Molecular Regulatory Mechanism Of Awn In Rice Residual Heterozygous Line

Awn of grains is an agronomic trait in rice（Oryza sativa L.）,which has undergone a long history of domestication.Cloning and studies on genes related to awn are helpful to elucidate the regulatory network of awn development,and also have great significance to reveal the evolution of rice.In this study,a set of mapping population named Residual Heterozygous Lines（RHLs）were constructed.Using high-throughput sequencing based on phenotypes,a high-density genetic map was constructed.The main results are as follows:（1）A line 402AF₈ showed segregation with long awn and short awn.Genetic analysis showed that this trait of awn length was controlled by a dominant locus,named Awn4-1.Based on the result of high-throughput sequencing,the molecular markers were designed to detect the heterozygous regions on chromosomes 4,6 and 9,and the genotypes of a molecular marker 425887 located on chromosome 4 was linked to the phenotypes.A line with 560 offspring（named 402AF₉）was used for verification.The results showed that the genotypes were linked to the phenotypes,indicating that 425887 was the linked marker in this line.Since 425887 is a molecular marker linked to the known awn-related gene An-2,this study suggests that the locus Awn4-1,may be an allele of An-2.（2）048AF₈ is another line with segregation for the awn length.In two F₉population048AF₉ and 048BF₉ derived from two 048AF₈plants,their separation ratios of long and short awn are 1:3 and 3:1,respectively.The results showed that the trait of awn length in048AF₉ was controlled by a recessive gene,and that of 048BF₉ was controlled by a dominant gene;the two genes were named Awn3-2 and Awn8-3,respectively.（3）A primary mapping with 10 plants of the 048AF₉ line found that Awn3-2 was linked to a marker 315921 on chromosome 3.Further mapping using 200 individuals of048AF₉ and 1380 individuals of 048AF₁₀located this recessive gene Awn3-2 to a 351 kb region.By expression and sequencing analyses of the 24 candidate genes in this mapped interval,a gene Os03g0407400 was identified as the most likely candidate for Awn3-2,which is the allelic gene of GS3 that is known for controlling the grain length.The gene Os03g0407400 has a single base mutation（C to A）in the second exon;all the recombinants with the Nipponbare type allele carrying the SNP of C base had no awn,while those with the 93-11 type allele carrying the SNP of A base had long awn.It is speculated that in the genetic background of this RHL,this defective-gene lossed its ability to inhibit the elongation of the awn,resulting the longer awn than that of 93-11.Therefore,Awn3-2 is a repressor gene of awn development.（4）Measurement of the grain weight,grain length and grain width of individuals of048AF₉with long awn and short awn phenotypes indicating that the long awn id associated with long grain.Therefore,Awn3-2 is a gene with pleiotropic effects controlling awn length and grain width.To comfirm the function of this gene,a CRISPR/Csa9 vector for knockout of Os03g0407400 was constructed,and used to transform the RHL.（5）High-throughput sequencing of pooled DNAs from 048BF₁₀（came from 048BF₉）individuals with long awn and those with short awn was used to the dominant locus Awn8-3.The result showed that Awn8-3 was located on chromosome 8 with a range of about 3 Mb between 23.2-26.2 Mb.In summary,this study rapidly identified one or more genes related to awn by using RHL and high-throughput sequencing.There findings are the foundation for further elucidating the genetic regulatory of awn development in rice.