Study On The Ascus Development And Mating-related Genes Of Morels

Morels(Morchella spp.)are sought after for their unique delicious taste,rich nutrition and high medicinal value.They are precious edible and medicinal fungi,and are also important trade wild mushroom in China.It is difficult to study morels’s genetic characteristics because of multinuclear in ascospores and the diversity of macroscopic and microscopic characterization of culture.Until now,many research findings of taxonomy and molecular biology,physiology and biochemistry about mycelium culture,sclerotia formation and fruiting body cultivation of morels had been obtained.However,researchers have many inconsistent views on them because of genetic variance.Research on sexual reproduction which may reveal the causes of morels’ genetic diversity could become an important breakthrough in morel study.Exploring the mating type of morels can provide solid theoretical basis for the further understanding of its sexual reproduction,especially development of fruiting body on artificial conditions,and then promote the development of morels commercialization,industrialization cultivation research effectively.M.importuna which grown in artificial condition was used for studying the ascus development and ascospore formation.Pheromone receptor genes and the mating type genes have isolated from representative strains of Elata Clade group(M.Elata,M.importuna and M.septimelata),Esculenta Clade group(M.crassipes)and Rufobrunnea Clade group(M.rufobrunnea)respectively.The sequence,structure,as well as expressing analysis have been carried out.The ascospore isolations from M.elata,M.importuna and M.crassipes strains also have been classified based on the distribution of the pheromone receptor genes and mating type genes.The ascus development of M.importuna was observed by Heidenhain’s hematoxylin and DAPI staining.We initially summarized the M.importuna ascus development and ascospore formation patterns.We found out that the spindle orients was "vertical--vertical--disorder" to ascus wall respectively in two meiotic division and mitosis processes.The nuclei were packaged randomly in the ascospore formation.M.importuna could produce diverse homokaryon and heterokaryon ascospores in non-sequential tetrad order.In addition,the nuclei in young spores was fewer than that of mature spores,so the multi-nucleic ascospores were the results of the mitosis after their formation.The 49.4Mb genome sequencing data were obtained from M.crassipes single spore strain NH057,and 12,340 genes with total length of 19,005,309 bp were predicated.There were ten genes were related to sexual reproduction,namely two pheromone receptor genes,M pheromones genes,mating type gene MAT1-1-1,MAT1-2-1 and MAT1-2-2,mating type conversion gene swi5 and swi10,and mating membrane fusion protein gene FIG1 and PRM1.The MAT1-2-2 was the newly discovered mating type genes.One of the most meaningful finding revealed by the sequence prediction analysis and sequence alignment on MAT1-1-1,MAT1-2-1 and MAT1-2-2 gene flanking sequences,was that mating type genes were not in conformity with their reverse sequences.On the basis of NH057 genomic analysis and M.rufobrunnea transcriptomic analysis,the pheromone receptors encoding genes--MPRa and MPRb in YAASM2688,YAASM2689,YAASMCH,YAASMBM and YAASMVR,and mating type genes--MAT1-1-1 and MAT1-2-2 in YAASM2688,YAASM2689 and YAASMCH were cloned and sequenced successfully by using amplification conservative section with degenerate primers and DNA Walking PCR amplification.The analysis on the conservation of all genes and their encoded amino acid sequences were processed,and the secondary structure of pheromone receptors were predicated and compared.The results showed that two types of genes and their coding products were specificity in relation to species.The comparing analysis showed that phylogeny based the sequences of MPRa and MPRb from 35 Morchella strains was mainly consistent with that of ITS rDNA,EF1-α and LSU rDNA.So,pheromone receptor genes could also be used for phylogenetic analysis.The 111,130,and 91 single spore isolations from fruiting body MCH,M2689 and M2688 were classified into 12,7,and 4 major types based on the MPRa,MPRb,MAT1-1-1,MAT1-2-1 and MAT1-2-2(M2689 only)gene respectively,They were classified into different kinds of homokaryon and heterokaryon.Compatibility test on different types of single spore isolations show that morels was heterothallic fungi,and compatibility is mainly determined by mating type genes.The mating type genes and the pheromone receptor genes expression analysis in five single spore heterokaryon strains from M2689 have been done by real-time quantitative PCR.In hypha--covered--plate stage,sclerotiaum--formation--vigorous stage and sclerotiaum--mature stage,the expression of MAT1-1-1 MAT1-2-2 and MPRa gene were significantly higher than that of the MAT1-2-1 and MPRb genes.The expression quantity of MPRa,MPRb,MAT1-1-1 and MAT1-2-2 gene in most strains in sclerotium--formation--vigorous stage were higher than in hypha--covered-plate stage,followed in sclerotiaum-mature stage.However,the expression quantity of MAT1-2-1 gene in all strains in hypha--covered--plate stage was higher than in sclerotium--formation--vigorous stage,followed in sclerotiaum--mature stage.In addition,There was no the gene expression rules in morels fruit body represents the five development stages for MPRa,MPRb,MAT1-1-1 and MA T1-2-2 gene,In the same fruit body,the expression quantity of mating type gene were higher than that of pheromone receptor gene expression,but the MPRb was not the main active gene for fruiting body development;In most of the fruiting body,the relative expression quantity of MPRa,MAT1-1-1 and MAT1-2-2 gene in ascocarp were higher than that in stipe,and that of MPRb expression in the stipe is higher than in ascocarp.