真姬菇AFLP分子标记的建立和遗传多样性分析及酿酒酵母细胞絮凝主效基因的定位克隆
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摘要
阐明复杂性状的遗传机制是动植物、微生物遗传改良以及人类复杂疾病致病机理研究的重要基础。复杂性状遗传解析的重点是定位控制复杂性状遗传的数量性状基因座(quantitative trait loci, QTL),检测QTL之间的相互作用,进而鉴定相应的候选基因。QTL定位分析要求整合遗传标记、表型数据和作图群体遗传结构3个方面的信息。本研究以真菌作为研究材料,首先从食用真菌真姬菇入手,通过建立AFLP分子标记,为真姬菇遗传图谱构建和QTL定位分析奠定基础；通过菌株遗传多样性分析,为揭示真姬菇群体遗传结构提供第一步参考,并为作图群体的亲本选择提供可靠依据。
由于食用菌基因组结构复杂且遗传信息匮乏,对真姬菇继续开展复杂性状遗传基础研究困难重重。为此,接下来本文创造性地利用酿酒酵母这一简单的模式真核生物对复杂性状遗传变异的分子基础进行解析,通过研究为系统解决数量遗传学的核心问题提供分析框架,并为包括真姬菇等食用菌品种在内的动植物及微生物复杂性状研究提供理论和方法参考。
真姬菇(Hypsizygus marmoreus (Peck) H.E.Bigelow),又称玉蕈、斑玉蕈、鸿喜菇和蟹味菇等,隶属于担子菌门、伞菌纲、伞菌目、白蘑科、玉蕈属。真姬菇是一种珍稀食用菌品种,具有味道鲜美、营养丰富的食用价值和抗真菌、抗肿瘤等药用价值,因此具有良好的市场前景。国内外对于真姬菇的研究主要集中在营养生理生化和药理作用等方面,对于真姬菇分子生物学和遗传学研究鲜有报导。1999年我国签署了《国际植物新品种保护法》,这要求我们在尊重其它国家的品种知识产权的同时也要加强保护本国的品种知识产权。建立有关真姬菇生产用菌种的分子遗传标记,并创立相应的菌种检测标准与方法,是保护我国真姬菇品种知识产权的重要途径。
本研究首先旨在建立一套可靠的AFLP (amplified fragment length polymorphism)分子标记实验方案对真姬菇进行基因分型,并对包括工厂化栽培品种和保藏品种在内的19个真姬菇菌株进行DNA指纹图谱构建和遗传多样性分析。主要研究结果如下：
1、真姬菇AFLP分子标记的建立
真姬菇多糖和蛋白含量较高,这为其基因组DNA的提取带来困难。为了制备符合AFLP标记技术要求的高质量DNA,我们对普遍应用于植物材料的十六烷基三乙基溴化铵(cetyltriethylammonium bromide, CTAB)法进行修改,建立了适合真姬菇的有效的DNA提取方法。
AFLP分析采用的限制性内切酶组合是EcoRⅠ知MseⅠ。通过对选择性碱基种类和数目以及引物组合进行筛选,最终确定10对引物组合(EcoRⅠ+2nt/MseⅠ+3nt)用于多态性检测。运用10对引物在19个基因型中共扩增得到609个AFLP标记位点,其中多态性位点为532个,多态性比率为87%。每对引物扩增的标记总数从47到80不等(平均标记数为60.9),但各引物组合多态信息量(polymorphic information content,PIC)很高,其中PIC最高达到92%。另外,除去由试验误差造成的缺失数据,10对引物在19个基因型中共检测到11184个标记数据点。以上数据表明：(1)真姬菇物种具有丰富的遗传差异和遗传多样性；(2)10对引物都具备较好的多态性检出能力,可作为今后试验(如遗传图谱构建)的候选引物；(3)丰富的多态性位点和标记数据点的获得证明本研究建立的AFLP标记技术能够有效地应用于真姬菇的遗传学研究。
2、19个真姬菇菌株遗传多样性评价
采用Dice相似系数分析和UPGMA法对19个真姬菇菌株的遗传相似性进行聚类分析。聚类结果表明,HmC1199等6个菌株在相似系数大于等于0.95处聚为类群一；另有HmC2637等7个菌株在相似系数大于等于0.92处聚为类群二；其余6个菌株与两个类群的遗传差异相对较大,尤其是菌株HmJ2632与HmC1199的相似系数仅为0.547。同时,采用主成分分析(principal component analysis, PCA)对19个真姬菇菌株的遗传多样性作进一步分析和验证,第一和第二主分揭示出与UPGMA聚类分析一致的结果。
将19个真姬菇菌株遗传分类的结果与形态学特征作比较,发现同一类群的菌株的子实体形态具有共同特点,如类群一,菇盖小、菌柄细；类群二,菇盖大、菌柄短而粗。据此可以把19个菌株划分为4个代表类别：即除两个类群外,还包括白色菇盖品种(HmJ3136)和瘤盖品种(HmJ2632)。尽管我们尚不了解真姬菇菇盖性状的遗传基础,但是克隆这个白色菇盖品种(HmJ3136)的白化基因并将其与其它菌株正常基因作比较,无疑将是一个正确的选择和起点。而瘤盖品种(HmJ2632)与其余18个菌株遗传差异非常大,借鉴其他菌种子实体结实温度的研究结论,我们认为,真姬菇可能存在与香菇类似的子实体结实类型(fruiting season type),其中瘤盖品种(HmJ2632)可能属于高温型,而其余18个菌株则可能分别属于中温或低温型。
目前,在复杂性状遗传结构解析方面,人类和动植物作图群体中有效减数分裂量的匮乏极大地限制了QTL定位的解析率,研究者将目光更多地投向模式生物。酿酒酵母(Saccharomyces cerevisiae)是适用于数量遗传学研究的一个重要的真核模式物种。酵母絮凝是一个可逆的、无性的且依赖于钙离子的过程,是指酵母细胞之间相互粘附形成聚集体(floes).絮凝特性与乙醇发酵生产密切相关,具有重要的工业应用价值。研究表明,酵母FLO基因家族的多个FLO基因的遗传变异和表达修饰可以导致絮凝表型的差异,这反映了絮凝这一性状受多基因控制的复杂遗传本质。本研究采用正向遗传学方法揭示酿酒酵母自然群体中细胞絮凝发生遗传变异的分子基础,通过研究为包括真姬菇等食用菌品种在内的动植物及微生物复杂性状研究提供理论和方法参考,并为用于发酵工业的酵母菌株的遗传改良提供可靠的理论依据。主要研究结果如下：
1、选择两个絮凝表型变异极其显著的酵母菌株YH1A(非絮凝)和YL1C(絮凝)作为亲本,通过两亲本杂交构建了由292个子代个体组成的F2代单倍体分离群体(Huet al.,2007)。同时,构建了以260个微卫星标记(STR)为主(Hu et al.,2007)、24个SNP标记为补充的酵母全基因组饱和分子标记连锁图谱,标记平均间隔在10-20cM范围内；
2、建立了一套简便可靠的实验方法对亲本及子代个体进行絮凝表型测定。以液体培养基中细胞沉淀至管底所用时间(T)作为判定标准,并以两个亲本作为对照,絮凝表型一共划分为四大类：第1类(T=0)；第Ⅱ类(0Understanding 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. marmoreus
The 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
由于食用菌基因组结构复杂且遗传信息匮乏,对真姬菇继续开展复杂性状遗传基础研究困难重重。为此,接下来本文创造性地利用酿酒酵母这一简单的模式真核生物对复杂性状遗传变异的分子基础进行解析,通过研究为系统解决数量遗传学的核心问题提供分析框架,并为包括真姬菇等食用菌品种在内的动植物及微生物复杂性状研究提供理论和方法参考。
真姬菇(Hypsizygus marmoreus (Peck) H.E.Bigelow),又称玉蕈、斑玉蕈、鸿喜菇和蟹味菇等,隶属于担子菌门、伞菌纲、伞菌目、白蘑科、玉蕈属。真姬菇是一种珍稀食用菌品种,具有味道鲜美、营养丰富的食用价值和抗真菌、抗肿瘤等药用价值,因此具有良好的市场前景。国内外对于真姬菇的研究主要集中在营养生理生化和药理作用等方面,对于真姬菇分子生物学和遗传学研究鲜有报导。1999年我国签署了《国际植物新品种保护法》,这要求我们在尊重其它国家的品种知识产权的同时也要加强保护本国的品种知识产权。建立有关真姬菇生产用菌种的分子遗传标记,并创立相应的菌种检测标准与方法,是保护我国真姬菇品种知识产权的重要途径。
本研究首先旨在建立一套可靠的AFLP (amplified fragment length polymorphism)分子标记实验方案对真姬菇进行基因分型,并对包括工厂化栽培品种和保藏品种在内的19个真姬菇菌株进行DNA指纹图谱构建和遗传多样性分析。主要研究结果如下：
1、真姬菇AFLP分子标记的建立
真姬菇多糖和蛋白含量较高,这为其基因组DNA的提取带来困难。为了制备符合AFLP标记技术要求的高质量DNA,我们对普遍应用于植物材料的十六烷基三乙基溴化铵(cetyltriethylammonium bromide, CTAB)法进行修改,建立了适合真姬菇的有效的DNA提取方法。
AFLP分析采用的限制性内切酶组合是EcoRⅠ知MseⅠ。通过对选择性碱基种类和数目以及引物组合进行筛选,最终确定10对引物组合(EcoRⅠ+2nt/MseⅠ+3nt)用于多态性检测。运用10对引物在19个基因型中共扩增得到609个AFLP标记位点,其中多态性位点为532个,多态性比率为87%。每对引物扩增的标记总数从47到80不等(平均标记数为60.9),但各引物组合多态信息量(polymorphic information content,PIC)很高,其中PIC最高达到92%。另外,除去由试验误差造成的缺失数据,10对引物在19个基因型中共检测到11184个标记数据点。以上数据表明：(1)真姬菇物种具有丰富的遗传差异和遗传多样性；(2)10对引物都具备较好的多态性检出能力,可作为今后试验(如遗传图谱构建)的候选引物；(3)丰富的多态性位点和标记数据点的获得证明本研究建立的AFLP标记技术能够有效地应用于真姬菇的遗传学研究。
2、19个真姬菇菌株遗传多样性评价
采用Dice相似系数分析和UPGMA法对19个真姬菇菌株的遗传相似性进行聚类分析。聚类结果表明,HmC1199等6个菌株在相似系数大于等于0.95处聚为类群一；另有HmC2637等7个菌株在相似系数大于等于0.92处聚为类群二；其余6个菌株与两个类群的遗传差异相对较大,尤其是菌株HmJ2632与HmC1199的相似系数仅为0.547。同时,采用主成分分析(principal component analysis, PCA)对19个真姬菇菌株的遗传多样性作进一步分析和验证,第一和第二主分揭示出与UPGMA聚类分析一致的结果。
将19个真姬菇菌株遗传分类的结果与形态学特征作比较,发现同一类群的菌株的子实体形态具有共同特点,如类群一,菇盖小、菌柄细；类群二,菇盖大、菌柄短而粗。据此可以把19个菌株划分为4个代表类别：即除两个类群外,还包括白色菇盖品种(HmJ3136)和瘤盖品种(HmJ2632)。尽管我们尚不了解真姬菇菇盖性状的遗传基础,但是克隆这个白色菇盖品种(HmJ3136)的白化基因并将其与其它菌株正常基因作比较,无疑将是一个正确的选择和起点。而瘤盖品种(HmJ2632)与其余18个菌株遗传差异非常大,借鉴其他菌种子实体结实温度的研究结论,我们认为,真姬菇可能存在与香菇类似的子实体结实类型(fruiting season type),其中瘤盖品种(HmJ2632)可能属于高温型,而其余18个菌株则可能分别属于中温或低温型。
目前,在复杂性状遗传结构解析方面,人类和动植物作图群体中有效减数分裂量的匮乏极大地限制了QTL定位的解析率,研究者将目光更多地投向模式生物。酿酒酵母(Saccharomyces cerevisiae)是适用于数量遗传学研究的一个重要的真核模式物种。酵母絮凝是一个可逆的、无性的且依赖于钙离子的过程,是指酵母细胞之间相互粘附形成聚集体(floes).絮凝特性与乙醇发酵生产密切相关,具有重要的工业应用价值。研究表明,酵母FLO基因家族的多个FLO基因的遗传变异和表达修饰可以导致絮凝表型的差异,这反映了絮凝这一性状受多基因控制的复杂遗传本质。本研究采用正向遗传学方法揭示酿酒酵母自然群体中细胞絮凝发生遗传变异的分子基础,通过研究为包括真姬菇等食用菌品种在内的动植物及微生物复杂性状研究提供理论和方法参考,并为用于发酵工业的酵母菌株的遗传改良提供可靠的理论依据。主要研究结果如下：
1、选择两个絮凝表型变异极其显著的酵母菌株YH1A(非絮凝)和YL1C(絮凝)作为亲本,通过两亲本杂交构建了由292个子代个体组成的F2代单倍体分离群体(Huet al.,2007)。同时,构建了以260个微卫星标记(STR)为主(Hu et al.,2007)、24个SNP标记为补充的酵母全基因组饱和分子标记连锁图谱,标记平均间隔在10-20cM范围内；
2、建立了一套简便可靠的实验方法对亲本及子代个体进行絮凝表型测定。以液体培养基中细胞沉淀至管底所用时间(T)作为判定标准,并以两个亲本作为对照,絮凝表型一共划分为四大类：第1类(T=0)；第Ⅱ类(0
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. marmoreus
The 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
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