Genetic Analysis And Gene Mapping Of Leaf Count In Tobacco Cultivar Jiucaiping 2

Leaf count is one of the crucial factors contributing to tobacco yield.To exploit the advantages of high leaf count,combined with yield and quality,in the tobacco cultivar Jiucaiping 2,this study employed the high-quality,low-leaf-count cultivar NC82 as the maternal parent and Jiucaiping 2 as the paternal parent.Six generations of genetic populations,including P₁,P₂,F₁,F₂,B₁,and B₂.A plant quantitative trait major gene+polygene mixed inheritance model was utilized to analyze the genetic patterns of leaf count in Jiucaiping 2.The BSA-seq sequencing and KASP technology were employed for gene mapping of leaf count in Jiucaiping 2,followed by the identification of key candidate genes through transcriptome sequencing.The main results of this study are as follows:（1）The average total number of leaves per plant for the tobacco variety Jiucaiping 2 was 29.33,while for NC82 it was 23.33.The leaf number between the two parents showed extremely significant differences,while the leaf number difference in the F₁ generation from reciprocal crosses was not significant,indicating that the leaf number trait is inherited by the nucleus.The total number of leaves per individual in the F₂ segregating population is a continuous distribution,indicating that the number of leaves in Jiucaiping 2 belongs to a quantitative trait inheritance.（2）The"major gene+polygene"mixed inheritance analysis of the six generations of genetic populations revealed that leaf count in Jiucaiping 2 is controlled by two pairs of additive-dominance-epistasis major genes,with the low-leaf trait being dominant over the high-leaf trait.The additive effects and dominance effects of the two pairs of major genes mainly exerted negative effects.In terms of epistasis,the additive×dominance,additive×additive,and dominance×dominance interactions positively regulated leaf count in Jiucaiping 2,while the dominance×additive interaction had a negative effect.The heritability（h²mg）of the two pairs of major genes in the B₁,B₂,and F₂ generations were 6.87%,45.51%,and 62.72%,respectively.（3）Using BSA-seq sequencing technology,the parental lines and extreme F₂ pools were sequenced.The preliminary mapping located the leaf count regulating gene within a 6.16 Mb interval on chromosome 9,containing 146 genes.Functional enrichment analysis of the candidate genes in the interval using GO and KEGG analysis indicated their involvement in plant respiration,photosynthesis,and the production and utilization of organic substances within the plant.（4）Utilizing KASP technology,five pairs of KASP primers were designed within the initial mapping interval and tested on all F₂ individuals,narrowing down the mapping interval to 2.00 Mb.Subsequently,two additional KASP primers were designed within this interval and tested on recombinant individuals,further narrowing down the candidate interval to 1.92 Mb,which contained 37 genes.Based on gene functional annotation,it is speculated that 11 genes within the candidate interval,such as Nitab4.5＿0004550g0110,Nitab4.5＿0002718g070,and Nitab4.5＿0008042g0010,are involved in regulating leaf count in tobacco.（5）Transcriptome analysis was performed on the parental lines,revealing a total of 4,112 DEGs（differentially expressed genes）between the two parents.These DEGs were mainly enriched in metabolic processes and hormone synthesis pathways.Among the 11 predicted genes within the fine mapping interval,four genes showed significant differences in expression levels:Nitab4.5＿0004550g0040,Nitab4.5＿0009025g0030,Nitab4.5＿0004622g0020（DHX16）,and Nitab4.5＿0004622g0050（K09264）.Nitab4.5＿0004550g0040 is annotated as a tobacco MYBX gene and is primarily involved in the regulation of flower color.Nitab4.5＿0009025g0030 is annotated with membrane protein function related to the SPX domain in tobacco,suggesting its involvement in maintaining phosphorus homeostasis within the plant.Nitab4.5＿0004622g0020（DHX16）is annotated as a tobacco pre-m RNA splicing factor ATP-dependent RNA helicase DEAH1,which plays a broad role in plant development,including leaf and floral organ development.Nitab4.5＿0004622g0050（K09264）is annotated as a member of the MADS-box gene family,which primarily participates in plant flowering time and floral organ development.Therefore,it is speculated that Nitab4.5＿0004622g0020（DHX16）and Nitab4.5＿0004622g0050（K09264）are key candidate genes involved in regulating leaf count in Jiucaiping 2.