糙皮侧耳优良杂交子筛选与侧耳属野生菌株驯化栽培研究
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摘要
本试验以糙皮侧耳(Pleurotus ostreatus)4个栽培菌株HP(华平特白)、PG30(平高30)、JG3(姬菇3号)、PG981(平菇981)为试验材料,采用单单杂交与双单杂交两种不同杂交方式获得杂交子进行育种试验,经过秋栽和春栽筛选农艺性状优良的杂交子。野外采集得到两野生菌株子实体并进行组织分离,基于ITS序列分析对两野生菌株进行鉴定,期望能驯化栽培出菇获得新的种质资源。
以锁状联合为遗传标记,利用原生质体单核化技术得到4个栽培菌株两种不同交配型的单核体,用单单杂交与双单杂交两种不同杂交方式将4个栽培菌株之间相互进行完全配对杂交,结合拮抗试验和ISSR分子标记鉴定杂交子。运用灰色关联度法对供试菌株的8个农艺性状进行综合比较,结合鲜菇总产量邓肯多重比较筛选杂交子。基于ITS序列构建两野生菌株的同源进化树,对两野生菌株进行鉴定。对未能出菇的张家界野生菌株利用原生质体技术再生出双核体,将再生双核体萌发的双核菌丝进行驯化栽培出菇试验。
试验结果表明,华平特白、平高30、平菇981两种交配型单核体的比例存在偏分离现象。4个供试菌株的8个单核体相互完全配对杂交,结合与双亲本的拮抗试验单单杂交配对得到13个杂交子。正反双单杂交完全配对得到13个杂交子。运用ISSR分子标记对杂交子进行了遗传分析,从40个引物中筛选出5个条带重复性好、谱带清晰的引物,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。聚类结果显示33个杂交子GS值变化范围0.31～0.94,进一步验证了双单杂交子的可靠性。灰色关联度与鲜菇产量邓肯多重比较结果确定了杂交菌株27号、26号、19号、37号、28号、30号等6个优良杂交子,春季栽培观察结果显示该6个杂交菌株性状表现稳定。采用PCR产物克隆测序获得两野生菌株ITS序列,测序后与其相似系数最高的前10位菌株进行同源性分析,鉴定出武当山野生菌株为佛罗里达侧耳,张家界野生菌株为凤尾菇。
上述研究表明单单杂交及双单杂交均可获得农艺性状优于亲本的杂交子。张家界野生菌株经过原生质体再生双核体分离可得以驯化。
The objectives of this study were to breed new excellent strains of Pleurotus ostreatus by the technique of Mon-Mon crossing and Di-Mon crossing breeding. HP, PG30, JG3 and PG981 were four test-strains. We cultivated the hybrids and parents in the fall and spring. Vegetative mycelium was isolated from tissue of fresh fruit body of wild strains. Based on ITS region sequence alignment identified two wild strains into species, this two wild strains would be the new genetic resource.
Non clamped monokaryons with two different mating types were isolated from HP, PG30, JG3 and PG981 by using protoplast monokaryogenesis techniques in this Mon-Mon mating and Di-Mon mating experiment. The hybrids of successful cross were preliminarily selected on the basis of antagonism test and ISSR markers. The grey correlation degree analysis of agronomic characters and Duncan multiple Comparison test were done of selected excellent hybrids for comparing their yield. Identified two wild strains through phylogenetic tree based on ITS region sequence alignment .one of wild strain can not domestication and cultivation; it is hope that using the heterokaryon from protoplast regeneration of this strain can be domestication.
The results showed that HP, PG30 and PG981 their ration of two types of mycelium protoplast monokaryons were not equality, According to the antagonism test we get thirteen hybrids from Mon-Mon crossing and thirteen hybrids from Di-Mon crossing breeding. ISSR markers were applied for study on genetic analysis of hybrids. Forty ISSR primers were screened and five of which could be differentiating hybrid strains effectively. The amplification results showed that genetic similarity coefficient ranged from 0.31 to 0.94, and to provide strong support that Di-Mon hybrids were reality hybrids. Compare grey correlation degree analysis of eight agronomic characters with Duncan multiple Comparison test for their yield. All hybrids including their parents and wild strains were cultivated twice in a year under control condition. The hybrid strains 27, 26, 19, 37, 28 and 30 performed better than that of others in both cultivation times. The two wild strains have been identified as Pleurotus floridanus and Pleurotus sajor-caju according to the results of internal transcription space (ITS) sequence, compare with the first 10 similar generous uses in GenBank and phylogenetic tree.
The results showed that both of Mon-Mon crossing and Di-Mon crossing breeding equally effective to generate new excellent strains. Application of protoplast technique was in Zhangjiajie wild strain for the fruitful cultivation.
以锁状联合为遗传标记,利用原生质体单核化技术得到4个栽培菌株两种不同交配型的单核体,用单单杂交与双单杂交两种不同杂交方式将4个栽培菌株之间相互进行完全配对杂交,结合拮抗试验和ISSR分子标记鉴定杂交子。运用灰色关联度法对供试菌株的8个农艺性状进行综合比较,结合鲜菇总产量邓肯多重比较筛选杂交子。基于ITS序列构建两野生菌株的同源进化树,对两野生菌株进行鉴定。对未能出菇的张家界野生菌株利用原生质体技术再生出双核体,将再生双核体萌发的双核菌丝进行驯化栽培出菇试验。
试验结果表明,华平特白、平高30、平菇981两种交配型单核体的比例存在偏分离现象。4个供试菌株的8个单核体相互完全配对杂交,结合与双亲本的拮抗试验单单杂交配对得到13个杂交子。正反双单杂交完全配对得到13个杂交子。运用ISSR分子标记对杂交子进行了遗传分析,从40个引物中筛选出5个条带重复性好、谱带清晰的引物,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。聚类结果显示33个杂交子GS值变化范围0.31～0.94,进一步验证了双单杂交子的可靠性。灰色关联度与鲜菇产量邓肯多重比较结果确定了杂交菌株27号、26号、19号、37号、28号、30号等6个优良杂交子,春季栽培观察结果显示该6个杂交菌株性状表现稳定。采用PCR产物克隆测序获得两野生菌株ITS序列,测序后与其相似系数最高的前10位菌株进行同源性分析,鉴定出武当山野生菌株为佛罗里达侧耳,张家界野生菌株为凤尾菇。
上述研究表明单单杂交及双单杂交均可获得农艺性状优于亲本的杂交子。张家界野生菌株经过原生质体再生双核体分离可得以驯化。
The objectives of this study were to breed new excellent strains of Pleurotus ostreatus by the technique of Mon-Mon crossing and Di-Mon crossing breeding. HP, PG30, JG3 and PG981 were four test-strains. We cultivated the hybrids and parents in the fall and spring. Vegetative mycelium was isolated from tissue of fresh fruit body of wild strains. Based on ITS region sequence alignment identified two wild strains into species, this two wild strains would be the new genetic resource.
Non clamped monokaryons with two different mating types were isolated from HP, PG30, JG3 and PG981 by using protoplast monokaryogenesis techniques in this Mon-Mon mating and Di-Mon mating experiment. The hybrids of successful cross were preliminarily selected on the basis of antagonism test and ISSR markers. The grey correlation degree analysis of agronomic characters and Duncan multiple Comparison test were done of selected excellent hybrids for comparing their yield. Identified two wild strains through phylogenetic tree based on ITS region sequence alignment .one of wild strain can not domestication and cultivation; it is hope that using the heterokaryon from protoplast regeneration of this strain can be domestication.
The results showed that HP, PG30 and PG981 their ration of two types of mycelium protoplast monokaryons were not equality, According to the antagonism test we get thirteen hybrids from Mon-Mon crossing and thirteen hybrids from Di-Mon crossing breeding. ISSR markers were applied for study on genetic analysis of hybrids. Forty ISSR primers were screened and five of which could be differentiating hybrid strains effectively. The amplification results showed that genetic similarity coefficient ranged from 0.31 to 0.94, and to provide strong support that Di-Mon hybrids were reality hybrids. Compare grey correlation degree analysis of eight agronomic characters with Duncan multiple Comparison test for their yield. All hybrids including their parents and wild strains were cultivated twice in a year under control condition. The hybrid strains 27, 26, 19, 37, 28 and 30 performed better than that of others in both cultivation times. The two wild strains have been identified as Pleurotus floridanus and Pleurotus sajor-caju according to the results of internal transcription space (ITS) sequence, compare with the first 10 similar generous uses in GenBank and phylogenetic tree.
The results showed that both of Mon-Mon crossing and Di-Mon crossing breeding equally effective to generate new excellent strains. Application of protoplast technique was in Zhangjiajie wild strain for the fruitful cultivation.
引文
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2.贺建超,贺榆霞.双孢蘑菇单孢子杂交育种研究.中国食用菌,2006,25(3):18-19
3.候集瑞,李玉,图力古尔,孙晓波.紫外线诱导对药用真菌菌丝体生长的影响.吉林农业大学学报,2002,24(6):20-24
4.贾定洪,郑用林,王波,郭勇,甘炳成,黄忠乾,彭卫红.五个野生木耳属菌株的ISSR分析.食用菌学报2008,15(3):7-12
5.贾身茂,秦淼.我国白阿魏蘑的驯化与栽培.中国食用菌,2006,25(3):3-7
6.江枝和,翁伯琦,黄挺俊.~(60)Co辐射诱变姬松茸突变株J3的氨基酸分析与比较研究.西北农业学报,2003,12(3):129-132
7.金玲,武艳霞,周宗俊.原生质体辐射诱变培育金针菇新菌株.园艺学报,2000,27(1):65-66
8.李翠新,何德.侧耳属蕈菌系统分类研究进展.中国食用菌,2007,26(5):8-10
9.李德舜,刘正学,张英,张长岂.平菇山大1号紫外诱变育种研究.食用菌,2002,3:9-11
10.李辉平,黄晨阳,陈强,张金霞.黑木耳栽培菌株的ISSR分析.园艺学报,2007,34(4):935-940
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