| The improvement of rice quality is currently an important goal of rice breeding work.Rice quality is composed of multiple traits,which are jointly influenced by genetic and environmental factors.By exploring and studying genes that control traits related to rice quality,a certain theoretical and material basis can be provided for a deeper understanding of the genetic basis of rice quality.Chalkiness is one of the key determinants of rice quality.The formation of chalkiness has multiple negative effects on rice quality,resulting in poor processing quality,appearance quality,and cooking and eating quality.At present,a large number of chalkiness related QTLs have been identified,but there are very few cloned chalkiness genes.Therefore,identifying rice chalkiness genes through map-based cloning can help solve the problem of high chalkiness in rice production of China.Master Zhang Hao,who has graduated from the laboratory,constructed a high-generation backcross population using the tropical japonica rice variety CYPRESS as the donor parent and the temperate japonica rice variety KY131 as the recurrent parent,and identified three chalkiness related QTLs:q BR2,qWCR3,and qWCR11.On this basis,this study conducted genetic validation and fine mapping of these three QTLs,to clone novel genes that affect rice chalkiness.In addition,eating and cooking quality are also key indicators of rice quality,and starch content is the main trait that controls eating and cooking quality of rice.Although a QTL related to amylose content was identified in the preliminary work,its genetic effects were not verified in the subsequent studies.The synthesis of amylose in rice grains is mainly controlled by Wx,while the synthesis of amylopectin is regulated by multiple genes.Currently,we lack a comprehensive understanding of the genetic network of amylopectin synthesis in rice grains.Therefore,we utilized CRISPR/Cas9 technology to create multiple mutant materials for rice amylopectin synthesis related genes and analyzed the comprehensive impact of these genes on rice quality,which is beneficial for us to understand and utilize these genes.The main results are as follows:1.Verification of genetic effects of three chalkiness QTLs: The genetic effects of a white back rate QTL q BR2 and two white core rate QTLs qWCR3 and qWCR11 were repeatedly verified in different environments.The high chalkiness phenotypes of q BR2 and qWCR11 in NILs were all derived from the KY131 genotype,while the high chalkiness phenotypes of qWCR3 were derived from the CYPRESS genotype.2.Fine mapping of qWCR3: We developed a population of 500 plants and identified 8 recombinant single plants between markers D3-6 and C3.26.6.Ten new molecular markers between D3-6 and C3.26.6 were added for progeny testing.As a result,we fine mapped qWCR3 to 100 kb interval between 25.49 and 25.59 and determined the candidate gene for qWCR3 by comparing sequencing and expression of the genes within this interval.3.Using CRISPR/Cas9 technology,14 genes related to amylopectin synthesis,OsSSI,OsSSIIa,OsSSIIb,OsSSIIc,OsSSIIIa,OsSSIIIb,OsSSIva,OsSSIVb,OsSBE1,OsSBE2 b,OsSBE4,OsISA1,OsISA2,and OsISA3,were knocked out and homozygous mutant materials were obtained from the japonica rice variety Nipponbare.Amylose content,total starch content,amylopectin content,four storage protein content,and taste value of these materials were measured.We found that the amylose content,total starch content,and amylopectin content were the main influencing factors on the taste value of rice.The OsSBE2 b and OsISA1 mutation materials showed seed development defects,but both can germinate normally,which may be essential for the development of rice endosperm.This study used both forward and reverse genetic methods to validate and clone new chalky QTLs,created gene editing materials for starch synthesis genes.The results of this study can lay the foundation for the cloning of new chalky QTLs and provide new genetic materials for the study of rice eating and cooking quality. |
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