地黄种质遗传关系及根际土壤微生物多样性研究
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摘要
地黄(Rehmannia glutinosa)为玄参科(Scrophulariaceae)多年生草本植物,其膨大的块根可以加工成鲜地黄、生地(黄)和熟地(黄)三种方式入药,是一种大宗常用传统滋补中药。地黄药材全部来源于人工栽培,在长期的人工栽培历史中,形成了众多的栽培品种或者变种。目前文献报道的地黄栽培品种就达50多个,然而只有少数品种的形态和农艺性状有比较详细的描述,多数是品种混杂,遗传关系不清楚。本研究根据地黄的来源、栽培品种、田间变异以及部分种子繁殖材料,建立了地黄种质资源圃。对所有材料从外观性状(叶型、叶色、叶缘、根重、根皮颜色、花冠和花药颜色,及植株形态等)方面作了研究分析;同时,应用RAPD、AFLP及rDNA-ITS等分子标记分析方法,对收集到的地黄种质从遗传物质的变异方面进行了深入研究。结果表明,89份地黄种质样本经过分子标记的遗传相似性聚类、外观形状分析和ITS序列比较,剔除重复后鉴定出二十一类共48份种质。其中种质2、20、39、50、53、71、82、86等既具有特异的基因型,又具有明显的表型性状,是挖掘地黄种质优异基因需要特别关注的新材料,也是根据遗传关系选择杂交育种工作需要考虑的“远缘”亲本。8对AFLP引物扩增出1019条带,10条RAPD引物扩增出了118条带,应用UPGMA和SHAN方法对这两组数据建立了地黄的遗传距离树状图。对根据AFLP-RAPD建立的遗传关系图上差异较大的种质进行了rDNA-ITS序列比较分析,参考GENBANK数据库地黄ITS序列和测定序列,应用Mega3.1软件建立了部分地黄种质进化树。地黄ITS1+5.8S+ITS2序列长度为611～613bp,其中有22个位点存在地黄属植物进化相关的碱基变化信息,C-T颠换是地黄ITS序列变异的主要碱基变化。
另外,生产实践表明,地黄种植生产中存在严重的连作障碍问题。我们推测,地黄连作问题可能和根系对土壤的微生物生态变迁有关。因此,本研究测定了地黄根际土壤和非根际土壤的土壤微生物生物碳(SMBC)、基础呼吸、代谢商和土壤pH值等基本理化数据,同时,采用基于Biolog唯一碳源利用的群落水平生理刨面(CLPP)和磷脂脂肪酸(PLFA)法,研究了地黄根际土壤微生物的群落功能和结构变化。地黄种植地田间调查表明,连作问题不仅在2年生田间发生,5年轮作田也存在。地黄根际土壤SMBC无明显变化,但是基础呼吸和代谢商显著高于非根际土壤;相反,基于Biolog碳源培养分析的地黄根际土壤微生物的AWCD和Shannon指数均有显著的下降,同时,AWCD和SMBC具有随季节变化的相似特征,在地黄生长发育的旺季高,而在收获季节低。应用主成分分析法对不同处理土壤样品在Biolog生态盘上的31种不同的碳源的代谢生理进行分析表明,主成分一(PC1)和主成分二(PC2)能够有效的区分不同处理的土壤,可以反映地黄根际土壤微生物群落的功能变化。主成分共解释了81.47%的变异,其中PC1为67.42%,PC2为14.05%。在所有33种PLFAs中,有18种PLFAs占88-90%的丰度,是地黄根际生态系统的主要成分。所有测试土壤中,真菌PLFA(18:2ω6,9)具有相对较高的丰度,并且发现比较一致的结果,地黄根际土壤真菌比对照土壤高1.36～2.62倍。总之,地黄根际土壤微生物生态研究表明,地黄种植改变了土壤微生物的代谢活动,降低了能量代谢的效率,同时,地黄根际土壤微生物的结构也发生明显的变化,这些变化会引起植物的不良生长,可能是引起地黄连作问题的主要原因。
Rehmannia glutinosa(named 'DiHuang' in Chinese),one of the most famous medicinal plants,is a perennial herbaceous plant of the Scrophulariacea family.Owing to its nourishing functions to human body, its tuberous root is frequently used for Traditional Chinese Medicines in three different forms,i.e.fresh,dry and boil processed.Many cultivars or varieties were selected and cultivated in China in a long history of R. glutinosa cultivation practices.According to the report,there are more than 50 cultivars of R.glutinosa in China,but only few of them are described in detail in agronomical and morphological characters.Most of them are promiscuous and with unclear genetic relationship.In this work,cultivars,varieties and some sexual raise up seeds of R.glutinosa were collected and planted in the garden of Institute of Medicinal Plant Development.Field investigation was carried out in the morphological diversity including the shape,color, edges of leaves,color of anther,cornal and root,and the yield of medicinal part of root.Two molecular markers,random amplified polymorphism DNA(RAPD ) and amplified fragment length polymorphism (AFLP ),were employed for determine the genetic relationship of R.glutinosa,and the ribosome DNA internal transcribed spacer(ITS ) sequences was also used for analyzing sequence variations and phylogenetic history.The results showed that 48 accessions belong to 21 clusters were identified from 89 collections of R.glutinosa by UPGMA cluster analysis using RAPD in combination with AFLP markers. Among the 48 accessions,the germplasms of 2,20,39,50,53,71,82 and 86 have differential genotype as well as obviously phenotype,and these materials might be pay more attention while mining specific gene or crossbreeding.According to the distances in the dendrogram of R.glutinosa established from 1137 polymorphic bands in RAPD and AFLP markers combination,rDNA ITS sequences in several accessions were amplified and sequenced.The analysis of ITS sequences indicated that ITS1-5.8S-ITS2 of R.glutinosa were informative in its.611-613bp long sequence and have 22 variable sites including mostly occurred C-T transversion.Two phylogenetic trees were generated by Neighbour-jioning and UPGMA methods based on ITS sequences and were compared.
In addition,R.glutinosa is an important medicinal plant,but there is a serious problem with its continuous cropping on the same land.We hypothesized some relationships between this problem and the disturbed soil ecosystem.In this work,two community-based microbiological measurements,community level physiological profiling(CLPP ) using Biolog sole C source utilization tests and phospholipid ester-linked fatty acid(PLFA) profiles,were used to evaluate soil microbial community function and composition of different R.glutinosa cropping soils.Field investigation showed that the problems with continuous cropping occurred not only in two-year continuous fields,but also in five-year rotation fields. Soil basal respiration and metabolic quotient were significantly higher in R.glutinosa cropping soils than in the non-cropping controls.On the contrary,the Shannon index and the average well color development (AWCD) from the Biolog data set were lower in R.glutinosa cropping soils,and we also found a seasonal change in AWCD and soil microbial biomass carbon(SMBC ),higher in cropping season but lower in harvest season.CLPP-based principal component analyses(PCA ) explained 87.47%variation of soil microbial communities,PC1 67.42%and PC2 14.05%,and these showed distinct groupings of soil microbial communities in R.glutinosa rhizosphere in PCA scatter plot.Eighteen PLFAs account for 88-90%aboundant in 33 PLFAs used in this work were main component in R.glutinosa rhizosphere soil.PLFA-based PCA also showed distinct composition of soil microbial communities in R.glutinosa rhizosphere,and 11 PLFAs representing different microbes were identified from the principal component scores of PLFAs.Among these, an abundance of PLFA 18:2ω6,9,which is a biomarker of soil fungi,was significantly higher (1.36-2.62times) in R.glutinosa cropping soils than control soils.These results suggest an alteration of soil microbial community following R.glutinosa cropping,and this might be an important reason for the constraints associated with continuous cropping.
另外,生产实践表明,地黄种植生产中存在严重的连作障碍问题。我们推测,地黄连作问题可能和根系对土壤的微生物生态变迁有关。因此,本研究测定了地黄根际土壤和非根际土壤的土壤微生物生物碳(SMBC)、基础呼吸、代谢商和土壤pH值等基本理化数据,同时,采用基于Biolog唯一碳源利用的群落水平生理刨面(CLPP)和磷脂脂肪酸(PLFA)法,研究了地黄根际土壤微生物的群落功能和结构变化。地黄种植地田间调查表明,连作问题不仅在2年生田间发生,5年轮作田也存在。地黄根际土壤SMBC无明显变化,但是基础呼吸和代谢商显著高于非根际土壤;相反,基于Biolog碳源培养分析的地黄根际土壤微生物的AWCD和Shannon指数均有显著的下降,同时,AWCD和SMBC具有随季节变化的相似特征,在地黄生长发育的旺季高,而在收获季节低。应用主成分分析法对不同处理土壤样品在Biolog生态盘上的31种不同的碳源的代谢生理进行分析表明,主成分一(PC1)和主成分二(PC2)能够有效的区分不同处理的土壤,可以反映地黄根际土壤微生物群落的功能变化。主成分共解释了81.47%的变异,其中PC1为67.42%,PC2为14.05%。在所有33种PLFAs中,有18种PLFAs占88-90%的丰度,是地黄根际生态系统的主要成分。所有测试土壤中,真菌PLFA(18:2ω6,9)具有相对较高的丰度,并且发现比较一致的结果,地黄根际土壤真菌比对照土壤高1.36～2.62倍。总之,地黄根际土壤微生物生态研究表明,地黄种植改变了土壤微生物的代谢活动,降低了能量代谢的效率,同时,地黄根际土壤微生物的结构也发生明显的变化,这些变化会引起植物的不良生长,可能是引起地黄连作问题的主要原因。
Rehmannia glutinosa(named 'DiHuang' in Chinese),one of the most famous medicinal plants,is a perennial herbaceous plant of the Scrophulariacea family.Owing to its nourishing functions to human body, its tuberous root is frequently used for Traditional Chinese Medicines in three different forms,i.e.fresh,dry and boil processed.Many cultivars or varieties were selected and cultivated in China in a long history of R. glutinosa cultivation practices.According to the report,there are more than 50 cultivars of R.glutinosa in China,but only few of them are described in detail in agronomical and morphological characters.Most of them are promiscuous and with unclear genetic relationship.In this work,cultivars,varieties and some sexual raise up seeds of R.glutinosa were collected and planted in the garden of Institute of Medicinal Plant Development.Field investigation was carried out in the morphological diversity including the shape,color, edges of leaves,color of anther,cornal and root,and the yield of medicinal part of root.Two molecular markers,random amplified polymorphism DNA(RAPD ) and amplified fragment length polymorphism (AFLP ),were employed for determine the genetic relationship of R.glutinosa,and the ribosome DNA internal transcribed spacer(ITS ) sequences was also used for analyzing sequence variations and phylogenetic history.The results showed that 48 accessions belong to 21 clusters were identified from 89 collections of R.glutinosa by UPGMA cluster analysis using RAPD in combination with AFLP markers. Among the 48 accessions,the germplasms of 2,20,39,50,53,71,82 and 86 have differential genotype as well as obviously phenotype,and these materials might be pay more attention while mining specific gene or crossbreeding.According to the distances in the dendrogram of R.glutinosa established from 1137 polymorphic bands in RAPD and AFLP markers combination,rDNA ITS sequences in several accessions were amplified and sequenced.The analysis of ITS sequences indicated that ITS1-5.8S-ITS2 of R.glutinosa were informative in its.611-613bp long sequence and have 22 variable sites including mostly occurred C-T transversion.Two phylogenetic trees were generated by Neighbour-jioning and UPGMA methods based on ITS sequences and were compared.
In addition,R.glutinosa is an important medicinal plant,but there is a serious problem with its continuous cropping on the same land.We hypothesized some relationships between this problem and the disturbed soil ecosystem.In this work,two community-based microbiological measurements,community level physiological profiling(CLPP ) using Biolog sole C source utilization tests and phospholipid ester-linked fatty acid(PLFA) profiles,were used to evaluate soil microbial community function and composition of different R.glutinosa cropping soils.Field investigation showed that the problems with continuous cropping occurred not only in two-year continuous fields,but also in five-year rotation fields. Soil basal respiration and metabolic quotient were significantly higher in R.glutinosa cropping soils than in the non-cropping controls.On the contrary,the Shannon index and the average well color development (AWCD) from the Biolog data set were lower in R.glutinosa cropping soils,and we also found a seasonal change in AWCD and soil microbial biomass carbon(SMBC ),higher in cropping season but lower in harvest season.CLPP-based principal component analyses(PCA ) explained 87.47%variation of soil microbial communities,PC1 67.42%and PC2 14.05%,and these showed distinct groupings of soil microbial communities in R.glutinosa rhizosphere in PCA scatter plot.Eighteen PLFAs account for 88-90%aboundant in 33 PLFAs used in this work were main component in R.glutinosa rhizosphere soil.PLFA-based PCA also showed distinct composition of soil microbial communities in R.glutinosa rhizosphere,and 11 PLFAs representing different microbes were identified from the principal component scores of PLFAs.Among these, an abundance of PLFA 18:2ω6,9,which is a biomarker of soil fungi,was significantly higher (1.36-2.62times) in R.glutinosa cropping soils than control soils.These results suggest an alteration of soil microbial community following R.glutinosa cropping,and this might be an important reason for the constraints associated with continuous cropping.
引文
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