Opposite Polarity Monospore Genome De Novo Sequencing and Comparative Analysis Reveal the Possible Heterothallic Life Cycle of Morchella importuna

Abstract

Morchella is a popular edible fungus worldwide due to its rich nutrition and unique flavor. Many research efforts were made on the domestication and cultivation of Morchella all over the world. In recent years, the cultivation of Morchella was successfully commercialized in China. However, the biology is not well understood, which restricts the further development of the morel fungus cultivation industry. In this paper, we performed de novo sequencing and assembly of the genomes of two monospores with a different mating type (M04M24 and M04M26) isolated from the commercially cultivated strain M04. Gene annotation and comparative genome analysis were performed to study differences in CAZyme (Carbohydrate-active enzyme) enzyme content, transcription factors, duplicated sequences, structure of mating type sites, and differences at the gene and functional levels between the two monospore strains of M. importuna. Results showed that the de novo assembled haploid M04M24 and M04M26 genomes were 48.98 and 51.07 Mb, respectively. A complete fine physical map of M. importuna was obtained from genome coverage and gene completeness evaluation. A total of 10,852 and 10,902 common genes and 667 and 868 endemic genes were identified from the two monospore strains, respectively. The Gene Ontology (GO) and KAAS (KEGG Automatic Annotation Serve) enrichment analyses showed that the endemic genes performed different functions. The two monospore strains had 99.22% collinearity with each other, accompanied with certain position and rearrangement events. Analysis of complete mating-type loci revealed that the two monospore M. importuna strains contained an independent mating-type structure and remained conserved in sequence and location. The phylogenetic and divergence time of M. importuna was analyzed at the whole-genome level for the first time. The bifurcation time of morel and tuber was estimated to be 201.14 million years ago (Mya); the two monospore strains with a different mating type represented the evolution of different nuclei, and the single copy homologous genes between them were also different due to a genetic differentiation distance about 0.65 Mya. Compared with truffles, M. importuna had an extension of 28 clusters of orthologous genes (COGs) and a contraction of two COGs. The two different polar nuclei with different degrees of contraction and expansion suggested that they might have undergone different evolutionary processes. The different mating-type structures, together with the functional clustering and enrichment analysis results of the endemic genes of the two different polar nuclei, imply that M. importuna might be a heterothallic fungus and the interaction between the endemic genes may be necessary for its complete life history. Studies on the genome of M. importuna facilitate a better understanding of morel biology and evolution.

Keywords: Morchella importuna; gene expansion and contraction; heterothallism; mating type; transcriptome.

Figure 1

Genomic comparison and characteristic analysis of the two opposite-polarity monospore strains of Morchella importuna. Genomic comparison and characteristic analysis of the top N50 sequences between the two monospore strains, M04M24 and M04M26. The outermost loop is the scaffold fragment of the two strains, with different scaffolds indicated in different colors; M04M24 is on the right, M04M26 is on the left. The scaffolds that were larger than the respective N50 values are numbered 1 to 17 for M04M26, and from 1 to 24 for M04M24. The innermost circle (f) is the gene collinearity between the two strain scaffolds of the outermost ring (a), and the inner color corresponds to the outer color of the scaffold. (b) shows the distribution of the four different types of repeat sequences in the genome, and from outside to inside are the three RNA repeat sequences: long terminal repeat (LTR) green lines, long interspersed nuclear element (LINE) purple lines, short interspersed nuclear element (SINE) blue lines, and DNA repeat sequences: red lines, with black lines in the innermost cycle displayed as the synthesis of four repeat sequences. (c) (the histogram) represents the percentage of GC in the genome. (d) shows the single-nucleotide polymorphism (SNP) frequency of the genome in the form of a histogram, and the ordinate is the SNP number per 20 kb of genome. (e) shows the gene expression of RNA sequencing (RNA-seq) samples in the form of heat map. Red indicates that FPKM (Fragments Per Kilobase Million) is more than 100, orange is 100 > FPKM > 10, green is 10 > FPKM > 0, and black indicates FPKM = 0.

Figure 2

Gene ontology (GO) annotation of endemic genes in the two monospores of Morchella importuna. (a,b) Biological process classification; (c,d) Cellular component and molecular function classification; (a,c) represent the M04M24 strain; (b,d) represent the M04M26 strain.

Figure 3

Evolutionary divergence time and statistical analysis of homologous genes in 17 species. (a) The maximum-likelihood method and LG + I + G + F model were used to construct the phylogenetic tree using 1072 single-copy genes identified from the two different monospore strains and 15 reference species. The abscissa unit was million years ago (Mya); the 15 species were schpo Schizosaccharomyces pombe, aspni Aspergillus niger, xylbe Xylona heveae, dipse Diplodia seriata, neucr Neurospora crassa, sclsc Sclerotinia sclerotiorum, artol Arthrobotrys oligospora, mlm24 Morchella importuna M04M24, mlm26 Morchella importuna M04M24, tubme Tuber melanosporum, pyrco Pyronema confluens, sacce Saccharomyces cerevisiae, pospl Postia placenta, pleos Pleurotus ostreatus, schco Schizophyllum commune, ustma Ustilago maydis, and rhoba Rhodotorula graminis (The reference genome used in this paper was downloaded from the Joint Genome Institute (JGI) website https://genome.jgi.doe.gov/programs/fungi/, Table S11). (b) The number of homologous genes of the corresponding 17 species is displayed by the stacking column pattern, and the number of homologous genes that appeared in the corresponding number of species is expressed in different colors.

Figure 4

Structural analysis of two opposite-polarity monospore strains mating-type genes in Morchella importuna. The fine structure sketch was drawn manually based on the annotation information. M04M24 and M04M26 represent the mating loci of the two monospore strains used in this study. The sequences KY782629.1 and KY782630.1 reported in Chai et al. (2017) were downloaded from the National Center for Biotechnology Information (NCBI). The red box shows mating-type genes. The orange box represents the hypothetical gene. The flanking conserved genes are represented by SLA2 with a light-gray box and the APN2 gene with a pale blue purple. The sequence between the two dashed lines represents the largest different region of the two mating-type genes, with a sequence length of 6.6–11.9 kb. The ruler is 3000 bp.