Identification And Validation Of The LAC6.1 Gene That Regulates Low Amylose Content In Rice

As an important food crop in China,it has been made important breakthroughs in rice production.However,with the improvement of peoples’living standards,there are increasingly higher requirements for the quality of rice.It is generally believed that the rice grain quality indicators are closely related to the amylose content in rice endosperm.It is well known that amylose in rice endosperm is mainly regulated by the waxy gene,and the natural allelic variation of this gene is an important reason for the differences in amylose content in different rice cultivars.Although the allelic variation of Wx gene is a good candidate for the gradient regulation of amylose content in rice,they are not fine enough on a small scale.Therefore,in order to achieve the goal of accurate design and utilization in rice breeding process.In the preliminary work,through the investigation of rice grain quality traits of a large number of rice germplasms,we screened two germplasms Wuxiangeng 113 and Xiangruanyu with low amylose content and excellent taste quality.Based on the sequence analysis of Wx gene in Xiangruanyu,we found that it may carry a new gene that is non-allele with the Wx gene to regulate low amylose content.Therefore,the quality characteristics of Xiangruanyu were compared with other soft rice varieties,and on the other hand,gene cloning and functional studies were carried out to indentify the new gene controling low amylose content.The above studies can provide useful genetic resources for high-quality design breeding of rice.The main results are as follows:1.The functional site of Wx gene were analyzed in 4 soft rice varieties,2 glutinous rice varieties and 2 normal japonica rice varieties,and the quality and starch structure of different varieties were systematically compared.The results showed that the soft rice varieties Xiangruanyu and Wuxianggeng 113 carried the normal Wxb allele,which indicate that Xiangruanyu and Wuxianggeng 113 might carry novel genes controling low amylose content.Besides,we found that rice grain from Xiangruanyu and Wuxiangeng 113 have lower amylose content than that of the other two soft rice varieties,and in terms of the grain gransparency,the two soft rice Xiangruanyu and Wuxiangeng 113 have less lower grain transparency compared with the other soft rice Nangeng5055 and Nangeng 9108.There were obvious cavities were found in the starch granulars of the four soft rice endosperms.It was found that amylose content had a negative correlation with the level of rice endosperm transparency,and the lower the amylose content,the larger and more the cavity within starch granules.Rice taste quality analysis showed that the soft rice with lower amylose content had a better taste value,which might be due that the rice with lower amylose content tended to have lower cool paste viscosity,setback value,and higher break down value.Starch fine structure analysis showed that endosperm starch form soft rice contains less amount of amylose and more of amylopectin short-chain compared with the two normal rice varieties.The results above provide a basis for the subsequent cultivation of new soft rice varieties and the cloning and utilization of candidate genes in future.2.To fine mapping the target gene controning low amylose content,an F2 population generated by the cross combination between Xiangruanyu and its sister line P17.Then 50 individiuls with extremely high and low amylose content were selected from the F2 population respectively,and the high and low amylose groups were mixed as a bulk.The two bulks and the corresponding parent rice varieties were then used for high-throughput re-sequencing analysis.The net reading after filtration is 83.87Gb,the Q30 reaches more than 90%,and the average GC content is 42.68%.The average comparison efficiency between the sample and the reference genome of japonica rice NIP was 98.52%,and the average coverage Depth was 51×.According to these results,the data volume of all samples is sufficient,the sequencing quality is confirmed,and the GC distribution is normal,which can be used for linkage analysis of subsequent traits.The results showed that there was a linkage region located on chromosome 6.We further analyzed the coding genes of the candidate region,and based on the prediction of variant sites and gene function,it was found that the candidate gene LOC_Os06g48530 encoded a zinc finger structural protein,where an SNP at exon 8 led to the early termination of its coding protein.Thus,the specific molecular marker of the SNP was developed and the genetic linkage analysis of a small population was performed,and it was found that the site was closely linked to the low amylose trait.Furthermore,the knockout rice line and complementary transgenic line of the target gene were constructed,and the results showed that the mutant line had a low amylose content phenotype,while the complementary transgenic line returned to normal amylose content.Subsequently,the gene was named as LAC6.1(Low Amylose Content 1 in Chromosome 6).3.In order to clarify the regulatory mechanism of LAC6.1 mutation on amylose synthesis,the expression profiles of Wx gene in the rice endosperm of Xiangruanyu and P17 were first analyzed.It was found that there was no significant difference in the total mRNA expression of Wx gene between the two lines,however,the abundance of mature mRNA of Wx gene in Xiangruanyu decreased significantly compared with P17.At the same time,the abundance of GBSSI protein in Xiangruanyu rice also decreased significantly.Then the splicing efficiency of Wx gene was further analyzed,and it was found that the abundance of Wx mature mRNA in Xiangruanyu was significantly lower than that P17,indicating that the loss of function of LAC6.1 gene caused a significant decrease in the splicing efficiency of Wx pr-mRNA,and also led to a decrease in the abundance of GBSSI.Besides,the grain quality of the LAC6.1 knockout line in Nip background was analyzed,and the results showed that the amylose content decreased significantly,and also rice grain showed a dark endosperm phenotype,though the taste value of rice increased significantly.As for the complementary transgenetic rice line,the amylose content recovered to about 15%,and the dark endosperm returned to transparency,and also the rice taste value decreased.In conclusion,in the present study,we identified a new type of soft rice with lower amylose content than the normal soft rice.What is more,by uing MutMap" method,gene LAC6.1,which controls low amylose content,was cloned and functionally verified.The results laid a foundation for the functional elucidation of LA C6.1.Besides,the LA C6.1 gene can serve as a potential target for the cultivation of new varieties of high-quality edible soft rice in rice breeding programs.