Fine Mapping Of Oat Dwarfing Gene Dw6

Oat（Avena sativa L.）is one of the most important cereal crops with high nutritional and economic values.However,most oat varieties have tall plants that are prone to lodging,leading to reduced yield and inferior quality.Dw6 is one of the eight officially reported oat dwarfing genes with the most potential for application in commercial breeding.Therefore,conducting a deep study on the influence of Dw6 on important agronomic traits and fine mapping of Dw6 are of great importance in further cloning the casual gene and the utilization in oat breeding.In this study,we used a recombinant inbred line（RIL）population along with two pairs of near-isogenic lines（NILs）derived from a cross between the tall variety“Caracas”and the dwarf oat variety“WAOA2132”carrying Dw6 to investigate the effects of Dw6 on oat agronomic traits.We further measured the active GA contents of the stem of the NILs at various developmental stages.Subsequently,we used the RIL population and an F₂population from the cross of one pair of NIL to genetically map the Dw6 gene.Finally,the candidate gene under the Dw6 locus was analyzed by the combination of the annotation information and transcriptome data from the peduncle of NILs at different developmental stages.The main results were as follows:1.Agronomic traits including plant height（PH）,hundred kernel weight（HKW）,kernel length（KL）,kernel width（KW）,kernel perimeter（KP）,productive tiller number（PTN）,spikelet number of the main tiller（SN）,and coleoptile length（CL）in RIL lines during 2019WJ,2019CZ and 2020WJ and in NIL isolines in 2022WJ were investigated.The results showed Dw6 significantly reduced the PH（34.87%-44.29%）,HKW（9.25%-30.14%）,KL（3.98%-14.35%）,and KP（1.71%-9.55%）,but increased the PTN.The effects of Dw6 on SN,KW,and CL were not uniform,depending on the tested environments or the genetic background of Dw6.The assessment of the root surface area（RSU）,length of all roots（LAR）,primary root number（PRN）,and maximum root length（MRL）of the NIL pairs showed that Dw6 had strong negative effects on RSU（24.44%-28.77%）and LAR（18.10%-20.96%）,but not affected the PRN and MRL.Further investigation of the internode number（IN）and length（IL）,and the cell size of the peduncle of the NIL pairs revealed that Dw6 reduced the length of all internodes and cell number of the peduncle,but not affected the IN,suggesting the reduced plant height is largely attributed to the decrease of cell length of the internode.2.The response of Dw6 to exogenous GA₃was investigated using the NILs.Exogenous GA₃significantly promoted internodes elongation of dwarf lines and restored the plant height of dwarf lines to a level similar to the tall lines,supporting Dw6 as a GA-sensitive gene.The GA₁,GA₃,GA₄,and GA₇contents in the stem of NILs at the jointing stage（Zadoks 33）,booting stage（Zadoks 43）,heading stage（Zadoks 53）,and filling stage（Zadoks 71）were determined,respectively.The results showed that GA₃and GA₄contents of dwarf lines were significantly lower than that of tall lines at all four developmental stages.The GA₇content of dwarf lines was significantly lower than that of tall lines at the booting,heading,and filling stages,but not at the jointing stage.No significant difference in the GA₁content was observed between the tall lines and dwarf lines at all except the filling stage,in which the GA₁content of dwarf lines was significantly lower than the tall lines.These results suggested that Dw6 reduced plant height by affecting the active gibberellin content in oat stem.3.The peduncles of the NIL pairs at flowering stage（Zadoks 65）and filling stage（Zadoks 71）were sampled and subjected to RNA sequencing.A total of 416 and 89common DEGs were detected in the two NIL pairs at the flowering stage and filling stages,respectively.GO and KEGG enrichment analysis of the common DEGs showed that the common DEGs were significantly enriched in the pathways the related to cell growth and development,which was consistent with the observation of the peduncle cell section.4.Genetic mapping of Dw6 based on phenotypes and genotypes of the RIL population localized Dw6 on chromosome 6D between markers SSR65 and SSR120.Further fine mapping using F₂population obtained from the NIL1-T/D and KASP markers developed based on transcriptome data finally localized Dw6 between markers SSR65 and KASP2,which are 0.15 Mb apart on the“”Sanfensan”reference genome sequence.Eight genes were annotated in this regions based on the annotation information from the“Sanfensan”reference genome.Of the eight genes,only A.satnud SFS6D01G001889 was differentially expressed between tall and dwarf lines.To further determine the candidate gene under the Dw6 locus,an attempt was made to obtain the coding sequences of the eight genes by Sanger sequencing.As of the completion of this thesis,sequencing work for five of these genes has been finished.Upon the CDS comparison between tall and dwarf isolines,it was found only A.satnud SFS6D01G001889 exhibited site variation.However,maker developed based on SNP site not accurately distinguish tall lines from different genetic backgrounds.Therefore,further studies are needed to comfirm whether the A.satnud SFS6D01G001889 gene is the candidate gene for Dw6.