| Volvariella volvacea has long been considered to be the primary homothallic fungus, but this genetic model itself can't explain many genetic mechanisms of Volvariella volvacea life cycle. So it has caused many difficulties in the genetic breeding and has seriously restricted the industry development of Volvariella volvacea. In this study, using recently developed protoplast technology and molecular markers, combined with traditional morphological and cytology cal methods, cultivars PY was used to reveal the life cycle and the genetic nature of Volvariella volvacea. The results of this study are as follows.1,New discovery of sex burden type and nuclear phase analysis of basidiospores Volvariella volvacea has long been considered to take four spores of a burden, and each basidiospore contains a nucleus.In this study, with the observation of the gills from Volvariella volvacea fruiting bodies of different periods by SEM, we described the development process of the burden and the basidiospores of sexual structure in Volvariella volvacea, and first time discovered that ,like the Agaricus bisporus, the burden is located by double spores,three spores and four spores, which the ratio is 9.06%,21.01% and 69.03% respectively;χ2 independent tests showed that there was no significant difference among the ratios of heterokaryotic basidiospores counted by three different methods (nuclear staining,burden observation by SEM and F1 generation test by co-dominant markers). Therefore, we believe that the theoretical expectation for the ratio of heterokaryotic basidiospores is 5.11%, and is 94.89% for homocaryotic basidiospores.2,To prove the existence of meiosis on Volvariella volvaceaThe primary homothallism itself can't explain why the single-spore isolates of Volvariella volvacea exist in wide variations.In this study, the 112 single-spore isolates of hybrid strains was used to genetic analysis by two SCAR markers obtained from different DNA bands of hybrid parents. χ2 test showed that the genetic separation ratios of the two SCAR markers (AB: Ab: aB: ab = 1:1:1:1) consistent with Mendel's law of free combination. This indicated that the SCAR marker is located on chromosome independent of each other, and also indicated that after the nuclear fusion, there is a meiotic process for Volvariella volvacea to make the genetic material of hybrid parents recombine and exchange sufficiently in single-spore isolates. Meiosis is the root of wide variation in single-spore isolates of Volvariella volvacea.3,Wide variant factor analysis of single-spore isolatesWide variant in single-spore isolates should be the quantitative traits determined by numerous minor genes.We tested the colony characterization indexes of 38 single-spore isolates from PY , including growth rate, the number of cell nucleus, length of cell, width of cell, the number of lateral branches , distance of lateral branch, as well as angle of lateral branch. Correlation analysis showed that seven phenotype indexes were significant, which illustrated that the differences of single-spore isolates were induced by genotype. Factor analysis showed that colony phenotype of Volvariella volvacea was mainly influenced by four main factors , which were named as aerial-type colony strong factor, prostrate type colony sparse factor, cytokines and colony to colony expansion of the nuclear phase factor, respectively. Starting from the main factors was not only conducive to the research of the paper on the phenotypic characteristics classification and of the cell biological characteristics determination of the single spore isolates from Volvariella volvacea, but also provided references to Volvariella volvacea's genetic and breeding research in the future.4,Interpretation of fruiting mechanism on single-spore isolatesThe primary homothallism is that single spore of Volvariella volvacea can complete life cycle without mating after the germination of basidiospores. However, it can't explain how mononuclear haploid single-spore strains complete its life cycle.The proportion of creeping type spores from 7 strains separated from different years was analyzed byχ2 test and the result showed no significant differences among them. The mathematical expectation proportion of creeping type spores is 76.77%, which indicated that creeping type spores was the dominant group. Creeping type spores couldn't fruit. The fruited proportion of aerial spores was less than 23.23%, so whether Volvariella volvaceas was homothallism or not was still needed to be verified.The result of genetic test of F2 generation and protoplast regeneration strains of aerial type and fruiting single spore PYd42 proved that single spore PYd42 was a heterokaryon; The result of genetic test of protoplast regeneration strains of aerial unfruiting spore PYYd41 and creeping unfruiting spores PYd15 and PYd21 were homokaryons. The genetic appraisal of fruiting and matching strains PYd15×PYd21 showed that PYd15 x PYd21 was a heterokaryons hybrid. Based on these analyses, we concluded that only heterokaryons hypha could fruit while monocyte or homokaryon couldn't fruit until mating. The heterokaryons which was 5.11% of Volvariella volvacea basidiospores had no more than 23.23% proportion of fruiting, which demonstrated the main sexual reproduction pattern of Volvariella volvacea was heterothallism.5,Identification of heterokaryotic mycelium on Volvariella volvaceaIn order to verify and explain the four conclusions above, we should analyze and identify whether PY is a heterocaryosis or not.Statistical analysis of protoplast showed that the expectation rate of prostrate type protoplast regeneration strain of Volvariella volvacea is 52.19%. Based on the comprehensive analysis of the colony morphology, growth rate, nuclei, nuclear genetic polymorphisms maps, mating and fruiting of 26 protoplast regeneration strains, we got two kinds of protoplast regeneration strains PYy14, PYy4 and PYy8 which were inter-heteronuclear .The further research showed that PYy14 and PYy8 were homokaryon.That was, PY was double heterokaryon.6,Test of the bipolar mating type system on Volvariella volvaceaThe result whether the monospores were mated or not was identified via the detection of self-breeding among aerial and unfruiting monospores, self-breeding among creeping monospores, backcrossing, inbreeding, which combined with SCAR-marker test. It also demonstrated that the mating system of Volvariella volvacea was controlled by a pair of A factor. The genetic mode in Volvariella volvacea was heterothallism and two-polarity.5.11% of heteronuclear basidiospore from Volvariella volvacea could fruit, whose monospore isolates were homothanllism; while the other monospore isolates of heteronuclear basidiospore from Volvariella volvacea are heterothallism result from being controlled by a pair of incompatibility A factor.7,Cloning of MIP gene closely linked with A factorUsing the degenerate primers reported to amplify MIP, we got a purpose fragment of 459bp by PCR from genomic DNA of Volvariella volvacea. Based on the fragment, three nested primers were designed to amplify downstream sequence by TAIL-PCR, and 445bp downstream sequence was got. After Sequence Assembly, we finally got 812bp of MIP gene sequences on Volvariella volvacea. |
