利用SSR标记鉴定香菇单核体及杂交后代

【目的】研究简单重复序列(Simple sequence repeat,SSR)分子标记方法用于香菇原生质体单核体、孢子单核体及其杂交后代的分离和鉴定。【方法】利用基于香菇全基因组序列信息开发的SSR标记,分析由香菇品种"L808"双核菌丝制备的原生质体单核体、孢子单核体及其杂交后代的SSR指纹。【结果】对制备的原生质体单核体的鉴定中,在不经过杂交配对的情况下,鉴定出"L808"的两种不同极性的原生质体单核体,其分离比例为191:1,该鉴定结果得到SSR标记、随机扩增多态性DNA(Random amplified polymorphismic DNA,RAPD)标记及传统方法的验证。另外,开发的香菇SSR标记还能以多位点组合的方式,用于对孢子单核体及其杂交后代的鉴定。【结论】应用SSR标记可加快香菇单核体的制备进程,并提高鉴定单核体及相关杂交菌株的准确性,促进香菇遗传育种研究。     

黑木耳亲本、单核、杂交菌株鉴定的研究

应用荧光核染色技术、酯酶同工酶鉴定技术,对黑木耳亲本双核菌株、原生质体单核菌株和单-单杂交菌株进行筛选鉴定。结果表明:荧光核染色技术可快速有效地鉴定获得菌株的核相,酯酶同工酶技术可准确地区分单核菌株及杂交新菌株的不同核型,研究得到1个酶带差异显著的新菌株。     

凤尾菇和裂褶菌原生质体非对称融合研究

具有双重营养缺陷型的裂褶菌单核体突变菌株的原生质体经过灭活处理后作为供体与同样带有营养缺陷型标记的风尾菇单核体原生质体融合,得到大量生长速度和菌落形态差异较大的多核体和双核体融合子．这些融合子主要显示了供体菌株的特性,数次转接后,有的融合子从多核体转变成双核体,有的融合子完全恢复了供体亲本双核体的遗传特性,同时获得了一些有意义值得继续探讨的新的生理特性,如菌丝体在木屑 皮以及废棉培养料上生长速度较快,在平板培养基和木屑培养料上均较早较快地形成原基和子实体等．结果表明原生质体非对称融合是一种效率较高的融合方式,可以作为食用菌育种的一种有效途经．     

凤尾菇和盖囊侧耳原生质体非对称融合的研究

盖囊侧耳的双核体原生质体经过灭活处理作为供体与带有营养缺陷型标记的凤尾菇单核体原生质体受体融合,得到大量生长速度和菌落形态差异较大的融合子,这些融合子主要显示了供体菌株的特性,数次转接和进行原生质体再分离后,有的融合子再生菌株恢复了供体亲本的遗传特性,同时获得了一些新的生理特证。结果表明原生质体非对称融合可以作为食用菌原生质体融合育种的一种有效方式。     

茯苓与凤尾菇目间原生质体融合研究初报

以无锁状联合的茯苓次级菌丝和具锁状联合的凤尾菇次级菌丝为亲本制备原生质体,以PEG为促融剂,在高钙、高pH条件下,将茯苓(或凤尾菇)原生质体热灭活,与凤尾菇(或茯苓)原生质体融合,从再生菌株中均能选择选择出能够再形成双核的单核菌株。栽培实验结果显示,单核菌株经诱导形成双核菌株后可以形成子实体或原基,若单核菌株不经诱导,则始终维持稳定的单核状态,且难以形成子实体。     

凤尾菇和裂褶菌原生质体非对称融合研究

具有双重营养缺陷型的裂褶菌单核体突变菌株的原生质体经过灭活处理后作为供体与同样带有营养缺陷型标记的凤尾菇单核体原生质体融合,得到大量生长速度和菌落形态差异较大的多核体和双核体融合子。这些融合子完全恢复了供体亲本双核体的遗传特性,同时获得了一些有意义值得继续探讨的新的生理特性,如菌丝体在木屑麸皮以及废棉培养料上生长速度较快,在平板培养基和木屑培养料上均较早较快地形成原基和子实体等。结果表明原生质体非对称融合是一种效率较高的融合方式,可以作为食用菌育种的一种有效途径。     

黑木耳交配型的研究*

从黑木耳Auricularia auricula沪3菌株的子实体收集、分离、鉴定得到157株单孢子萌发的单核菌丝体,以其中73株孢子单核体为材料进行交配实验,经过三轮交配反应,结果表明,木耳的交配型符合四极性交配系统的分布规律。根据四种交配反应情况把73株孢子单核体分为四组,并从四组中挑选出B17、B177、B101、B65作为四种交配型的标准菌株;与此同时,从沪3菌株的双核菌丝体制备的原生质体中获得53株原生质体单核体,对这些单核体进行交配,确定出两种亲本交配型,以Y150(A1B1)和Y121(A2B2)作为标准菌株,将两个亲本交配型标准菌株与四个孢子单核体标准菌株进行交配反应,初步确定出四种孢子单核体的交配型分别为: B17 (A2B2)、B177(A1B1)、B101(A2B1)和B65(A1B2)。     

凤尾菇和盖囊侧耳原生质体非对称融合的研究

盖囊侧耳的双核本原生质体经过灭活处理作为供体与带有营养缺陷型标记的凤尾菇单核体原生质体受体融合,得以大量生长速度和菌落形态差交大的融合子,这些融合子主要显示了供体菌株的特性,数次转接和进行原生质体再分离后,有的融合子再生菌株恢复了供体亲本的遗传特性,同时获得了一些新的生理特证。结果表明原生质体非对称融合可以作为食用菌原生质体融合育种的一种有效方式。     

香菇亲本菌株及其杂交后代的RAPD分析

运用随机扩增多态性ＤＮＡ技术对源于两个香菇双核菌的孢子单核体,原生质体单核体及其杂交后代进行了基因组ＤＮＡ多态性分析,用９个随机引物共扩增出１１６条ＤＮＡ片段,其中８２．５％具有多态性,综合分析９个随机引物的扩增谱带,可将所有供试亲本单核本清楚地工,且单聚类分析的结果与其来源及遗传背景相吻合。此外,用两个双核亲本菌株的各４个不同酱 孢子单核体两两酱所得的所有杂交组合,也均可与双核亲本菌株明确地区分     

ISSR标记在黑木耳单核体遗传分析中的应用

采用原生质体单核化和单孢分离法分别获得黑木耳Auricularia auricula栽培菌株新科5号两个亲本单核体(T1、T2)和33个F1代孢子单核体。从73条ISSR引物中筛选出13条可区分两个亲本单核体(T1、T2)的引物,对新科5号及F1代孢子单核体进行扩增,共扩增出70条带,其中63条在供试菌株间表现出多态性,多态位点百分率为90.0%。根据扩增结果采用软件NTsys2.10e计算菌株间的遗传相似系数并进行聚类分析,36个菌株间的遗传相似系数(GS值)的变化范围为0.2500～0.8382,其中T1和T2之间的GS值最小,遗传相似程度最低;F1代33个孢子单核体中24个与T1聚为一类,其余9个与T2聚为另一类,但并非严格按照交配型进行归类。试验表明黑木耳子实体上各个担子在减数分裂中染色体交换极具多样性,F1代孢子单核体表现出偏向其中一个亲本的现象,ISSR标记在黑木耳杂交育种中具有潜在的应用价值。     

香菇135菌株特异SCAR标记在其原生质体单核中的分布

以香菇保藏菌种庆元135以及庆元出菇的135新鲜子实体组织分离得到的菌丝为材料制备原生质体,分别获得58和83个原生质体单核体;经交配型鉴定后,用已获得的135特异SCAR引物对单核进行PCR扩增,统计SCAR条带的分布。结果显示：原生质体单核体分为A1B1和A2B2两种交配型,而特异条带仅存在于后者中,证明135特异SCAR标记是稳定遗传的,也为鉴定以带标记核为亲本之一的杂交后代提供了依据,这就进一步拓宽了分子标记应用于菌株鉴定的适用范围。     

香菇135菌株特异SCAR标记在其原生质体单核中的分布

以香菇保藏菌种庆元135以及庆元出菇的135新鲜子实体组织分离得到的菌丝为材料制备原生质体,分别获得58和83个原生质体单核体;经交配型鉴定后,用已获得的135特异SCAR引物对单核进行PCR扩增,统计SCAR条带的分布。结果显示:原生质体单核体分为A1B1和A2B2两种交配型,而特异条带仅存在于后者中,证明135特异SCAR标记是稳定遗传的,也为鉴定以带标记核为亲本之一的杂交后代提供了依据,这就进一步拓宽了分子标记应用于菌株鉴定的适用范围。     

Development of SCAR Markers to Determine the Mating Types of Lepista nuda Protoplast Monokaryons

Lepista nuda (Bull. ex Fr.) Cooke belongs to Tricholomataceae and is an edible fungus with both economic and medical value. Mycelia were isolated from the fruiting bodies of L. nuda and were used to prepare the protoplast monokaryons. One hundred and fifteen monokaryons were obtained and their mating types were determined using somatic incompatibility tests. Protoplast monokaryons segregated into either the A₁B₁ or the A₂B₂ mating types. Inter-simple sequence repeats and sequence-related amplified polymorphism fingerprinting were used to analyse the mating types of these protoplast monokaryons and 16 sequence-characterised amplified region primers were developed to efficiently differentiate between the monokaryon mating types. Multiplex PCR analyses were also established. The data presented here outline a method for the precise and rapid identification of protoplast monokaryon mating types, which has the promise to shorten the period required for conventional crossbreeding.     

Isolation and characterization of a sporeless mutant in Pleurotus eryngii

A sporeless mutant dikaryon, completely defective in sporulation, was isolated from mycelial protoplasts of Pleurotus eryngii mutagenized by UV irradiation. Newly established dikaryons between one component monokaryon from the mutant, and 12 different wild type monokaryons from 3 other wild type dikaryons, all exhibited the sporeless phenotype, whereas those between the other monokaryon and the same wild type monokaryons all produced normal fruiting bodies. These results indicated that the sporeless mutation was induced in one of two nuclei of the mutant and was dominant. In the wild type basidia, the pattern of nuclear behavior during sporulation corresponded to the pattern C nuclear behavior as defined by Duncan and Galbraith. Cytological observation revealed that in the sporeless mutant meiosis was blocked at the meta-anaphase I in most basidia and hence basidiospores and sterigmata were not produced. Although fruiting bodies of the sporeless mutant showed a somewhat leaning growth, their gross morphology and its fruiting body productivity were comparable to that of the original wild type strain. Based on these results, it was considered that the sporeless mutant could serve as a potential material in breeding of sporeless P. eryngii commercial strains.     

香菇交配型因子次级重组体的鉴定

对13个香菇菌株的担孢子后代进行了交配型分析,其中8个菌株非亲和反应与亲和反应之比与预期的3∶1的比例无显著差异。另外5个菌株非亲和反应与亲和反应之比不符合3∶1,其中4个菌株在0.05显著水平的X2值仅略高于理论值,而另一菌株HL01具有特殊的表现,其单核体132个随机配对的非亲和反应与亲和反应之比为82∶50,X2值显著偏离3∶1的临界值。用4个标准测试菌株鉴定了来自HL01同一子实体的189个孢子单核体的交配型,在189个单核体中,161个单核体归于4种正常交配型(A1B1,A2B2,A1B2,A2B1)之一。而另外28个可能源于次级重组的单核体可分成另外4个类群。通过以所有可能的组合进行配对杂交,进一步分析了28个单核体的交配型。结果表明,次级重组同时在A因子和B因子中发生,重组值分别为8.5%和11.6%。A因子至少由2个亚基组成而B因子可能由不止2个亚基组成。随后的出菇试验表明,至少含有1个重组体的所有可亲和配对均具有结实能力。     

Kraft Pulp Bleaching and Delignification by Dikaryons and Monokaryons of Trametes versicolor

The ability of 10 dikaryotic and 20 monokaryotic strains of Trametes (Coriolus) versicolor to bleach and delignify hardwood and softwood kraft pulps was assessed. A dikaryon (52P) and two of its mating-compatible monokaryons (52J and 52D) derived via protoplasting were compared. All three regularly bleached hardwood kraft pulp more than 20 brightness points (International Standards Organization) in 5 days and softwood kraft pulp the same amount in 12 days. Delignification (kappa number reduction) by the dikaryon and the monokaryons was similar, but the growth of the monokaryons was slower. Insoluble dark pigments were commonly found in the mycelium, medium, and pulp of the dikaryon only. Laccase and manganese peroxidase (MnP) but not lignin peroxidase activities were secreted during bleaching by all three strains. Their laccase and MnP isozyme patterns were compared on native gels. No segregation of isozyme bands between the monokaryons was found. Hardwood kraft pulp appeared to adsorb several laccase isozyme bands. One MnP isozyme (pI, 3.2) was secreted in the presence of pulp by all three strains, but a second (pI, 4.9) was produced only by 52P. A lower level of soluble MnP activity in one monokaryon (52D) was associated with reduced bleaching ability and a lower level of methanol production. Since monokaryon 52J bleached pulp better than its parent dikaryon 52P, especially per unit of biomass, this genetically simpler monokaryon will be the preferred subject for further genetic manipulation and improvement of fungal pulp biological bleaching.     

Identification of Degenerate Nuclei and Development of a SCAR Marker for Flammulina velutipes

Flammulina velutipes is one of the major edible mushrooms in the world. Recently, abnormalities that have a negative impact on crop production have been reported in this mushroom. These symptoms include slow vegetative growth, a compact mycelial mat, and few or even no fruiting bodies. The morphologies and fruiting capabilities of monokaryons of wild-type and degenerate strains that arose through arthrospore formation were investigated through test crossing. Only one monokaryotic group of the degenerate strains and its hybrid strains showed abnormal phenotypes. Because the monokaryotic arthrospore has the same nucleus as the parent strain, these results indicated that only one aberrant nucleus of the two nuclei in the degenerate strain was responsible for the degeneracy. A sequence-characterized amplified region marker that is linked to the degenerate monokaryon was identified based on a polymorphic sequence that was generated using random primers. Comparative analyses revealed the presence of a degenerate-specific genomic region in a telomere, which arose via the transfer of a genomic fragment harboring a putative helicase gene. Our findings have narrowed down the potential molecular targets responsible for this phenotype for future studies and have provided a marker for the detection of degenerate strains.     

高等担子菌双重营养缺陷突变菌株的分离

带有单一营养缺陷的凤尾菇和裂褶菌的单核体菌株经亲和性交配,各自交配产生后代,从中分离出遗传特性稳定,生理特征表型正常的双重缺陷营养害变型菌株,为原生质体融合育种研究提供了可靠的亲本菌株。