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The prominent problem of "uncertainty" has been frustrating morel farming since the commercial production of Morchella mushrooms was realized in 2012 in China. Spawn aging may be the main underlying reason. In this paper, aging in cultivated strains of M. importuna T4 and M. sextelata T6 was achieved by successive subculturing. Oxidative stress, mycelial growth rate, biomass, sclerotial formation, pigmentation, and yield of different subcultures were determined. The results suggested that M. importuna T4 and M. sextelata T6 exhibited systemic senescence manifested as 24 and 17 subcultures and lifespan of 3048 and 2040 h, respectively. Aging showed a close relevance to oxidative stress. Regression analysis revealed a strong positive correlation between time of subculturing (aging) and lipid peroxidation (oxidative stress) in both morels. In addition, pigmentation tended to increase, while the number of sclerotia tended to decrease, with the rise of subculturing times in both morels. Moreover, the mycelial growth rate and biomass of the last two subcultures were significantly lower than those of others, indicating that cultural characteristics may be used as signs of seriously aging culture. Finally, the yield of subcultures was significantly lower than that of the original strains in artificial cultivation. Regression analysis showed a strong negative correlation between time of subculturing and yield in two morels. This work will improve the understanding of "uncertainty" and thus be beneficial for stable development of morel farming.

The commercial production of Morchella mushrooms, especially M. importuna , has been realized since 2012 in China, which facilitates the fundamental studies of Morchella spp. In this paper, the cytological characteristics at three stages of ascocarpic development and the nuclear behavior during meiosis and ascosporogenesis in cultivated strain M. importuna 1 # was visualized by confocal laser scanning microscopy. The results suggested that the strain sporulated at sporulation stage of ascocarpic development. A total of six nuclear divisions typically took place during ascosporogenesis. The first and second divisions were meiotic in which the single diploid nucleus divided into four haploid nuclei. The subsequent mitosis gave rise to eight nuclei, and eight incipient ascospores with one nucleus in each spore were formed after spore delimitation in the clavate ascus. Then, the nucleus in most of the young ascospore underwent three successive mitoses producing 6–8 haploid nuclei in each mature spore, and thus the multinucleate ascospores in each ascus were all homokaryons. To the best of our knowledge, this is the first dynamic tracing study of nuclear behavior during meiosis and ascosporogenesis in cultivated morels, and the spore delimitation time is also the first report. The study will be beneficial for the genetics study and strain breeding of Morchella mushrooms.

Morchella crassipes (Vent.) Pers., a typical yellow morel species with high economic value, is mainly distributed in the low altitude plains of Eurasia. However, rare research has been performed on its genomics and polarity, thus limiting its research and development. Here, we reported a fine physical map of the nuclear genome at the subchromosomal-scale and the complete mitochondrial genome of M. crassipes. The complete size of the nuclear genome was 56.7 Mb, and 23 scaffolds were assembled, with eight of them being complete chromosomes. A total of 11,565 encoding proteins were predicted. The divergence time analysis showed that M. crassipes representing yellow morels differentiated with black morels at ~33.98 Mya (million years), with 150 gene families contracted and expanded in M. crassipes versus the two black morels (M. snyderi and M. importuna). Furthermore, 409 CAZYme genes were annotated in M. crassipes, containing almost all plant cell wall degrading enzymes compared with the mycorrhizal fungi (truffles). Genomic annotation of mating type loci and amplification of the mating genes in the monospore population was conducted, the results indicated that M. crassipes is a heterothallic fungus. Additionally, a complete circular mitochondrial genome of M. crassipes was assembled, the size reached as large as 531,195 bp. It can be observed that the strikingly large size was the biggest up till now, coupled with 14 core conserved mitochondrial protein-coding genes, two rRNAs, 31 tRNAs, 51 introns, and 412 ncORFs. The total length of intron sequences accounted for 53.67% of the mitochondrial genome, with 19 introns having a length over 5 kb. Particularly, 221 of 412 ncORFs were distributed within 51 introns, and the total length of the ncORFs sequence accounted for 40.83% of the mitochondrial genome, and 297 ncORFs had expression activity in the mycelium stage, suggesting their potential functions in M. crassipes. Meanwhile, there was a high degree of repetition (51.31%) in the mitochondria of M. crassipes. Thus, the large number of introns, ncORFs and internal repeat sequences may contribute jointly to the largest fungal mitochondrial genome to date. The fine physical maps of nuclear genome and mitochondrial genome obtained in this study will open a new door for better understanding of the mysterious species of M. crassipes.

The complete mitochondrial genome of Morchella importuna, the famous edible and medicinal mushroom, was assembled as a 272,238 bp single circular dsDNA. As the largest mitogenome among fungi, it exhibits several distinct characteristics. The mitogenome of M. importuna encoded 14 core conserved mitochondrial protein-coding genes and 151 mitochondrial non-conserved open reading frames (ncORFs) were predicted, of which 61 were annotated as homing endonuclease genes, and 108 were confirmed to be expressed during the vegetative growth stages of M. importuna. In addition, 34 introns were identified in seven core genes (cob, cox1, cox2, cox3, nad1, nad4 and nad5) and two rRNA genes (rrnS and rrnL) with a length from 383 bp to 7453 bp, and eight large introns with a length range of 2340 bp to 7453 bp contained multiple intronic mtORFs. Moreover, 34 group I (IA, IB, IC1, IC2, ID and derived group I introns) and four group II intron domains were identified for the 34 introns, including five hybrid ones. Furthermore, the M. importuna mitogenome showed the presence of about 18.7% mitogenomic interspersed repeats. These and the aforementioned ncORFs and introns, contributed to the enlarged size of the mitogenome.

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.

Sclerotial formation is a key phase of the morel life cycle and lipids have been recorded as the main cytoplasmic reserves in sclerotia of Morchella fungi without any experimental verification. In this study, the ultrastructural features of the undifferentiated mycelia stage (MS) and three main sclerotial differentiation states (sclerotial initial [SI], sclerotial development [SD] and sclerotial maturation [SM]) were compared by transmission electron microscopy. The nature of the energy-rich substance in hypha and sclerotium of Morchella importuna was qualitatively investigated by confocal laser scanning microscopy and quantitatively studied by extraction of lipids. Sclerotia were observed to form from the repeated branching and enlargement of either terminal hyphae or subordinate hyphal branches, indicating a complex type of sclerotial development. Autophagy and apoptosis were involved in the sclerotial metamorphosis of the cultivated strain of M. importuna. During the SI phase, the characteristic features of autophagy (vacuolation, coalescence of small vacuoles, existence of autophagosomes and engulfment of autophagosomes by vacuoles) were observed. At the SD phase, apoptotic characteristics (condensation of the cytoplasm and nucleus, shrinkage of plasma membrane, extensive plasma membrane blebbing and existence of phagosomes) could be seen in some developing sclerotial cells. In the final stage of sclerotial morphogensis, the sclerotial cells showed a necrotic mode of cell death. In addition, confocal laser imaging studies of live cells indicated that the energy-rich substance in morel hyphae and sclerotia was lipid. The lipid content in sclerotia was significantly more than that in hyphal cells. To the best of our knowledge, this is the first detailed ultrastructural description highlighting the involvement of autophagy and apoptosis in sclerotial metamorphosis of Morchella species and lipid accumulation during morel sclerotial development was also first experimentally verified. This work will promote a better understanding of the mechanism of morel sclerotial metamorphosis.

In recent years, the artificial cultivation of Morchella mushrooms that belong to Elata Clade, including Morchella elata, has been developed rapidly in China. However, the prominent problem of spawn aging has been frustrating the morel farming. In this paper, aging in M. elata was achieved from 12 to 17 subcultures and lifespan of 1536-2256 h by successive subculturing. The lifespan can be roughly divided into juvenile phase and senescent phase with respect to the mycelia linear growth rate. After a certain period of rapid growth with almost constant rate at the juvenile phase, the isolate entered the senescent phase characterized by slow down of mycelia growth, producing pigments ahead of time and final death of the apical hyphae. The period of the senescent phase was definitely 240-288 h; while that of the juvenile phase was diverse relying on different isolates. Moreover, microscopic study showed that angles between the leading and primary hyphae increased constantly with aging. In senesced hyphal cells of M. elata, the typical characteristics of autophagy (enlargement of vacuoles and existence of organelles sequestrated lysosomes) and apoptosis (condensation of the cytoplasm and nuclear and plasmolysis) were observed. In addition, in the final stage of senescence, the apical hyphae collapsed with the plasma membrane and all the cellular organelles disrupted, indicated a necrotic mode of cell death. Taken together, these data revealed the involvement of autophagy, apoptosis and necrosis in senescence of M. elata. The characterization and molecular mechanism of autophagy, apoptosis and necrosis need further study and the systematic study of morel aging will be beneficial for the healthy development of morel farming.