Organization Of Mating Type And Relationship Analysis Between Mating Pathway And Fv-hmg Genes In Flammulina Velutipes

Flammulina velutipes(Curt.ex Fr.)Sing,is a wide cultivated edible mushroom.There are abundant germplasm resources of F.velutipes in China which is the cradle and most cultivation country.The industry is still growing in high speed especially in industrialization that is an important direction.The number and quality of primordia are basis of high yield.But the regulatory mechanism of fruiting body formation is still unknown.F.velutipes is typical tetrapolar basidiomycetes and has clear stages in development.The genome of F.velutipes was published in 2014.Our lab also re-sequenced genomes of monokaryons L11 and W23 and sequenced different stages’transcriptomes of dikaryon H1123 which crossed by L11 and W23.So it is a good candidate model for fruiting body formation in edible mushroom.In this study,we analyzed the mating type genes of F.velutipes which controlling plasmogamy based on genomes and transcriptomes.We also obtained two transcription factors(TFs)that are up-regluated in primordia formation.Further,RNAi and overexpression transformants of these two TFs were constructed.Then,quantitative real-time PCR(RT-qPCR)was performed to explore the relationship between mating pathway and these two Fv-hmg genes.The main results are as follows:(1)We identified the mating type genes in monokaryons L11 and W23,which sequenced by our lab,using homologous alignment.Then,the gene models were corrected by transcriptome data to find the exon and intron boundary.Both MAT-A and MAT-B contain two subloci.Sublocus MAT-Aa is linked to Mitochondrial Intermediate Peptidase(MIP)gene.And this sublocus only can encode one HD2 protein;MAT-Ab containing a pair of Hd1/Hd2 genes located～73 kb to MAT-Aa.The distant between MAT-Ba and MAT-Bb is about 181 kb.Both of these two subloci are constituted of several pheromone precursors and one pheromone receptor gene.Besides,pheromone receptor-like genes were also identified(STE3.s1-STE3.s6).Segregation analysis of mating type genes was performed in the single spores isolates(SSIs)of H1123.The results showed a recombination site between MAT-Aa and MAT-Ab,and it near the MAT-Ab sublocus.However,this is a small probability event(1/31).There was no recombination happened between MAT-Ba and MAT-Bb subloci.(2)The synteny analysis among L11,W23 and the published KACC42780 genomes showed the high conserved MAT-Aa sublocus(DNA:99.77%;Protein:100%).The polymorphism was detected in MAT-Ab sublocus.(3)We selected 15 single spore isolates of 5 dikaryons from different area.Then MAT-Aa and MAT-Ab subloci were cloned using general primers which were digned based on genomes alignment.In most strains(11/15),there was only a conserved Hd2 gene in MAT-Aa sublocus.They may lose the MAT-Aa already.The pairs of Hd1/Hd2 genes were identified in all strains in MAT-Ab.(4)We re-sequenced the genome of Fv25-3,which show no bands on MAT-Aa,to study the structure and function of MAT-Aa and MAT-Ab further.At the same time,we analyzed the mating type of two monokaryotic genomes from Nongjin6.The crossing test were performed to study function of MAT-A by checking clamp cells.Results showed that the MAT-Aa contains incomplete Hd1/Hd2 gene pair,only one Hdl or Hd2,in strains we tested.This is a strange and degraded sublocus comparing with other mushrooms.But it still could active MAT-A pathway when the genes are compatible.MAT-Ab is the major variant sublocus with polymorphism.Certainly it can active the MAT-A pathway.(5)Both MAT-Ba and MAT-Bb were constituted of pheromone precursor and pheromone receptor genes with polymorphism.All the six pheromone receptors have 7 transmembrane domains(7-TM),but there is no pheromone precursor in the flanking area.And these six pheromone receptors are equal in all the F.velutipes genomes.It means that they are not involved in mating process.The phylogenetic analysis of pheromone receptors and pheromone receptor-like proteins showed two major clades.Pheromone receptors of MAT-Ba were distributed in different clades implied that they came from different original pheromone receptors.However,pheromone receptors of MAT-Bb were distributed in the same clade even in the small group.This suggested they may come from the same original pheromone receptors.By the same way,STE3.s1-STE3.s5 may have a common origin but STE3.s6 did not.(6)Genome and digital gene expression profiling(DGE)of Volvariella volvacea were combined to genome and transcriptome data of F.velutipes.Two differential expressed transcription factors(Fv-hmgl and Fv-hmg2)containing HMG-box domains were obtained.Integration behavior was analyzed by high-throughput sequencing in Fv-hmgl transformants(1382OE1 and 1382Ri3).We also obtained two RNAi and two overexpression transformants of gene Fv-hmg2 using agrobacterium-mediated transformation(ATMT).RT-qPCR was performed to detect the expression of target genes in transformants and the relationship between mating pathway and Fv-hmg genes.The results showed that the overexpression of Fv-hmgl can prevent the clp1 gene from expressing,which located downstream of MAT-A pathway.But Fv-hmgl gene can be inhibited by actived MAT-B pathway.The organization and function of vital factor,which named mating type,in mating were deeply analyzed in this study.This is an advance for understanding the mating type system in mushroom forming fungi.Two transcription factors as candidates were obtained and the relationship with mating pathway was also analyzed.This can be useful to further research on fruiting body formation mechanism,molecular assisted breeding and production of edible mushrooms.