Genetic And Evolution Analysis Of Extrafloral Nectary In Cotton

The extrafloral nectary is considered as an indirect defensive trait,plays an important role in plant-animal interaction.Although the extrafloral nectary are important in the system evolution of plant-animal and biological control.Extraflral nectary,known as the“the sleeping beauty”of plant science,has been focused on its roles in plant-animal interactions.However,there are few reports on its heredity and evolution.we used more than 20 wild cotton species and the cultivated species G.arboreum to study the origin,evolution,inheritance,expression regulation and metabolism of the extrafloral nectaries.The main experimental results are as follows:1.The nectariless is generated by strong artificial or natural selectionWe checked for foliar nectaries in 27 cotton species,All of the investigated species had extrafloral nectaries,except for the diploid cotton G.gossypiodes?Ulbrich?Standley?D₆?and allotetraploid G.tomentosum?AD₃?.We analysed the population differentiation?Fst?of nectary and nectariless groups of G.arboreum at the whole-genome scale,and found a strong chromosome difference between nectary and nectariless groups.We found the sweep region on Chr12 overlapped with the strong association signal of GWAS of nectary in?19.5 to?20.1 Mb region,indicating that the nectariless trait was artificially or naturally selected during improvement.2.GaNEC1 is a key gene to regulate nectary developmentWe selected an EFN-bearing accession and a non-EFN accession from the GWAS population as parents for construction of an F₂ population through hybridization and selfing.Next,we used a bulk segregation analysis?BSA?strategy to identify the potential nectary locus for primary mapping.As expected,the locus controlled nectary formation was also located on chromosome 12.Using a large F₂population,we encrypted the density of molecular markers,and eventually narrowed the nectary locus?nec1?into to a 96.6 kb region that spans three annotated genes.We further cloned and sequenced the three candidate genes,and found that only GaNEC1,encoding a PB1 domain-containing protein,harbering a 15-bp InDel and a nonsynonymous substitution,which were predicted to cause 3D structural changes in GaNEC1 protein that probaly caused the function divergent and confer the nectariless phenotype.3.The regulatory network of expression analysisTo gain insight into the network underlying nectary development,mRNA-Seq of EFN and non-EFN foliar mid-veins was performed to compare their gene expression.we constructed a regulatory network for nectary development,mRNA-Seq analysis showed that JA-related genes are up-regulated and cell wall-related genes are down-regulated in the nectary.Further analysis showed that GaNEC1 is connected to multiple hormone signalling pathways and Wounding-induced,suggesting that plant hormones may play a improtant role in indirect defensive of the extrafloral nectary.4.VIGS of GaNEC1 resulted in nectary cell enlargementTo knock down the expression of GaNEC1 in nectary accession,we constructed virus-induced gene silencing?VIGS?vector.GaNEC1 expression was significantly depressed with a smaller nectaries in the midrib of true leaves.We next used histochemical visualization to inspect the nectary structure.VIGS plants display shallower nectary tissue grooves and larger nectary parenchyma cells,induce morphoogical changes,GaNEC1 may be the key gene controlling nectary formation.5.Composition of cotton nectar is a complex mixtureWe measured the metabolic composition of nectar,EFN foliar mid-veins and non-EFN foliar mid-veins.The composition of the necatar is so complicated with more than 400 metabolites identified.These metabolites The expected sugars and amino acids,some unreported secondary metabolites are also uncorved in nectar.Phenolic acids metabolites?cinnamic acid,sinapyl alcohol and coniferyl alcohol?involved in phenylpropanoid biosynthesis are enriched in the nectar,indicating lignin may be involved in nectary development.Through the above studies,we revealed the relatioship of genetic and evolution in cotton extrafloral nectary,which play significant roles in plant-aninals interaction and pest-resistance breeding via biological controls.