length-width ratio after harvesting and drying. Average values for each SSSL and parent were used for QTL analysis. Detection and effect analysis of QTLs QTLs were identified based on significant differences between the SSSLs and the recipient parent Nipponbare, and determined by variance analysis using SPSS The threshold of QTL was detected at the 1% level of significance. The additive effect and the percentage of additive effect of QTLs for grain shape were estimated according to the method of Eshed and Zamir (1995). Additive effect = (Phenotypic value of homozygous single segment substitution lines Phenotypic value of control) / 2; Percentage of additive effect = (Additive value / Phenotypic value of control) 100%. Substitution mapping of the regions of QTLs for grain shape was determined by using SSSLs and accorded to the method of Paterson et al (1990). RESULTS Detection of QTLs for rice grain shape by SSSLs The result of ANOVA showed that 22 QTLs for grain shape were detected in 14 SSSLs, including 7 QTLs for grain length, 6 QTLs for grain width, 5 QTLs for length-width ratio and 4 QTLs for grain thickness. These QTLs distributed on eight chromosomes except chromosomes 6, 9, 11 and 12 (Table 2). In this study, different QTLs for grain shape were detected in an SSSL. For example, the QTLs for grain length (qgl-3) and length-width ratio (qlwr-3) were Table 1. Single segment substitution lines used in the experiment. Code Chromosome Substitution segment C1 1 LSTS1-40 LSTS1-41 RM129 RM11160 RM11189 RM11229 S1-7 RM11410 C2 1 RM11160 RM11189 RM11229 S1-7 RM11410 C3 2 RM12521 RM12705 RM12769 C4 2 RM13617 RM13672 C5 2 RM13034 ZSTS2-5 RM3443 ZSTS2-6 ZSTS2-7 RM13263 S2-5 S2-6 C6 2 RM13034 ZSTS2-5 RM3443 ZSTS2-6 ZSTS2-7 C7 2 S2-5 S2-6 C8 3 S3-1 RM14360 ZSTS3-2 RM5819 C9 3 S3-1 RM14360 C10 3 S3-1 RM14360 ZSTS3-2 C11 3 RM3291 ZSTS3-8 ZSTS3-9 S3-8 ZSTS3-11 C12 4 S4-2 S4-3 C13 5 RM3089 RM3170 RM19199 C14 5 RM3170 RM19199 C15 5 S5-1 S5-12 S5-2 RM7293 S5-3 C16 7 RM21767 RM21856 C17 7 RM5672 RM21344 RM5499 S7-3 RM21529 C18 8 RM23642 C19 10 RM25626 RM25735 C20 10 RM24865 RM24934 C21 10 RM24865 C22 10 RM24865 RM24934 RM25041

3 LI Sheng-qiang, et al. QTL Detection for Rice Grain Shape Using SSSLs 275 Table 2. Additive effects of QTLs for the traits of grain shape in the single segment substitution lines in rice (P<0.01). Trait Code of SSSL Chromosome QTL Mean ± SD A R 2 (%) Grain length (mm) Nipponbare 7.33±0.21 C3 2 qgl ± C11 3 qgl ± C12 4 qgl ± C15 5 qgl ± C16 7 qgl ± C17 7 qgl ± C19 10 qgl ± Grain width (mm) Nipponbare 3.35±0.11 C1 1 qgw ± C4 2 qgw ± C13 5 qgw ± C15 5 qgw ± C16 7 qgw ± C22 10 qgw ± Grain length-width ratio Nipponbare 2.20±0.12 C5 2 qlwr ± C11 3 qlwr ± C15 5 qlwr ± C17 7 qlwr ± C18 8 qlwr ± Grain thickness (mm) Nipponbare 2.26±0.08 C8 3 qgt ± C13 5 qgt ± C15 5 qgt ± C16 7 qgt ± A, Indicates additive effect; R 2, Indicates percentage of additive effect. simultaneously detected in the SSSL C11. The QTLs for grain width (qgw-5-1) and grain thickness (qgt-5-1) were detected in C13. The QTLs for grain length (qgl-7-1) and length-width ratio (qgw-7) were detected in C16. The QTLs for grain length (qgl-7-2) and length-width ratio (qlwr-7) were detected in C17. The QTLs for grain shape including grain length (qgl-5), width (qgw-5-2), length-width ratio (qlwr-5) and thickness (qgt-5-2) were all detected in C15. The QTLs controlling one trait of grain shape were also detected in different SSSLs. The QTLs for grain length were detected in C3, C11, C12, C15, C16, C17 and C19, and distributed on chromosomes 2, 3, 4, 5, 7, 7 and 10, respectively. The QTLs for grain width were detected in C1, C4, C13, C15, C16 and C22, and distributed on chromosomes 1, 2, 5, 5, 7 and 10, respectively. The QTLs for length-width ratio were detected in C5, C11, C15, C17 and C18, and distributed on chromosomes 2, 3, 5, 7 and 8, respectively. The QTLs for grain thickness were identified in C8, C13, C15 and C16, and distributed on chromosomes 3, 5, 5 and 7, respectively. The results showed that the grain shape were complex quantitative traits controlled by several genes. Substitution mapping of QTLs for rice grain shape Using phenotypical and genotypical data of the overlapping SSSLs, we conducted the substitution mapping of chromosome regions affecting grain shape. Six regions carrying putative QTLs for grain shape were narrowed (Fig. 1 and Fig. 2). The QTL qgw-1 for grain width was detected in C1, but not in C2. The QTL was determined on the non-overlapping region of the two SSSLs spanning 4.7 Mb. The QTL qlwr-2 for length-width ratio was detected in C5, but not in C6 and C7. The QTL was determined on the non-overlapping region of the three SSSLs spanning 2.05 Mb. The QTL qgt-3 for grain thickness was detected in C8, but not in C9 and C10. The QTL was determined on the non-overlapping region of the three SSSLs spanning 2.2 Mb. The QTLs qgt-5-1 for grain thickness and qgw-5-1 for grain width were detected in C13, but not in C14. The QTLs were determined on the non-overlapping region of the two SSSLs spanning 0.75 Mb. The QTL qgw-10 for grain width was detected in C22, but not in C20 and C21. The QTL was determined on the nonoverlapping region of the two SSSLs spanning 2.9 Mb.

4 276 Rice Science, Vol. 18, No. 4, 2011 Fig. 1. Substitution mapping of QTLs for the traits of rice grain shape. The substituted segments are represented by horizontal dark bars with the C code. The regions to which the substituted segments best map QTL are shown by two vertical dotted lines. Fig. 2. Chromosomal loci of QTLs for the traits of rice grain shape. Molecular markers are indicated on the right of the chromosomes. Physical locations (Mb) are shown on the left of the chromosomes. Black bars in each chromosome refer to the intervals with the QTL identified on the right. DISCUSSION Rice grain shape is an important trait for both appearance quality and yield. Many studies observed that QTLs for grain shape were distributed on 12 rice chromosomes. The QTLs for grain length were distributed on 11 rice chromosomes except chromosome 11. The QTLs for grain width were detected on chromosomes 1, 2, 3, 4, 5, 7, 8 and 10 (Xing et al, 2001; Shinya et al, 2002; Wu et al, 2002; Li et al, 2003; Yan et al, 2003; Li et al, 2004; Liu et al, 2004; Zhang et al, 2004; Wan et al, 2005; Zeng et al, 2006; Zhou et al, 2006; Yu et al, 2008; Zhao et al, 2008). Using two F 6 populations developed from two residual heterozygous lines (RHLs), the QTLs for grain length and grain width were located at the intervals RMl0390 RMl344 and RMl0376 RMl0398 on chromosome 1, respectively (Yu et al, 2008). In the backcross population developed from Balilla and Nantehao, a QTL for grain length was detected at the interval RM101 RM270 on chromosome 12, and two QTLs for grain width were detected at the intervals RM154 RM21 on chromosome 2 and RM257 RM175 on chromosome 3, respectively (Yan et al, 2003). GL2(t), a grainlength dominant gene, was located at the interval

5 LI Sheng-qiang, et al. QTL Detection for Rice Grain Shape Using SSSLs 277 RM13955 RM530 on chromosome 2 using near isogenic lines (NILs) (Zhao et al, 2008). QTL for grain weight and grain shape (gw3.1) was located in a 93.8-kb region using NILs (Zhou et al, 2006). Using a segregating population derived from SSSLs, QTL for grain length (gl3) was located at the interval RM6146 PSM377 on chromosome 3, and QTL for grain width (GW8) was located at the interval RM502 RM447 on chromosome 8 (Zeng et al, 2006). LK-4(t) controlling grain length was located between the molecular markers P1-VeoRV and P2-ScaI using BC 2 F 2 (Zhou et al, 2006). Two genes (GW3 and GW6) controlling grain weight were mapped at the intervals WGW16 WGW19 on chromosome 3 and RM7179 RM3187 on chromosome 6 (Guo et al, 2008). At present, some QTLs for grain shape have been cloned, such as GS3 for grain length and grain weight (Fan et al, 2006), GW2 for grain width (Song et al, 2007) and GW5 for grain weight (Weng et al, 2007). In this study, using 22 SSSLs developed from a backcross between the japonica recipient Nipponbare and the indica donor Guanglu ai 4, 22 QTLs for grain shape were detected on eight chromosomes except chromosomes 6, 9, 11 and 12, including 7 QTLs for grain length, 6 for grain width, 5 for length-width ratio and 4 for grain thickness. Compared with the results of previous studies, it is obvious that qgw-1 is differed from the locus identified by Xu et al (2008); qgl-2 is near the QTL for grain width but not the same locus identified by Yan et al (2003); qgw-2 is a different locus identified from GL2(t) by Zhao et al (2008); qlwr-8 is near GW-8 but not the same locus identified by Zeng et al (2006); QTLs qgl-3 and qlwr-3 were detected in the SSSL C11, which spanned gl3.1, gl3 and LK4(t). It remains to determine whether they are different alleles on the same loci. Grain shape is indicated by length-width ratio. In fact, grain shape is a comprehensive trait including grain length, grain width and grain thickness. Some QTLs affecting different grain shape traits were detected on the same segment. In this study, both QTLs for grain length and grain width were detected in C11 (qgl-3 and qlwr-3) and C17 (qgl-7-2 and qlwr-7), respectively. QTLs for grain width and grain thickness were both detected in C13 (qgw-5-1 and qgt-5-1). QTLs for grain length (qgl-7-1), grain width (qgw-7) and grain thickness (qgt-7) were detected in C16. In C15, QTLs for grain length (qgl-5), grain width (qgw-5-2), grain length-width ratio (qlwr-5) and grain thickness (qgt-5-2) were all detected. According to the directions of QTLs effect, qgl-5 was contrary to qgw-5-2 and qgt-5-2, and qgl-7-1 was contrary to qgw-7 and qgt-7. The results showed that although the grain length was negatively correlated with grain width, the QTLs with the same direction for the components of grain shape were detected, and these loci might increase the grain length, grain width and grain thickness simultaneously. It is speculated that QTLs for grain length was tightly linked to those for grain width or grain thickness, and the pleiotropy were also observed between QTLs for grain width and grain thickness. In SSSLs, each substitutive segment contains only one substituted segment from the donor in the genetic background of the recipient, and all the genetic variation between it and the recipient is considered to be associated with the substituted segment. Compared with traditionally primary mapping populations, the QTLs with relatively small individual effect of each gene on phenotype could be detected, because the SSSLs might avoid gene interaction and minimize genetic background noise, and increase the accuracy and sensitivity for QTL detection. In rice, a series of SSSLs were developed with different donors and genetic backgrounds using various indica-japoncia crosses. Many QTLs for various phenotypic traits were detected by SSSLs, such as QTLs affecting eating quality of cooked rice (Wan et al, 2004), endosperm chalkiness characteristics (Wan et al, 2005), rice grain length (Wan et al, 2006), panicle architecture (Ando et al, 2008), and tiller number (Liu et al, 2009). The results of the studies indicate that SSSLs are considered as powerful tools for detecting QTLs for complex agronomic traits and breeding in rice. ACKNOWLEDGEMENT This work was supported by the National Basic Research Program of China (Grant No. 2005CB120807). REFERENCES Ando T, Yamamoto T, Shimizu T, Ma X F, Shomura A, Takeuchi Y,

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