松杨栅锈菌种内群体分化及SCAR标记
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摘要
松杨栅锈菌(Melampsora larici-populina Kleb.)是青杨派、黑杨派及其杂交杨上重要的病原之一,在世界范围内均有发生。该菌种内群体分化明显,遗传背景复杂。欧洲、北美曾报道过该菌的5个生理小种,我国也相继发现该菌生理小种分化现象。为摸清该菌在我国的发生状况和遗传背景,准确的进行小种鉴别,本文采用致病性测定、ITS序列分析、RFLP、RAPD、RAMs、SCAR标记、细胞核染色等技术对该菌进行了研究,取得了如下成果:
1.通过对幼苗温室接种,对来自全国各地的松杨栅锈菌致病性分化进行了系统研究,建立了我国该菌小种鉴别的技术体系,确定了我国该菌小种鉴别的寄主杨树谱,以及扩繁寄主杨树(太白杨),首次提出我国松杨栅锈菌5个生理小种的观点。
2.在对比分析的基础上,创建了锈菌夏孢子微量快速提取技术,并对该技术及其在PCR操作中的应用进行了优化研究。该方法克服了常规分子操作中大量需要夏孢子材料的困难,使得DNA提取时间缩短在30min内,夏孢子用量10㎎以下或单个夏孢子堆,产率达882μg/g。对纯化夏孢子DNA提取原液稀释倍数进行了研究,该结果有利于DNA的保存和有效利用。在ITS扩增中,稀释倍数可达6倍,随机引物扩增中优化稀释倍数为16~32倍。采用Gardes & Bruns (1993)设计的担子菌特异性ITS引物,可以从感病(显症和不显症)杨树叶片DNA提取液中特异性的检测到松杨栅锈菌ITS片段,该结果对指导杨(柳)树叶锈病检疫和系统学研究,具有重要意义。
3.对该菌全国范围内5个小种的11个菌样遗传分化做了初步研究。初步确立了我国该菌西部和北方两大地理菌群的观点。RAPD解析可将高山森林生态型和平原生态型分开,而RAMs解析则不能。遗传多样性指数T检验结果表明,C2小种遗传多样性指数与C4、C3、C1小种的差异显著,其余小种间不显著。我国平原生态型菌群平均遗传多样性指数高于高山森林生态型菌群的平均遗传多样性指数,推测杨树引种或锈菌的其他行为(如“异核体现象”)可能在该菌变异中起着重要作用。小种致病性分化与遗传分化不一定相关。来自C4小种的Qh、Gl菌样常被聚在同一个遗传型中,而来自C4、C5小种的Cjq、Bq菌样也常被聚在同一个遗传型中。致病性表达不仅决定于遗传背景,也与环境条件密切相关。核糖体ITS区段研究表明:该菌ITS序列(ITS1,5.8SrDNA,ITS2)全长581bp,且高度保守,不适合该菌种内群体分化研究。来自德国、法国、加拿大、英国、中国的菌系,ITS序列同源性高达99%以上。ITS序列变异主要发生在个别碱基的颠换上,与已报道的其他病原菌ITS变异方式不同。ITS的RFLP解析也得到相同的结果,根据ITS序列和RFLP结果,首次得到了该菌ITS内切酶限制性图谱。
Melampsora larici-populina Kleb. is the important pathogen occurred in the Sect.Tacamachacae, Sect. Aigerios and their hybrids poplars in the worldwide. There has distinguished pathogenic division found within species , and the genetic background is complex. 5 races were reported in European and North America. To understand the occurrence condition and the genetic background of the pathogen in China, this dissertation focused on studying the rust fungus by pathogenesis measure, ITS sequence analysis, RFLP, RAPD, RAMs, SCAR markers and nucleates DAPI dying, and achieved results bellowing:
1. Pathogenicity division study is done by inoculation M. larici-populina with poplar saplings, and the identification system of race division is founded, which includes host poplar ranges, propagation host (Poplus purdomii) and technology standards. 5 races are reported in China firstly.
2. Based on compare and analysis, a rapid and minimum DNA extraction technology is established, and the optimization study of PCR operation is done. 6-fold diluted extraction liquid DNA can be used for ITS-PCR operation, and 16×-32×is the optimism dilution multiples for random primer PCR, the parameters is benefit for DNA reservation and high usage efficiency. By this DNA extraction method, the extracting operating time can be reduced into 30 min with fewer than 10㎎ urediospores,even with a single mono-uredinium and the DNA production rate can be about 882μg/g. With the Basidiomycetes specified primers designed by Gardes and Bruns in 1993, ITS of M. larici-populina happened on symptom or no symptom poplar leaves can be amplified specifically, which is important for future quarantine and polygenetic study of Melampsora spp fungi occurred in Salicaceae trees.
3. Genetic division based on 11 isolates of 5 Races, which are collected in throughout China, is studied preliminarily by ITS sequence, ITS-RFLP,RAPD and RAMs molecular technologies. The results indicate the genetic division is correlated to the originate locus of isolations. The tested isolations can be separated into Northern geographic population and Western geographic population, and the latter can be separated into Hi-mountain forest ecologic type and western plain ecologic type. Genetic division must not correspondent with the race's pathogenic.The same genentic background isolates may belong to the different races. T test shows the genetic index of Race C2 is significantly different to the Race C4,C3,C1,no
1.通过对幼苗温室接种,对来自全国各地的松杨栅锈菌致病性分化进行了系统研究,建立了我国该菌小种鉴别的技术体系,确定了我国该菌小种鉴别的寄主杨树谱,以及扩繁寄主杨树(太白杨),首次提出我国松杨栅锈菌5个生理小种的观点。
2.在对比分析的基础上,创建了锈菌夏孢子微量快速提取技术,并对该技术及其在PCR操作中的应用进行了优化研究。该方法克服了常规分子操作中大量需要夏孢子材料的困难,使得DNA提取时间缩短在30min内,夏孢子用量10㎎以下或单个夏孢子堆,产率达882μg/g。对纯化夏孢子DNA提取原液稀释倍数进行了研究,该结果有利于DNA的保存和有效利用。在ITS扩增中,稀释倍数可达6倍,随机引物扩增中优化稀释倍数为16~32倍。采用Gardes & Bruns (1993)设计的担子菌特异性ITS引物,可以从感病(显症和不显症)杨树叶片DNA提取液中特异性的检测到松杨栅锈菌ITS片段,该结果对指导杨(柳)树叶锈病检疫和系统学研究,具有重要意义。
3.对该菌全国范围内5个小种的11个菌样遗传分化做了初步研究。初步确立了我国该菌西部和北方两大地理菌群的观点。RAPD解析可将高山森林生态型和平原生态型分开,而RAMs解析则不能。遗传多样性指数T检验结果表明,C2小种遗传多样性指数与C4、C3、C1小种的差异显著,其余小种间不显著。我国平原生态型菌群平均遗传多样性指数高于高山森林生态型菌群的平均遗传多样性指数,推测杨树引种或锈菌的其他行为(如“异核体现象”)可能在该菌变异中起着重要作用。小种致病性分化与遗传分化不一定相关。来自C4小种的Qh、Gl菌样常被聚在同一个遗传型中,而来自C4、C5小种的Cjq、Bq菌样也常被聚在同一个遗传型中。致病性表达不仅决定于遗传背景,也与环境条件密切相关。核糖体ITS区段研究表明:该菌ITS序列(ITS1,5.8SrDNA,ITS2)全长581bp,且高度保守,不适合该菌种内群体分化研究。来自德国、法国、加拿大、英国、中国的菌系,ITS序列同源性高达99%以上。ITS序列变异主要发生在个别碱基的颠换上,与已报道的其他病原菌ITS变异方式不同。ITS的RFLP解析也得到相同的结果,根据ITS序列和RFLP结果,首次得到了该菌ITS内切酶限制性图谱。
Melampsora larici-populina Kleb. is the important pathogen occurred in the Sect.Tacamachacae, Sect. Aigerios and their hybrids poplars in the worldwide. There has distinguished pathogenic division found within species , and the genetic background is complex. 5 races were reported in European and North America. To understand the occurrence condition and the genetic background of the pathogen in China, this dissertation focused on studying the rust fungus by pathogenesis measure, ITS sequence analysis, RFLP, RAPD, RAMs, SCAR markers and nucleates DAPI dying, and achieved results bellowing:
1. Pathogenicity division study is done by inoculation M. larici-populina with poplar saplings, and the identification system of race division is founded, which includes host poplar ranges, propagation host (Poplus purdomii) and technology standards. 5 races are reported in China firstly.
2. Based on compare and analysis, a rapid and minimum DNA extraction technology is established, and the optimization study of PCR operation is done. 6-fold diluted extraction liquid DNA can be used for ITS-PCR operation, and 16×-32×is the optimism dilution multiples for random primer PCR, the parameters is benefit for DNA reservation and high usage efficiency. By this DNA extraction method, the extracting operating time can be reduced into 30 min with fewer than 10㎎ urediospores,even with a single mono-uredinium and the DNA production rate can be about 882μg/g. With the Basidiomycetes specified primers designed by Gardes and Bruns in 1993, ITS of M. larici-populina happened on symptom or no symptom poplar leaves can be amplified specifically, which is important for future quarantine and polygenetic study of Melampsora spp fungi occurred in Salicaceae trees.
3. Genetic division based on 11 isolates of 5 Races, which are collected in throughout China, is studied preliminarily by ITS sequence, ITS-RFLP,RAPD and RAMs molecular technologies. The results indicate the genetic division is correlated to the originate locus of isolations. The tested isolations can be separated into Northern geographic population and Western geographic population, and the latter can be separated into Hi-mountain forest ecologic type and western plain ecologic type. Genetic division must not correspondent with the race's pathogenic.The same genentic background isolates may belong to the different races. T test shows the genetic index of Race C2 is significantly different to the Race C4,C3,C1,no
引文
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