Study On The Phenomenon And Genetic Basis Of Segregation Distortion Of Mating-type Factors In Lentinula Edodes

This study made 28 strains of Lentinula edodes, including 16 wild strains collected from center-, northwest- and southwest-China and 12 cultivated strains which well-popularized around China as materials, statistically analyzed the ratios of protoplast monokaryons to spores from different aspects via mating types analysis and OWE-SOJ technique. Results from this study firstly systematically identified skewed expected distribution of mating-type factors segregation in Lentinula edodes has commonly statistical meanings between protoplast and spore monokaryons in wild and cultivated strains. Meanwhile, the genetic basis of this specific phenomenon was discussed through protoplast monokaryotization; mon-mon and di-mon crosses; mating-type analysis of progeny from positive and negative parental-recombined fruiting as well as amplified fragment length polymorphism-bulked segregation analysis. The main results are summarized as follow: 1 The distribution ways and statistical meanings of segregation distortion ofmating-type factors(1) In all 28 tested strains two mating types were recovered from dikaryotic strain in 27 strains except 1 strain, which was determined as only one mating type among 56 randomly selected protoplast monokaryons. According to x2 statistic analysis, ratio of two nuclear types varied and did not show a 1:1 ratio in 17 strains out of 28 tested strains, 61.71% of all strains tested; ratio of 4 mating types unbalanced 1:1:1:1 expected ratio in 11 out of 17 fruiting strains tested, 64.71% of all strains tested.(2) According to x2 statistic results, 8 out of 12 cultivated strains, 66.67% of cultivated strains tested, and 9 out of 17 wild strains,56.25%of wild strains tested, displayed unbalanced distribution between two nuclear types after dedikaryotization. Meanwhile, 11 strains tested, 64.71% of 17 fruiting strains tested, in which including 9 out of 12 cultivated strains (75%) and 2 out of 5 wild strain (40%), exhibit distorted Mendelian's segregation. To some extent, the degree of skewed ratios of mating type between protoplast monokaryons and spores in tested wild strains is lower than that of cultivated strains tested.(3) According to analyzed aspects, we can class the distribution among spores into 4 classes: ratio among four mating type spores, ratio between two types' parental spores, ratio between two types' recombinant spores and ratio between parental andrecombinant spores. Statistical results revealed that in 17 strains tested, 6 strains (35.29% of 17 strains tested) follow expected segregation according to above classes, including 3 out of 12 cultivated strains(25% of cultivated strains tested) and 3 out of 5 wild strain(40% wild strains tested). Among 11 strains skewed Mendelian's segregation, 2 strains exhibit balanced distribution in 3 types but one type skewed distribution; 5 strains coincide with Mendelian's segregation in 2 types and 2 types skewed distribution; 3 strains satisfy with theoretically segregation in one type but 3 types skewed distribution; furthermore, one strain exhibits extremely skewed distribution of spores, in which only two parental nuclear types are isolated from 64 spores 2 The genetic basis of the phenomenon of segregation distortion of mating-typefactors in Lentinula edodes(1) Observation showed that segregation distortion of mating-type factors didn't correlate with colony shapes, cultivation conditions and mating-reaction polymorphisms. The differences of colony shape, media types, pH value, culture temperature as well as compatible mating polymorphism between two monokaryons have no correlation with distribution of mating types.(2) Statistical results revealed that the ability of average regeneration per day and sustained growth time between two types of protoplast monokaryon exist difference after dedikaryotization in those strains skewed distribution of mating-type factors. The difference of survival and regenerated ability between two nuclear types directly caused the occurrence of segregation distortion.(3) Genetic analysis to various heterokaryon recombined suggested that specificity of B mating-type factor, or of a gene linked to the B factor, appears to influence ratio of dedikaryotized nuclei. Specificity of the A factor has no apparent effect on this phenomenon. This is to say, skewed ratios of one type of nucleus to the other mainly attributed to the difference of B-mating-type alleles, it influence nuclear survival. Specificity of B loci has been shown to correlate with percentage survival of the component nuclear types recovered from both kinds of heterokaryon, suggesting differential effects of these series of multiple alleles on nuclear function in monokaryons isolated from heterokaryons. The alleles can be put in a hierarchical order with respect to this function. A nucleus carrying a higher specificity B gene is more likely to survive dedikaryosis than is a nucleus carrying a lower specificity B gene in both A^ B^ heterokaryons and A= B^ heterokaryons, this function of Bmating-type gene was more obviously in last case. Compared to the result of the difference of survival and regenerated ability between two nuclear types directly caused the occurrence of segregation distortion, the function of B factor has further genetic background. The hierarchical trend of Bl to B4 factors tested was Bl> B3> B4> B2.(4) Genetic analysis to fully compatible di-mon mating showed that the monokaryons with acceptor nuclear type outnumbered exclusively those pre-entrance nuclear type after dedikaryotization in all secondary derived homocytoplasm heterokaryon consisted of acceptor cytoplasm and acceptor and donor karyons via di-mon cross, suggesting that some factors in cytoplasm are probably responsible for influencing survival of nuclei recovery from dedikaryotization. Moreover, both nuclei of donor dikaryon will be able to dikaryotize the monokaryon, the nucleus migrated from a dikaryon to a monokaryon can be the same one or two different one while a dikaryon mating with different monokaryons, in fact, the nucleus type migrated into acceptor is the nucleus with stronger compatibility between different B factors, then they form derived dikaryon, this showed that a pre-entrance nucleus exists in donor and pre-entrance nucleus mainly dependent on the compatible specificity of B mating-type factor between acceptor and donor nucleus; the higher the specificity, the faster the migration of pre-entrance nucleus.(5) Genetic analysis to positive and negative parental-recombined fruiting showed that nuclear type of Fl progeny spores among those strains segregated theoretically distribution mainly depended on combined state of parental dikaryons. Whether dikaryotic mycelia with mating type AxBx+AyBy or those AxBy+AyBx generated by mating recombinant spores served as parental strain, the ratio between parental nuclei and recombinant nuclei among spores isolated from Fl progeny always not display 1:1 distribution. Predominant nuclei types were those with mating type identical to that of the dikaryotic parent in both cases, indicating that the genetic basis of segregation distortion of spores is different from that of protoplast monokaryons in which B factor takes predominant responsibility for that phenomenon, it cooperate A factor with B factor to influence the ratio of spores.3 Segregation ways of AFLP markers among different gene pools of mating typesEight selective primer combinations were used to analyze polymorphisms and segregated ways of these polymorphism bands among parental (IBOlx IB15) and 4 gene pools of mating types of Fl progeny spores. Results showed that thepolymorphism of the AFLP markers in the Fl population was high, and the average frequency of the segregation loci was 29.19%. The segregation patterns included Mendelian, deviation from Mendelian and abnormal segregation models, and their number and frequency were, respectively, 566,27.13%, 27,12.99% and 49, 2.31%; 53.99% of the segregation loci were in Mendelian model. The total frequency of distorted distribution among AFLP marks is up to 15.3%. Among different gene pools of mating types of Fl progeny spores, there exist difference in ratio of polymorphism bands from corresponding parent and existence and number of specifically bands. Result suggested that the difference of activity degree and of possibility of mutation among different chromosomal including B mating genes, such as rearrangement, deletion, insertion, could be responsible for segregation distortion of AFLP markers linked with mating-type factors. This result further proof the conclusions resulted from cytological experiments: the phenomenon of segregation distortion of mating-type factors in Lentinula edodes is caused by mating-type gene itself, no artificial factors or environmental factors involved.