Omics On The Growth And Development Of Morchella Importuna And The Morchella

True morels（Morchella spp.）are famous and rare edible fungus with unique taste and fragrance,but wild morel only occurs in a specific season every year,so the domestication and cultivation has always been the goal of global mushroom lovers.With the efforts of researchers and fans of morel,China’s morel field cultivation technology has made great progress that the cultivated region and area have been expanded rapidly in recent years.However,due to the lack of basic biological research on the polarity,life cycle,development and nutrition metabolism,70%of the growers are unable to make steady profits every year,which greatly limits the steady development of morel industry.Based on the basic problems faced by morel industry,the purpose of this project is to analyze the life cycle,growth and development of M.importuna and the genome of the main species in Morchella genus by means of modern molecular biology and genomics.1. The F₁ monosporic population of M.importuna（F₀）was prepared.The cultivation test was carried out with seven randomly monosporic strains and 10 random pairwise mixing of 7 monosporic strains.The results showed that 6 monosporic strains and all the pairwise combinations could produce ascocarp normally.In order to further study the phenomenon of monosporic fruiting,two ascomycetes（F₂）were randomly selected from each test,and two F₂ ascomycetes were used for the preparation of new monosporic population F₃,and then the mating genotypes of F₀,F₁,F₂ and F₃ were identified by using specific primers of mating gene.The results showed that although the monosporic strains with single mating genotype could produce ascocarp normally,their progenies F₂ were all added an opposite mating genotype,which might be caused by the propagation of"asexual spore"and"mating"in the cultivation process.Two different mating monosporic strains M04M24 and M04M26 were used to genome sequence,de novo assemble and annotate.The genome of M.importuna was obtained.Based on the whole genome,gene structure and function of differential gene,the differences between the two mating strains were analyzed.The results showed that they were different and complementary in gene sequence,mating gene structure and function of species-specific genes,which indicated that the cooperation between them should be necessary to complete the whole life cycle.M.importuna is a kind of heterothallic life cycle fungus.2. On the basis of reference genomic,the main stages of vegetative and reproductive growth（mycelium,sclerotia initiation,sclerotia maturation,primordium,young and mature ascocarp）in the life cycle of M.importuna were analyzed by means of transcriptomics and proteomics.The results of transcriptome analysis showed that 97.55%genes were expressed in the above six stages,the number of genes expressed in three sexual reproduction stages was significantly more than that in the vegetative growth stages,and the mature ascocarp stage was the most,which matched with the complex morphogenesis and internal physiological metabolism during the formation of sexual ascocarps.Under the standard of p<0.001 and log2>1,a total of 6421 differential expressed genes were identified and analyzed according to the expression pattern.The results showed that 87.2%（257）transcription factors in the genome were differentially expressed in these six stages.Some transcription factors annotated as ste11,cnjb,APSES,forkhead,MYB,Zn（II）₂cys₆ might be directly involved in the development and regulation of morel’s ascocarp or sclerotia.3. Five stages of M.importuna were analyzed by LC-MS/MS,including mycelium,sclerotia initiation,sclerotia maturation,young and mature ascocarp,and the differential proteomic analysis was carried out with label-free relative quantitative method.A total of2060 credible proteins were identified,including 1935 in the vegetative growth stage and1622 in the sexual reproductive growth stage,with 437 and 124 proteins specific to two stages,respectively.With the continuation of the growth and development process,the number of differential proteins continued to increase.Functional analysis showed that the continuously down-regulated proteins were mainly enriched in three basic metabolism,and the up-regulated proteins were also corresponding to physiological phenotype.In addition to the high expression of lectin（11.07%）and elongation factor 1α（2.72%）,a woronin body protein was also highly expressed,accounting for 3.75%of the total protein abundance.The mRNA level of these highly abundant proteins was verified by qRT-PCR,and some levels were highly consistent between the mRNA level and protein abundance.4.17 species of Morchella and 1 species of Verpa were used to genome sequence and de novo assemble.The results showed that the average genome size of 18 species was56.37Mb,the average number of contig and N50 were 96 and 1.3Mb,respectively;the average predicted encoded protein was 11150,and of 95.75%could be annotated,and99.26%of the BUSCO conservative genes could be predicted.5. The mitochondrial genomes of 17 species of Morchella and 1 species of Verpa were assembled and annotated.The results showed that they were all circular double-stranded DNA,showing significantly different characteristics from other fungi species in the following four aspects:1)They were the largest of all fungi,ranging in the size from 244 kb to 569 kb,with GC%ranging from 39.95%to 47.15%.2)Different species had different numbers of introns,with the total length of introns ranging from98.34 kb to 237.36 kb,accounting for 36.07%-70.73%of the total genome,which was one of the main reasons for the increase of mitochondrial genome.3)There were a large number of non conservative ORF（nc ORF）hidden in mitochondrial genome,ranging from 152 to 499,and the proportion of nc ORF sequence length in genome varies from25.74%to 50.71%.4)There were a large number of interspersed repetitive sequences,with the number of repeat units ranging from 333 to 770,and the proportion in genome ranges from 15.00%to 51.31%.The differentiation of mitochondrial genomic structural features corresponded to the classification of Morchella as a whole,implying their evolutionary independence and particularity.6. Based on the whole genome data,the phylogeny of Morchella was constructed.The evolutionary by using whole genome conserved protein,divergence time,mitochondrial conserved protein and mitochondrial genome sequence all showed that two clades were constructed in Morchella,rather than earlier three groups（Elata,Esculenta and Rufobrunea clade）based on multigene phylogenetic analysis.The results showed that although M.rufobrunnea,the representative species of Rufobrunnea clade,had a certain evolutionary distance with Esculenta clade,they were still classified under an independent branch,named Yellow clade,which was sister group with Black clade.The divergence time of the two groups was 102.71MYA.7. The collinearity analysis of mitochondrial genome and conservative ORF showed that although they had experienced rapid evolution,the collinearity among similar evolutionary groups was relatively high,and there was no obvious gene rearrangement phenomenon.Which indicated that they should originate from a common ancestor,and in the evolution it was mainly the insertion of repeat sequences from unknown sources in intron region and gene interval region,which eventually led to the formation of these enlarged mitochondrial genomes.The origin of nc ORF was still a mystery.Based on the fact that 45.57%of nc ORF could be annotated as HGEs,60.24%of nc ORF was in active expression,and the high GC%content of repetitive sequences corresponded to the high GC%content of CDS of nuclear genes,and there was no collinearity between mitochondrial genome and nuclear genome,it was speculated that the repetitive sequences in mitochondrial genome were most likely through"cut to insert"from nuclear genome to mitochondrial genome.However,the function of these nc ORF still needs further study.