Fine-mapping And Candidate Analysis For QPC-10, Controlling Protein Content In Milled Rice

Rice is one of the main food crops in China, its planting area accounts for 30% in all food crops and its yield accounts for 40% in total food yield. So the rice production plays an essential role in ensuring grain safety in our country. However, as people’s living standard improves, grain quality is becoming much more popular to rice consumers. In recent decades, rice breeders have done a great deal of research on genetic analysis and improvement of rice grain quality. Some important progresses have been made, especially on starch quality improvement. However, the genetic basis of grain protein content remains unclear to date, the another main factor which having a great effect on rice cooking and eating quality. Therefore, to reveal the genetic basis underlying rice protein content is urgent for the improvement of rice quality in the rice breeding practice.In this study, the variation of protein content in milled rice in 334 rice germplasm resources was analyzed. And the analysis of quantitative trait locus (QTL) conferring the protein content was conducted through charactering a set of chromosome segment substitution lines (CSSL), which deriving from the cross between a japonica cultivar Sasanishiki and an indica cultivar Habataki. The results of the study are as follows:1. The milled rice protein content (PC) in 334 accessions was measured in two environments. The result showed that there was a wide range of variation in different accessions ranged from 6.67% to 14.33%. It also showed that the PC were generally higher in indica than those in japonica, most of rice accessions have intermediate PC, while there was very small number of varieties with either extreme high or low PC.2. A CSSLs population derived from the cross of Sasanishiki/Habataki was employed for PC measurement and related QTL analysis in three environments. The result showed that qPC-10 can be repeatedly detected in all three environments as well as qPC-1, indicating that qPC-10 is stably inherited. The allele from Habataki can significantly increase PC of milled rice in Sasanishiki background.3. The CSSL SL431 carrying the indica allele qPC-10 was used to cross with the recurrent parent Sasanishiki to develop BC1F2 segregation population. And the BC1F2 together with their parents were planted in the summer of Yangzhou. The PC and the marker genotypes of population were measured for further QTL analysis. The QTL analysis result indicated that qPC-10 was located between marker RM5758 and RM467 on chromosome 10, and the genetic distance between them is about 3.85 cM.4. In order to further narrow the target region, more polymorphic markers were developed in this region. On the other hand, some individual with heterozygous genotype in qPC-10 target region were selected to generate BC1F3 population for fine-mapping of qPC-10. Finally, qPC-10 was delimited to a region governed by the marker Y1 and Y3, the physical distance between them is about 35 kb.5. A set of nearly isogenic lines (NIL) for qPC-10, namely NIL-qPC-10H and NIL-qPC-10s, were developed on the basis of genotyping BC1F3 population. The agronomic traits and PC were measured in NILs. The result clearly showed that there was a significant difference in PC of milled rice, but no significant differences between Nils were found in the agronomic traits including tiller number,1000-grain weight, grain number per panicle, plant height, grain weight, grain length and heading date. Furthermore, the storage protein fraction contents of NIL were also measured, and the result showed that there was no significant difference in albumin, globulin, prolamin content other than glutelin content, indicating that the increase in glutelin content may be the main reason causing the higher total PC in NIL-qPC-10H.6. Bioinformatics information analysis indicated that four open reading frames (ORF) were located in this 35Kb-long DNA region, of which, LOC_Os10g26060 is predicted to function in 1 the synthesis of the glutelin. The sequencing result indicated that there were 7 in total deletion/insertion variation in the promoter region and 3’UTR between Sasanishiki and Habataki, no difference was found in the coding region. RT-PCR was used to investigate the expression pattern of LOC_Os10g26060. The LOC_Os10g26060 transcripts were abundant in endosperm and could be detected from 5 to 25 DAF (day after flowering), while no expression was detected in vegetative tissues examined. Additionally, the significant difference of expression level was observed on 15 DAF, with more strongly in NIL-qPC-10H than in NIL-qPC-10S during endosperm development. This result implied that the LOC_Os10g26060 may be the candidate gene of qPC-10 and the sequence variations in promoter may be the reason causing the different expression level.7. Real-time PCR analysis revealed that the genes,17KD-Prolamin、Glutelin-λ、GluAl、 GluA2、GluBl and GluB4, all of which are involved in synthesis of storage protein, were up-regulated in NIL-qPC-10H. On the contrary, 10KD-Prolamin and 13KD-Prolamin were down-regulated in NIL-qPC-10H. The result indicated that LOC_Os10g26060 may have positive or negative effects on these storage protein synthesis related genes. In addition, it was found that the expression level of several starch synthesis related genes were slightly altered between NILs, but not statistical significant.8. In order to distinguish the different alleles on qPC-10 in Habataki and Sasanishiki, a cleaved amplified polymorphic (CAPs) marker was developed on the basis of sequence variation in the promoter region of LOC_Os10g26060. This marker can be severed as an efficient tool in the molecular marker-assisted selection for qPC-10in rice grain quality improvement.