Analysis Of QTL For Important Agronomic Traits Based On Rice CSSL-Z431 With Short-wide Grain And Multiple Panicles Per Plant And Candidate Gene Analysis Of QPN3,qGL3 And QGW5

The continuous increase of rice yield is of great practical significance to the food security of the people,especially in the case of the decrease of cultivated land and the increase of population.Therefore,improving rice yield is still main goal of rice breeding,especially with the growth of population and the decreasing of available field.With the completion of rice whole genome sequencing,the scientific research and variety breeding of rice have entered into the post-genome era.It is necessary to create bundful natural variation and identification of favorable quantitative trait loci（QTLs）for agronomic traits.Chromosome substitution segment lines can create abundant natural variations and are ideal mapping populations for identifying QTLs,and they can also be used directly in molecular breeding by design.Here,we identified a rice CSSL-Z431,which is mainly characterized by short-wide grain and multiple panicles per plant,derived from Xihui18 as recipient and Huhan3 as donor parent.In this study,Z431 was used as materials to carry out a list of studies.1.Analysis of substitution segments,phenotypype,and cell morphology in glumes of Z431Rice CSSL-Z431 contained six substitution segments from Huhan 3 based on the genetic background of Xihui18.The total length of these substitution segments was estimated as 12.71 Mb.Among them,length of the longest substitution segment of Z431was estimated as 3.76 Mb,the shortest one was 0.95 Mb and average length of these substitution segments was 2.12 Mb.Compared with Xihui18,the panicle length,number of grains per panicle,number of spikelets per panicle,grain length and ratio of length to width in Z431 decreased significantly by 20.37%,38.60%,37.77%,1.37%and33.24%,respectively.The number of panicles per plant,grain width,1000-grains weight and yield per plant of Z431 increased significantly than Xihui18 by 80.95%,30.00%,16.55%and 34.11%,respectively.There was no significant difference in seed setting rate（86.40%）between Z431 and that（87.99%）of Xihui18.The cell length of the glume in Z431 was shorter than that of Xihui18.While the cell width in Z431was wider than that of Xihui18.There was no significant difference in total cell number between Xihui18 and Z431 in the outer epidermis of the glume along the longitudinal axis.2.QTLs for important agronomic traits carried by the substitution segments of Z431A total of 13 QTLs for yield-related traits were detected in a secondary F₂ population constructed from Xihui 18/Z431,explaining phenotypic variation from 2.10%to 30.75%.Among them,there were one QTL for panicle length,number of panicles per plant,number of spikelets per panicle,number of grains per panicle,grain length and grain width,respectively,and 3 QTLs for ratio of length to width and 1000-grains weight,respectively.The short panicle of Z431 was controlled by qPL3,which decreased the panicle length by 0.89 cm.qPN3 increased the number of panicles per plant of Z431 by0.51,explained 6.75%of the phenotypic variation.qGL3 could decrease the grain length of Z431 by0.27 mm,explained 30.75%of the phenotypic variation.qGW5 increased the grain width of Z431 by0.09 mm,explained 2.10%of the phenotypic variation.The grain shape of Z431 is controlled by qRLW2,qRLW3 and qRLW5.The the 1000-grain weight of Z431 was controlled by two major QTL（qGWT3,qGWT-5-2）and one minor QTL（qGWT-5-1）.qGWT3 reduced the 1000-grain weight of Z431 by 1.09 g,while qGWT-5-1 and qGWT-5-2 increased by 0.84 and 0.76 g,respectively.In addition,we found some QTLs displayed in cluster and linked with the same marker.we analyzed the Pearson correlation coefficient for 10 yield-related traits in F₂ population by IBM SPSS Statistics 26.The number of panicles per plant（PN）displayed significantly positive correlations with the number of grains（GPP）and spikelets per panicle（SPP）,while no correlation with panicle length（PL）and Grain size related traits.The number of grains（GPP）exhibited significantly positive correlation with number of spikelets per panicle（SPP）and seed setting rate（SSR）.Grain size related traits（GL,GW,GWT,RLW）displayed significant correlation.Thus,qPN3 should not be controlled by same gene with qPL3,qGL3,qRLW3 and qGWT3.While qGL3,qRLW3,qGWT3 belonged to pleiotropy.Also,qGPP12 and qSPP12 were pleiotropy,similarly qSSR5,qRLW5 and qGWT-5-1should belong to pleiotropy.3.Development of secondary SSSLs,DSSLs and TSSLs with target QTLsBased on the QTL mapping,six SSSLs（S1～S6）,three DSSLs（D1～D3）and two TSSLs（T1 and T2）were further developed by MAS in F₃.4.Validation of QTLs using SSSLs and their analysis of additive effectsAmong 13 QTLs identified by F₂ population,eleven QTLs（qPN3,qGL3,qRLW3,qGWT3,qPL3,qRLW5-1,qGW5,qGWT5,qGWT5-2,qGPP12 and qSPP12）could be validated by SSSLs,but two minor QTLs（qSSR5,qGWT5-1）were not validated.In addition,fifteen QTLs（qGL1,qGW1,qRLW1,qGWT1,qYD1,qGL2,qGWT2,qYD2,qYD3,qGL5,qGW5-1,qPN5,qRLW5-2,qYD5 and qYD12）were detected by according SSSLs,indicating that SSSLs had a higher efficiency of QTL detection than F₂ population.5.Analysis of pyramid of QTLs for important agronomic traits based on DSSLs,TSSLs and according SSSLsSix DSSLs,2 TSSLs togegther with 6 SSSLs were used to analyze the additive and epistatic effects of QTLs and pyramiding effects.The result showed that The results showed that qGW1 and qGW5,qGWT2 and qGWT3,qGWT2 and qGWT5,qYD2 and qYD5 belonged to independent inheritance.While the other QTLs showed epistatic interaction.For example,pyramiding of qGL2（a=-0.47）and qGL3（a=-0.31）in D1 yielded an epistatic effect of 0.62,which reduced grain length genetically by 0.16 mm.D1 showed longer grain（10.03）than those（9.39 and 9.72）of S2（qGL2）and S3（qGL3）,displaying significant difference with S2.Pyramiding of qYD2（a=-1.69）,qYD3（a=6.72）and qYD5（a=7.45）in T2 produced an epistatic effect of-7.79,which increased the yield genetically by 4.69 g.The yield per plant（37.18 g）of T2 was significant higher than that（24.41）of S2 and that（27.66 g）of Xihui18,and no significant differences with those（41.25 and 42.70）of S3（qYD3）and S5（qYD5）.Pyramiding of qPN3（a=1.63）and qPN5（a=1.43）in D3 yielded an epistatic effect of-1.86,thus the genetic effect of PN in D3 was 1.20.D3 had less number of panicles（6.75）than those（7.60and 7.20）of S3（qPN3）and S5（qPN5）,however,with no significant differences among them.6.Analysis of candidate genes of qPN3,qGL3,and qGW5qPN3 was dissected into the single segment substitution Line S3,whose estimated substitution length was 1.48 Mb.By prediction of candidate genes and their DNA sequencing at the interval,reported gene（Os IAGLU）which influence the tiller formation by negatively regulating IAA were found existing four differences of single nucleotide polymorphisms（SNPs）in the CDS between Xihui18 and S3.Furthermore,qRT-PCR analysis showed that the expression levels of Os IAGLU in root,stem,leaf,sheath and panicle in S3 was higher than that in Xihui18.Thus,Os IAGLU should be the candidate gene for qPN3.qGL3 was also dissected into S3.By analysis of all the genes related to the regulation of grainsize at the substitution interval.We found six potential candidate genes.DNA sequencing showed that there were two SNP differences and two base deletion in candidate gene 2 between S3 and Xihui 18.However,qRT-PCR analysis showed that the expression level displayed no significant differences between S3 and Xihui18.For the candidate 4,we found differences of 4 SNPs between Xihui18 and S3.Intrigually,qRT-PCR analysis showed that expression levels of the gene in S3 were significantly higher than in Xihui18.About the candidate gene 6,there was a deletion of base and3 SNP differences.Simultaneously,qRT-PCR analysis showed that the expression levels of the gene in S3 were significantly higher than in Xihui 18.In conclusion,candidate gene 4 and 6 should be the best candidate genes of qGL3,and candidate gene 2 also acted as its candidate gene.qGW5 was dissected into single segment substitution line S5,whose estimated substitution interval of 1.68 Mb.At the interval we found 6 possible candidate genes.By DNA sequencing,for the candidate 1（RING-type E3 ubiquitin transferase）,there were 2 SNP differences in the CDS between Xihui18 and S5.qRT-PCR analysis showed that expression levels of the gene were significantly higher in panicle,while lower in root,stem,leaf and sheath of S5 than those in Xihui18.With regard to the candidate 2（Eukaryotic translation initiation factor 3 subunit）,there were 3 SNP differences in the CDS between Xihui18 and S5.Furthermore,qRT-PCR analysis showed that its expression levels were significantly higher in stem,leaf,sheath and panicle of S5 than that of Xihui18.The other four genes could not be found actual differences between S5 and Xihui18.In conclusion,the candidate gene 1（RING-type E3 ubiquitin transferase）and candidate gene 2（eukaryotic translation initiation factor 3subunit）should be the best candidate genes for qGW5.