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Abstract To provide new experimental materials for QTL analysis of rice yield trait， we constructed a mapping population of 150 1ines （recombination inbred lines， R1L） derived from a cross between rice varieties V20B and CPSLO17， and localized QTLs and evaluated the genetic effects in the two parents and 150 RILs for thousand??grain weight trait by using internal mapping method of software MapQTL5 combining thousand??grain weight phenotypic data of the RILs. The results showed that a new QTL （qTGW??3） related to thousand??grain weight trait was detected. Individual QTL （LOD=4.14） explained 11.9% of the observed phenotypic variance. And the QTL alleles came from the parent V20B.
Key words Rice； RIL； Thousand??grain weight； Mapping population； QTL
Thousand??grain weight （TGW） is a very important agronomic trait， which is one of the three components deciding rice yield， as well as a factor with the highest heritability the easiest to be controlled manually. Progresses have been made in the research field of important economic traits in rice， and the molecular mechanism controlling thousand??grain weight has started to take shape. Grain weight is controlled by subtraits such as grain width， grain length， grain thickness and grain plumpness， while these subtraits are quantitative traits controlled by multiple genes， and might counterbalance mutually to control thousand??grain weight together.
Researches have shown that thousand??grain weight is a complicated quantitative trait controlled by polygenes， and multiple major QTL genes have been cloned successfully， while these major QTL genes also control other traits. Gs3 gene[3-43] is a major QTL gene controlling rice grain length and grain weight， which is obtained through cloning by map??based cloning. GW2 gene[5] and qSW5 gene[6] are major QTL genes controlling rice grain width and grain weight， while GIF1 gene[7] is a major QTL gene controlling rice grain filling rate and grain weight. Currently， studies on QTLs of thousand??grain weight are mainly conducted on the background of indica??indica hybridization or indica??japonica hybridization. In this study， RILs newly constructed on the background of indica??japonica hybridization with widely??compatible CPSLO17 and indica V20B with strong combining ability as parents were collected as materials， and QTL mapping and genetic effect analysis for thousand??grain weight were performed using thousand??grain weight as phenotypic data combining high??density SLAF linkage map. This study provides new materials for fine mapping of QTLs of thousand??grain weight trait in rice and cloning of genes controlling yield， and lays a theoretical basis for marker??assisted selection （MAS） of high??yield rice varieties. Materials and Methods
Tested materials
V20B/CPSLO17 recombination inbred lines （RILs） were obtained by single seed descent method from the F1 generation. Parents and RILs were planted in experimental field of Guizhou Rice Research Institute （Guiyang） according to 20 plants per material. The seeds of two parents and 150 RIL materials were harvested at maturation stage， dried and preserved， for QTL analysis of thousand??grain weight of rice.
Determination of thousand??grain weight
Each material was collected and stored， and thousand??grain weight was determined three months later. During the determination， 50 preserved full grains were weighed with an electronic balance. For each material， three replicates were done， and the values were averaged， obtaining the average value which was converted to thousand??grain weight as the phenotypic value of this trait.
QTL analysis
Molecular data of SLAF tags of V20B/CPSLO17 RILs （not reported） were obtained by Biomarker Technologies with SLAF??seq （Specific??Locus Amplified Fragment Sequencing） technique[8] and software HighMap[9]. This map have 8 602 high??quality SLAF tags in total， which are uniformly distributed on 12 chromosomes； and this map covers 2 508.65 cM of rice genome， with a mean distance of 0.292 cM between markers. QTL analysis of thousand??grain weight was performed by Internal Mapping method with software MapQTL5， during which the scanning step and LOD value were set as 1.0 cM and 3.9， respectively， and the contribution rate and additive effect of each QTL were calculated. QTLs were named according to the method put forward by McCouch et al.[10]. A positive additive effect value indicates that the increasing effective allele is from cold??tolerant parent V20B， while a negative value means that the increasing effective allele is from non??tolerant parent CPSLO17.
Results and Analysis
Phenotypic data of thousand??grain weights of parents and RIL populations
Each material was determined for thousand??grain weight three months after collection and preservation. Parent V20B had large grains with a thousand??grain weight of 27.71 g （left arrow in Fig. 1）. Parent CPSLO17 had thin long grains with a thousand??grain weight of 21.47 g （right arrow in Fig. 1）. The thousand??grain weights of 150 V20B/CPSLO17 RILs were determined， and their distribution is shown in Fig. 1. The 150 RILs had the thousand??grain weights in continuous distribution from 15 to 35 g （Fig. 1）， with an average value of 24.98. Thousand??grain weight exhibited the genetic characteristics of quantitative traits controlled by polygenes. It could be seen from the QTL analysis of rice thousand??grain weight that a QTL of rice thousand??grain weight was detected on chromosome 3 and designated as qTGW??3. This QTL was corresponding to the linkage group position of Marker823024??Marker900904， and its additive effect and dominant effect were 1.263 25 and 0.586 75， respectively qTGW??3 had an LOD value of 4.14， and explained 11.9% of the observed phenotypic variance. The grain weight QTL allele came from parent V20B.
Results and Discussion
It could be seen from studies at home and abroad that grain weight trait in rice is a complicated quantitative trait controlled polygenes， with complicated genetic background and extensive genetic diversity. In this study， a high??density linkable map was constructed according to phenotypic data of thousand??grain weight trait in 150 V20B/CPSLO17 RILs using 8 602 high??quality SLAF tags， and QTL analysis of grain weight trait was performed by Internal Mapping method in software MapQTL5， during which one grain weight QTL （qTGW??3） was detected on chromosome 3. Through searching of grain weight genes in China Rice Data Center （http：//www.ricedata.cn/gene/）， three grain weight genes TGW3b[11]， gw3.1[12] and GS3[3-4] were found on chromosome 3， among which TGW3b is preliminarily located in the interval between RM15885 and W3D16 with a genetic distance of 2.6 cM，gw3.1 is fine mapped in the physical interval between JL123/RM640 and JL109/RM636 with a genetic distance of 93.8 kb， and only GS3 gene （RAP??DB： Os03g0407400） is cloned successfully. Through blast alignment on Gramene Markers Database， NCBI and RGAP， it was found that the physical location interval of qTGW??3 on chromosome is different from TGW3， gw3.1 and GS3 genes， suggesting it is a new grain weight QTL controlling rice grain weight， while whether qTGW??3 gene participates in the regulation of other yield traits still needs further study.
References
[1] XING YZ， ZHANG QF. Genetic and molecular bases of rice yield[J]. Annu Rev Plant Biol， 2010， 61：421-442
[2] MIURA K， ASHIKARI M， MATSUOKA M. The role of QTLs in the breeding of high??yielding rice [J]. Trends Plant Sci， 2011， 16： 319-326
[3] FAN CC， YU XQ， XING YZ， et al. The main effects， epistatic effects and environmental interactions of QTLs on the cooking and eating quality of rice in a doubled??haploid line population [J]. Theor Appl Genet， 2005， 110（8）： 1445-1452
[4] MAO HL， SUN SY， YAO JL， et al. Linking differential domain functions of the GS3 protein to natural variation of grain size in rice [J]. Proc Natl Acad Sci USA， 2010， 107（45）： 19579-19584 [5] SONG X， HUANG W， SHI M， et al. A QTL for rice grain width and weight encodes a previously unknown RING??type E3 ubiquitin ligase [J]. Nat Genet， 2007， 39： 623-630
[6] SHOMURA A， IZAWA T， EBANA K， et al. Deletion in a gene associated with grain size increased yields during rice domestication [J]. Nat Genet， 2008， 40：1023-1028
[7] WANG E， WANG J， ZHU X， et al. Control of rice grain??filling and yield by a gene with a potential signature of domestication [J]. Nat Genet， 2008a， 40： 1370-1374
[8] SUN X， LIU D， ZHANG X， et al. SLAF??seq： an efficient method of large??scale De novo SNP discovery and genotyping using high??throughput sequencing [J]. PLoS One， 2013， 8（3）： e58700
[9] LIU D， MA C， HONG W， et al. Construction and analysis of high??density linkage map using high??throughput sequencing data [J]. PLoS One， 2014， 9（6）： e98855
[10] MACOUCH SR， CHO YG， YANG M， et al. Report on QTL nomenclature [J]. Rice Genet Newslett ， 1997， 14：11-13
[11] LIU TM， SHAO D， KOVI MR et al. Mapping and validation of quantitative trait loci for spikelets per panicle and 1 000??grain weight in rice （Oryza sativa L.）[J]. Theoretical and Applied Genetics， 2010， 120（5）： 933-942.
[12] LI JM， THOMSON M， MCCOUCH SR. Fine mapping of a grain??weight quantitative trait locus in the pericentromeric region of rice chromosome 3[J]. Genetics， 2004， 168（4）： 2187-2195.
Key words Rice； RIL； Thousand??grain weight； Mapping population； QTL
Thousand??grain weight （TGW） is a very important agronomic trait， which is one of the three components deciding rice yield， as well as a factor with the highest heritability the easiest to be controlled manually. Progresses have been made in the research field of important economic traits in rice， and the molecular mechanism controlling thousand??grain weight has started to take shape. Grain weight is controlled by subtraits such as grain width， grain length， grain thickness and grain plumpness， while these subtraits are quantitative traits controlled by multiple genes， and might counterbalance mutually to control thousand??grain weight together.
Researches have shown that thousand??grain weight is a complicated quantitative trait controlled by polygenes， and multiple major QTL genes have been cloned successfully， while these major QTL genes also control other traits. Gs3 gene[3-43] is a major QTL gene controlling rice grain length and grain weight， which is obtained through cloning by map??based cloning. GW2 gene[5] and qSW5 gene[6] are major QTL genes controlling rice grain width and grain weight， while GIF1 gene[7] is a major QTL gene controlling rice grain filling rate and grain weight. Currently， studies on QTLs of thousand??grain weight are mainly conducted on the background of indica??indica hybridization or indica??japonica hybridization. In this study， RILs newly constructed on the background of indica??japonica hybridization with widely??compatible CPSLO17 and indica V20B with strong combining ability as parents were collected as materials， and QTL mapping and genetic effect analysis for thousand??grain weight were performed using thousand??grain weight as phenotypic data combining high??density SLAF linkage map. This study provides new materials for fine mapping of QTLs of thousand??grain weight trait in rice and cloning of genes controlling yield， and lays a theoretical basis for marker??assisted selection （MAS） of high??yield rice varieties. Materials and Methods
Tested materials
V20B/CPSLO17 recombination inbred lines （RILs） were obtained by single seed descent method from the F1 generation. Parents and RILs were planted in experimental field of Guizhou Rice Research Institute （Guiyang） according to 20 plants per material. The seeds of two parents and 150 RIL materials were harvested at maturation stage， dried and preserved， for QTL analysis of thousand??grain weight of rice.
Determination of thousand??grain weight
Each material was collected and stored， and thousand??grain weight was determined three months later. During the determination， 50 preserved full grains were weighed with an electronic balance. For each material， three replicates were done， and the values were averaged， obtaining the average value which was converted to thousand??grain weight as the phenotypic value of this trait.
QTL analysis
Molecular data of SLAF tags of V20B/CPSLO17 RILs （not reported） were obtained by Biomarker Technologies with SLAF??seq （Specific??Locus Amplified Fragment Sequencing） technique[8] and software HighMap[9]. This map have 8 602 high??quality SLAF tags in total， which are uniformly distributed on 12 chromosomes； and this map covers 2 508.65 cM of rice genome， with a mean distance of 0.292 cM between markers. QTL analysis of thousand??grain weight was performed by Internal Mapping method with software MapQTL5， during which the scanning step and LOD value were set as 1.0 cM and 3.9， respectively， and the contribution rate and additive effect of each QTL were calculated. QTLs were named according to the method put forward by McCouch et al.[10]. A positive additive effect value indicates that the increasing effective allele is from cold??tolerant parent V20B， while a negative value means that the increasing effective allele is from non??tolerant parent CPSLO17.
Results and Analysis
Phenotypic data of thousand??grain weights of parents and RIL populations
Each material was determined for thousand??grain weight three months after collection and preservation. Parent V20B had large grains with a thousand??grain weight of 27.71 g （left arrow in Fig. 1）. Parent CPSLO17 had thin long grains with a thousand??grain weight of 21.47 g （right arrow in Fig. 1）. The thousand??grain weights of 150 V20B/CPSLO17 RILs were determined， and their distribution is shown in Fig. 1. The 150 RILs had the thousand??grain weights in continuous distribution from 15 to 35 g （Fig. 1）， with an average value of 24.98. Thousand??grain weight exhibited the genetic characteristics of quantitative traits controlled by polygenes. It could be seen from the QTL analysis of rice thousand??grain weight that a QTL of rice thousand??grain weight was detected on chromosome 3 and designated as qTGW??3. This QTL was corresponding to the linkage group position of Marker823024??Marker900904， and its additive effect and dominant effect were 1.263 25 and 0.586 75， respectively qTGW??3 had an LOD value of 4.14， and explained 11.9% of the observed phenotypic variance. The grain weight QTL allele came from parent V20B.
Results and Discussion
It could be seen from studies at home and abroad that grain weight trait in rice is a complicated quantitative trait controlled polygenes， with complicated genetic background and extensive genetic diversity. In this study， a high??density linkable map was constructed according to phenotypic data of thousand??grain weight trait in 150 V20B/CPSLO17 RILs using 8 602 high??quality SLAF tags， and QTL analysis of grain weight trait was performed by Internal Mapping method in software MapQTL5， during which one grain weight QTL （qTGW??3） was detected on chromosome 3. Through searching of grain weight genes in China Rice Data Center （http：//www.ricedata.cn/gene/）， three grain weight genes TGW3b[11]， gw3.1[12] and GS3[3-4] were found on chromosome 3， among which TGW3b is preliminarily located in the interval between RM15885 and W3D16 with a genetic distance of 2.6 cM，gw3.1 is fine mapped in the physical interval between JL123/RM640 and JL109/RM636 with a genetic distance of 93.8 kb， and only GS3 gene （RAP??DB： Os03g0407400） is cloned successfully. Through blast alignment on Gramene Markers Database， NCBI and RGAP， it was found that the physical location interval of qTGW??3 on chromosome is different from TGW3， gw3.1 and GS3 genes， suggesting it is a new grain weight QTL controlling rice grain weight， while whether qTGW??3 gene participates in the regulation of other yield traits still needs further study.
References
[1] XING YZ， ZHANG QF. Genetic and molecular bases of rice yield[J]. Annu Rev Plant Biol， 2010， 61：421-442
[2] MIURA K， ASHIKARI M， MATSUOKA M. The role of QTLs in the breeding of high??yielding rice [J]. Trends Plant Sci， 2011， 16： 319-326
[3] FAN CC， YU XQ， XING YZ， et al. The main effects， epistatic effects and environmental interactions of QTLs on the cooking and eating quality of rice in a doubled??haploid line population [J]. Theor Appl Genet， 2005， 110（8）： 1445-1452
[4] MAO HL， SUN SY， YAO JL， et al. Linking differential domain functions of the GS3 protein to natural variation of grain size in rice [J]. Proc Natl Acad Sci USA， 2010， 107（45）： 19579-19584 [5] SONG X， HUANG W， SHI M， et al. A QTL for rice grain width and weight encodes a previously unknown RING??type E3 ubiquitin ligase [J]. Nat Genet， 2007， 39： 623-630
[6] SHOMURA A， IZAWA T， EBANA K， et al. Deletion in a gene associated with grain size increased yields during rice domestication [J]. Nat Genet， 2008， 40：1023-1028
[7] WANG E， WANG J， ZHU X， et al. Control of rice grain??filling and yield by a gene with a potential signature of domestication [J]. Nat Genet， 2008a， 40： 1370-1374
[8] SUN X， LIU D， ZHANG X， et al. SLAF??seq： an efficient method of large??scale De novo SNP discovery and genotyping using high??throughput sequencing [J]. PLoS One， 2013， 8（3）： e58700
[9] LIU D， MA C， HONG W， et al. Construction and analysis of high??density linkage map using high??throughput sequencing data [J]. PLoS One， 2014， 9（6）： e98855
[10] MACOUCH SR， CHO YG， YANG M， et al. Report on QTL nomenclature [J]. Rice Genet Newslett ， 1997， 14：11-13
[11] LIU TM， SHAO D， KOVI MR et al. Mapping and validation of quantitative trait loci for spikelets per panicle and 1 000??grain weight in rice （Oryza sativa L.）[J]. Theoretical and Applied Genetics， 2010， 120（5）： 933-942.
[12] LI JM， THOMSON M， MCCOUCH SR. Fine mapping of a grain??weight quantitative trait locus in the pericentromeric region of rice chromosome 3[J]. Genetics， 2004， 168（4）： 2187-2195.