Characterization Of CSSL-Z796 With Shortened Grain Length And Increased Grain Width And Gene Discovering Of Purple Hull In Rice

Grain shape related traits serve as important components in controlling the performance of grain weight,while grain weight presents as one of the main factors of the final grain yield in rice（Oryza sativa L.）,exhibiting complex genetic inherentance patterns.In rice or other crops,the complex traits can be dissected into simple patterns through the constructing of choromosomal segment substitution lines（CSSLs）,series of specific isogenic lines with more similar genetic background except special genomic segment（s）from donor that linked tightly to the interest traits.In the present study,we screened out CSSL-Z796 from the isogenic descdents carried chromosomal segments from donor of Huhan 3 under the genetic background of Xihui 18,an elite restorer line in rice.Shorteded grain length,increased grain width,and purple glume exhibited as the most outstanding phenotypic performacen of CSSL-Z796.Focused on the characterization of CSSL-Z796,we carried out two main work,including the QTL mapping of intertesed traits,and gene discovering of purple glume.The main resuls are as follows:1.Characterization of CSSL-Z796 and QTL mapping of intertested traits（1）Molecular and phenotypic characterization of CSSL-Z796Results showed that Z796 carries 6 substitution segments from Huhan3,which were located on chromosome 2（Chr2,one segment）,Chr3（two segments）,Chr5（one segment）,Chr6（Chr6,one segment）,and Chr12（Chr12,one segment）respectively.The total replacement fragment length is 26.84Mb,and the average length is 4.47Mb（1.41Mb-10.1 Mb）.Compared with the receptor Xihui 18,the grain width,seed setting rate,and 1000-grain weight of CSSL-Z796 were significantly increased by 1.14 mm,3.48%,and 2.74 g,respectively（P<0.05）.However,panicle length,grain length,and ratio of grain length to width were correspondingly decreased by 1.16 mm and 5.71 mm,and0.93,statistically lower than those of backbround Xihui 18（P<0.05）.（2）Cytological analysis of glumeThe results of scanning electron microscope（SEM）showed that the length of glume epicuticle cells of CSSL-Z796 was 17.64μm,shorter than that of Xihui 18（P<0.01）,and the width was increased by 5.04μm（P<0.01）,while there was no significant difference in the number of cells within the same scanning view between CSSL-Z796 and Xihui 18,indicating that the shortened and widened grains of CSSL-Z796 was mainly caused by the transverse expansion of cells.（3）QTL detection of interested traitsFourteen QTLs controlling grain length（GL）,grain width（GW）,ratio of length-width（RLW）,panicle length（PL）,1000-grain weight（GWT）,and seed setting rate（SSR）were detected through genotyping of F₂ population constructed by Xihui 18and CSSL-Z796.Three QTL were detected for GL,i.e.q GL2-1 and q GL2-2 increased performance of GL by 0.15 mm and 0.22 mm,respectively,while q GL3-1 reduced GL by 0.41 mm,with corresponding contribution rates（CRs）of 13.31%,29.43%,and50.67%.Two QTL were detected for GW,including qGW3-1 and qGW5,increased GW by 0.08 mm and 0.20 mm,respectively,with CRs of 12.68%and 79.68%,respectively.Another two QTL were detected for RLW,with CRs of 38.93%（q RLW3-1）and 37.09%（q RLW5）,which reduced RLW by 0.12 and 0.11,respectively.Only one QTL（q PL3）was detected for PL,with CR of 8.35%to shorten PL by 0.73 cm.Four QTL were detected for GWT,among which qGWT2,qGWT3-1,and qGWT5 increased GWT by2.12 g,1.03 g,and 1.36 g,respectively,with CRs of 55.60%,28.72%,and 38.29%,while qGWT3-2 reduced GWT by 1.55 g,with CR of 39.40%.Two QTL were detected for SSR,i.e.q SSR2-1 decreased SSR by 7.19%with CR of 13.00%,q SSR2-2 increased SSR by 13.95%with CR of 24.11%.（4）Interaction effect dissection of detected QTLBased on the QTL mapping results,molecular marker-assisted selection was performed to screening target individuals from F₃ population,and 11 further substitution lines were identified,including four single-segment substitution lines（S1-S4）,four double-segment substitution lines（D1-D4）,and three treble-segment substitution lines（T1-T3）.The genetic stability of q GL3-1,qGW3-1,and qGW5 was verified by S1-S4,and the estimated additive effects of the QTL were 0.07mm,0.14mm,and 0.15mm,respectively.The double-fragment substitution line,D3,carried both fragments from Chr3 and Chr5,containing the corresponding QTL of qGW3-1（additive effect was 0.11mm）and qGW5（additive effect was 0.26 mm）.The estimated epistatic effect of these two QTL to GL was-0.14mm.Therefore,the theoretical genetic effects of qGW3-1 and qGW5 pyramiding within D3 could lead to the increasing of GW by 0.23 mm（0.11+0.26-0.14=0.23）.Phenotyping results showed that the GW of D3 was wider than that of S2（only containing qGW3-1）,but narrower than that of S3（only containing qGW5）,indicating that the pyramiding of qGW3-1 and qGW5 did not produce wider grains.Similarly,D3 carried q GL3-1（additive effect was-0.14）and q GL5（additive effect was-0.15）,with the epistatic effect to GL was 0.12 mm.Therefore,the genetic effect of D3 reduced the grain length by 0.17 mm,suggesting that the pyramiding of q GL3-1 and q GL5 would produce shorter grains.2 Gene discovering of purple glume via CSSL-Z796（1）Anthocyanin content within grain and glume of CSSL-Z796The color of both Xihui 18 and Luhan 3 was yellow,while that of CSSL-Z796 was purple,the anthocyanin content within both grain and glume of CSSL-Z796 was determined at the maturity stage.The results showed that the content of anthocyanin in the glume of CSSL-Z796 was 0.018 mg/g higher than that of Xihui 18（P<0.05）,indicating that the purple color of CSSL-Z796 glume was mainly caused by the accumulation of anthocyanin.（2）Discovering and determining of candidate gene for purple glumeThrough liankage analysis of SSRs,the candidate gene for purple glume of CSSL-Z796 was preliminarily mapped in the6.52 Mb region between RM247-RM101on Chr12,containing 30 annotated genes.Results from the candidate gene prediction and DNA sequencing analysis showed that LOC＿OS12G07640,one of the annotated genes,possessed a deletion of CCG（coden for amino acid of Pro）at position 520,resulting in the differences for both DNA and amino acid sequences between CSSL-Z796 and sativa line of Xihui 18,while presenting the same to those of japonica lines in rice.Summarily,LOC＿OS12G07640 was finally determined as the candidate controlling purple glume of CSSL-Z796.