Genetic Diversity Assay With AFLP Markers In Hypsizygus Marmoreus And Map-based Cloning Of Major Genes Affecting Cell Flocculation In Yeast

Understanding the genetic mechanism of complex traits is of key importance for genetic improvements of crops and domestic animals, and is also helpful to elucidate the genetic aetiology of human complex diseases. The essential issues in genetic analysis of complex traits is to map quantitative trait loci (QTLs) that affect the inheritance of complex trait, to detect the interaction among QTLs (epistasis), and consequently to identify the candidate genes underlying complex trait.Analysis of QTL mapping integrates three sources of basic information, genetic markers, phenotype of the target trait and genetic structure of mapping population. The research materials in the present study are fungi and an edible fungi Hypsizygus marmoreus is firstly studied. Development of AFLP molecular markers in Hypsizygus marmoreus provides basis for genetic linkage map construction and QTL analysis in this species. Also, the present study provides an initial but useful step towards an understanding of population genetic structure of the species and, a reliable reference for screening parental strains to construct mapping population.It is difficult to carry on genetic study of complex traits in Hypsizygus marmoreus due to its complex genome structure and to lack of useful genetic imformation. Therefore, the eukaryotic model organism Saccharomyces cerevisiae is subsequently used to dissect the molecular basis of genetic variation in complex trait. The results in the study provide and experimental and analytic framework in which core issues in quantitative genetics can be addressed systematically, and provide theoretical and method reference for studying complex traits in animals, plants and microbes including edible fungi.Hypsizygus marmoreus is a basidiomycete fungus which is not only a delicious and nutritious food but also of great medicinal value of antifungus and antitumor activity. Therefore, H. marmoreus has good market prospects. Although the literature on this fungus species has been foused on the biochemistry of nutrition and nutritious physiology and pharmacology, the molecular and genetic study of H. marmoreus is rare. China subscribed to an agreement for the protection of new cultivars of plants including mushrooms in 1999, which required protection of intellectual property rights in our own country particularly. It is necessary to develop molecular makers and provide new standards and tests for cultivated strains of H. marmoreus.The present study was designed to develop a reliable experimental protocol for detecting and genotyping amplified fragment length polymorphism (AFLP) markers in H. marmoreus and to evaluate the genetic polymorphisms and diversity among 19 stains including both commercial cultivars and stock strains based on AFLP markers. The results of the present study were listed below.1、Development of AFLP markers in H. marmoreusThe high concentration of polysaccharides and proteins in H. marmoreus makes it difficult to extract pure genomic DNA. In order to prepare good quality DNA samples as required for a successful AFLP analysis, we established an appropriate protocol for DNA extraction in H. marmoreus by modifying the CTAB method commonly used in plants.EcoRⅠand MseⅠwere used to digest DNA in AFLP analysis.10 primer pairs (EcoRⅠ+2nt/ MseⅠ+3nt) were used for detecting genetic polymorphisms. The AFLP analysis with 10 primer pairs has generated a total of 609 markers, of which 532 (87%) were polymorphic. The capability of different primer pairs to generate AFLP markers varied significantly, from 47 to 80 (average 60.9) markers per primer pair over all gentypes. However, the polymorphic information content (PIC) was high and reached up to 92%. In total,11184 marker data points were scored across all the 19 genotypes. These results had three implications:(1) The richness of genetic divergence and polymorphisms could be found in H. marmoreus; (2) A number of markers were generated from each of the 10 primer pairs, which could be used as good candidacy for further studies; (3) The abundant polymorphic sites and marker data points achieved in present study showed that the AFLP protocol established could be effective and reliable in genetic study of H. marmoreus.2、Estimation of genetic diversity of H. marmoreus Similarities among samples were calculated as Dice coefficient. A dendrogram was produced based on the similarity coefficient by UPGMA. The results showed that six stains including HmC1199 et al. were clustered as groupⅠat a genetic similarity of>=95%; and the second group containing 7 genotypes including HmC2637 et al. shared a genetic similarity of>=92%; and the remaining six genotypes showed a higher level of genetic difference than the other two groups, in particular, similarity coefficient between HmJ2632 and HmC1199 was only 0.547. We further estimated the genetic diversity among 19 H. marmoreus strains by principal component analysis (PCA). The first and second principal components reveals a very similar pattern of divergence to that obtained by the UPGMA clustering analysis.A comparison of the genetic diversity of the 19 H. marmoreus strains with their morphologic characters indicated that the strains in the same cluster shared a common fruiting body character, e.g. small caps and thin stipes in groupⅠand wide caps but short stipes in groupⅡ. All the 19 genotypes basically represented 4 different morphological phenotypes:in addition to the two groups mentioned above, there were one genotype with white pileus(HmJ3136)and another one with tumor-baring caps(HmJ2632). Although the genetic basis of the pileus trait is still not established in H. marmoreus, it is certainly a good starting point to clone and compare the albino gene from the strain HmJ3136 with that from others. The stain with malformed caps was most genetically distant from the others. According to previous information in fruiting season types in other fungus species, we suppose that similar fruiting season types exist in H. marmoreus, in particular, the strain HmJ2632 may be of the H type, and the remaining 18 stains would be possibly classified as either M or L type.Presently, the resolution of QTL mapping is restricted due to lack of informative meiosis in humans, animals and plants mapping populations. Therefore, researchers are trying to study complex traits in model organisms. Saccharomyces cerevisiae is an important model organism in quantitative genetics. Yeast flocculation is a reversible, asexual and calcium-dependent process in which cells adhere to each other to form flocs. Yeast flocculation properties are closely related to ethanol fermentation and therefore are of great industrial values. Several published researches have identified a series of FLO genes of which either genetic variants or modified expression may change the flocculation phenotype, strongly supporting the polygenic nature of its genetic control. In the present study, we describe the development of yeast flocculation as a model for quantitative genetics. Through a combination of genetic mapping and functional verification, we finally identified a quantitative trait nucleotide (QTN) variant that control yeast flocculation differences. We also investigated the genetic interactions between the three QTL genes. This study represented the first forward genetic approach for dissecting genetic basis underlying yeast flocculation and the significance of this study in theory and practice could be as follows:First, the study provides theoretical and method reference for genetic dissection of complex traits in animals, plants and microbes including edible fungi.Second, the study provides a reliable theoretical basis for genetic improvement of yeast strains used in fermentation industry. The results of the present study were listed below:1、Two yeast strains YH1A (nonflocculent) and YL1C (flocculent) with highly divergent trait phenotype were chosen as parental strains. A F2 haploid segregating population comprising 292 segregants from crossing the two parental strains was developed (Hu et al.,2007). Meanwhile, a saturated linkage map across yeast genome with mainly 260 STR markers(Hu et al.,2007), supplemented by 24 SNP markers, was constructed. The average map distance between markers is 10-20cM.2、We developed a simple but reliable experimental method for scoring the flocculation phenotype of both parents and individuals. The sediment time(T)for cells sinking to the bottom of tubes in liquid culture was used as criterion. We classified the parental and offspring stains into 4 flocculation categories:CategoryⅠ(T=0),CategoryⅡ(0<T<=2.5), CategoryⅢ(2.5<T...