榆黄蘑单孢杂交及后代筛选
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摘要
榆黄蘑属于食药兼用菌,具有很高的营养价值和药用价值,榆黄蘑的人工栽培开始于20世纪70年代,现在已经能够进行周年栽培。但是到目前为止,在栽培上大都将采集的野生菌株直接用于生产,或者经过筛选驯化后作生产用菌种,新品种选育的报道较为少见。从世界各先进国家育种工作现状来看,杂交育种仍是食用菌育种中应用广泛、效果显著的一种方法,鉴于此,本文采用单孢杂交的手段对榆黄蘑新品种的选育工作进行了研究,以期获得优良新菌株。
1采用垂直板聚丙烯酰胺凝胶电泳技术,利用酯酶同工酶和过氧化物同工酶酶谱的相似性和差异性,计算了8个榆黄蘑供试菌株间的Pearson相似系数;并采用类间平均法(Between-groups linkage)进行系统聚类,对供试菌株的亲缘关系进行分析。结果表明,所有供试菌株在2个同工酶系统中均有共同特征谱带,同时又体现了一定的多态性。菌株pc-5与其它各菌株遗传差异均较大。另外,酯酶同工酶酶谱在Rf 0.07,Rf 0.27和Rf 0.51处存在的三条稳定的共同酶带和过氧化物同工酶酶谱分别在Rf 0.39,Rf 0.64和Rf 0.81处存在的三条稳定清晰的共同酶带均可以作为表达榆黄蘑这个种基本特征的基础酶带。
2本实验对8个榆黄蘑供试菌株进行了拮抗试验,除菌株Pc-1和Pc-7、菌株Pc-3和Pc-8两个组合的拮抗线不明显外,其它菌株间拮抗线都比较明显,经同工酶分析,拮抗线不明显的菌株组合在两种同工酶酶谱中谱带类型均相同或相近。
3对8个榆黄蘑供试菌株菌丝体的生长情况及农艺性状进行了比较,并对某些性状的相性关进行了分析,结果表明:母种菌丝生长速度与栽培种菌丝长速呈正相关;各菌株栽培种菌丝生长速度与平均生物学效率的相关性不显著;菌株的产量与质量没有相关性。
4通过以上对供试菌株的菌丝生长情况、生物学效率、子实体农艺性状等进行比较,经拮抗试验、同工酶酶谱、相似系数及聚类分析等筛选出亲缘关系较远,农艺性状优良且能互补的杂交亲本组合,分别是菌株Pc-5和菌株Pc-8。
5通过对亲本进行单饱分离、培养、镜检,获得单核体64个,然后进行不完全双列杂交,共获得1008对杂交组合,经镜检发现有锁状联合的菌株937个,经拮抗试验和液体出菇试验,筛选出95个与其双亲拮抗线明显且出现原基的杂交菌株。对亲本及95个杂交子代的菌丝体进行酯酶同工酶分析,在杂交子代中选出了具有“杂种新酶带”或“互补型酶带”的强优势杂交新菌株15个,对这15个新菌株的菌丝生长情况、多糖含量、抗绿菌能力农艺性状和低温出菇特性等与亲本进行了比较分析,最后筛选出具有利用推广价值的菌株5个。
Pleurotus citrinopileatus Sing. is a famous Fungus of Basidiomycetes for its high food value and many kinds of medicinal functions. Its manual cultivation tarted from1970s.Up to the present,it can be produced yearly. But the breeding of P. citninopileatus is still not conducted in the world; so the Pleurotus citrinopileatus used for cultivation was wild strain.In this study, the breeding of P. citninopileatus was carried out by monospore crossand several new strains were obtained preliminarily. The main results were showed as follows:
1.The differences and similarity of esterase isoenzymes(EST)and Peroxidase isoenzymes (POD)of 8 Pleurotus citrinopileatus strains were analyzed by between -groups linkage Cluster Method. The results showed that the basic characteristics and polymorphism of EST and POD isozyme bands were evident. The relationship between strains pc-5 and others was distant. The three stable common EST bands at Rf 0.07, Rf 0.27 and Rf 0.51 and the three stable common POD bands at Rf 0.39, Rf 0.64 and Rf 0.81 could be considered as the foundation enzyme bands of P. citninopileatus.
2 . In the antagonistic experiment of 8 Pleurotus citrinopileatus strains, except the combinations of Pc-1×Pc-7 and Pc-3×Pc-8, the others exhibited antagonism obviously. Isozyme analysis showed that the types of bands were same or similar in the combinations of Pc-1×Pc-7 and Pc-3×Pc-8.
3.The mycelium growth conditions, agronomic characters and fruitbody yields of the 8 Pleurotus citrinopileatus strains were compared.The results showed that there existed the positive correlation relationship between the mother culture growth rate and culture spawn growth rate, but no correlation between the production and mycelial growth rate and also no correlation between the fruitbody yields and fruitbody quality.
4. According to the above experimental date and combined with the isozymecluster dendrogram and similarity index of 8 Pleurotus citrinopileatus strains,the two strains with complementary agronomic characters were screened out.
5.By isolation and identification of individual spore from parent strains, 64 monokaryotic mycelium were obtained, and then by hybridization of incomplete diallel cross, examination under the microscope, identification of antagonistic reaction and analysis of complementary enzyme bands, mycelium growth rate, polysaccharide content and cryogenic fruiting characteristics, 5 new strains which have application value were obtained.
1采用垂直板聚丙烯酰胺凝胶电泳技术,利用酯酶同工酶和过氧化物同工酶酶谱的相似性和差异性,计算了8个榆黄蘑供试菌株间的Pearson相似系数;并采用类间平均法(Between-groups linkage)进行系统聚类,对供试菌株的亲缘关系进行分析。结果表明,所有供试菌株在2个同工酶系统中均有共同特征谱带,同时又体现了一定的多态性。菌株pc-5与其它各菌株遗传差异均较大。另外,酯酶同工酶酶谱在Rf 0.07,Rf 0.27和Rf 0.51处存在的三条稳定的共同酶带和过氧化物同工酶酶谱分别在Rf 0.39,Rf 0.64和Rf 0.81处存在的三条稳定清晰的共同酶带均可以作为表达榆黄蘑这个种基本特征的基础酶带。
2本实验对8个榆黄蘑供试菌株进行了拮抗试验,除菌株Pc-1和Pc-7、菌株Pc-3和Pc-8两个组合的拮抗线不明显外,其它菌株间拮抗线都比较明显,经同工酶分析,拮抗线不明显的菌株组合在两种同工酶酶谱中谱带类型均相同或相近。
3对8个榆黄蘑供试菌株菌丝体的生长情况及农艺性状进行了比较,并对某些性状的相性关进行了分析,结果表明:母种菌丝生长速度与栽培种菌丝长速呈正相关;各菌株栽培种菌丝生长速度与平均生物学效率的相关性不显著;菌株的产量与质量没有相关性。
4通过以上对供试菌株的菌丝生长情况、生物学效率、子实体农艺性状等进行比较,经拮抗试验、同工酶酶谱、相似系数及聚类分析等筛选出亲缘关系较远,农艺性状优良且能互补的杂交亲本组合,分别是菌株Pc-5和菌株Pc-8。
5通过对亲本进行单饱分离、培养、镜检,获得单核体64个,然后进行不完全双列杂交,共获得1008对杂交组合,经镜检发现有锁状联合的菌株937个,经拮抗试验和液体出菇试验,筛选出95个与其双亲拮抗线明显且出现原基的杂交菌株。对亲本及95个杂交子代的菌丝体进行酯酶同工酶分析,在杂交子代中选出了具有“杂种新酶带”或“互补型酶带”的强优势杂交新菌株15个,对这15个新菌株的菌丝生长情况、多糖含量、抗绿菌能力农艺性状和低温出菇特性等与亲本进行了比较分析,最后筛选出具有利用推广价值的菌株5个。
Pleurotus citrinopileatus Sing. is a famous Fungus of Basidiomycetes for its high food value and many kinds of medicinal functions. Its manual cultivation tarted from1970s.Up to the present,it can be produced yearly. But the breeding of P. citninopileatus is still not conducted in the world; so the Pleurotus citrinopileatus used for cultivation was wild strain.In this study, the breeding of P. citninopileatus was carried out by monospore crossand several new strains were obtained preliminarily. The main results were showed as follows:
1.The differences and similarity of esterase isoenzymes(EST)and Peroxidase isoenzymes (POD)of 8 Pleurotus citrinopileatus strains were analyzed by between -groups linkage Cluster Method. The results showed that the basic characteristics and polymorphism of EST and POD isozyme bands were evident. The relationship between strains pc-5 and others was distant. The three stable common EST bands at Rf 0.07, Rf 0.27 and Rf 0.51 and the three stable common POD bands at Rf 0.39, Rf 0.64 and Rf 0.81 could be considered as the foundation enzyme bands of P. citninopileatus.
2 . In the antagonistic experiment of 8 Pleurotus citrinopileatus strains, except the combinations of Pc-1×Pc-7 and Pc-3×Pc-8, the others exhibited antagonism obviously. Isozyme analysis showed that the types of bands were same or similar in the combinations of Pc-1×Pc-7 and Pc-3×Pc-8.
3.The mycelium growth conditions, agronomic characters and fruitbody yields of the 8 Pleurotus citrinopileatus strains were compared.The results showed that there existed the positive correlation relationship between the mother culture growth rate and culture spawn growth rate, but no correlation between the production and mycelial growth rate and also no correlation between the fruitbody yields and fruitbody quality.
4. According to the above experimental date and combined with the isozymecluster dendrogram and similarity index of 8 Pleurotus citrinopileatus strains,the two strains with complementary agronomic characters were screened out.
5.By isolation and identification of individual spore from parent strains, 64 monokaryotic mycelium were obtained, and then by hybridization of incomplete diallel cross, examination under the microscope, identification of antagonistic reaction and analysis of complementary enzyme bands, mycelium growth rate, polysaccharide content and cryogenic fruiting characteristics, 5 new strains which have application value were obtained.
引文
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