厚朴良种选择及其RAPD种源鉴别研究
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摘要
厚朴主要来源厚朴Magnolia officinalis及其变种凹叶厚朴M.officinalis var biloba。历史上按产地分为川朴(产于湖北和四川,来源于厚朴)和温朴(产于浙江和福建,来源于凹叶厚朴)[1]。真实性是品种质量评价的重要标准之一,其鉴定技术也是解决新品种权益保护、种子(苗)经济纠纷问题的技术基础。选择优良品种,建立厚朴优良性状综合评价体系,收集保存优良种质资源,完善丰产栽培技术是当前大力发展中药材厚朴前提工作。
本文对福建省不同种源地厚朴调查及形态指标研究的基础上,筛选出综合评价高的优良种源区及有效成分含量高、性状优良的优树,并对全国不同种源区厚朴进行RAPD分子标记及遗传多样性等进行分析研究。主要研究结果如下:
1、厚朴酚类提取按正交设计L9(34)进行试验研究,表明乙醇浓度是厚朴酚类提取测定的关键因素,其次是超声处理时间。其提取最优方法为:乙醇浓度为90%,浸泡时间为50min,浸泡温度为60℃,超声时间为20min。
2、调查显示厚朴在福建省基本上呈零星分布状态,主要分布于福建省西部和北部。对24个县市的147株初选厚朴优树进行聚类分析,不同地理种源的厚朴酚含量差异较大,147株厚朴初选优树中酚含量最低为0.0907 mg.g-1,最高5.4759 mg.g-1。以采样地的基本气候因子对厚朴酚类含量影响分析表明,年均温和无霜期对厚朴酚类物质含量影响最大。
3、季节的变化对植物生长次生代谢物质的含量具有影响,枝皮中厚朴酚与和厚朴酚含量在1月份达到最高,后降低再升高,到9月份又下降的趋势。在厚朴叶中和厚朴酚含量始终高于厚朴酚含量,且嫩叶期趋势更明显;总的酚含量由嫩叶到老叶呈增加趋势,直到落叶前含量达到最高。
4、不同器官部位厚朴酚类物质含量存在一定的差异,但同一器官中厚朴酚含量与和厚朴酚含量相差不大。不同器官中酚类物质含量大小顺序依次是:根皮>干皮>枝皮>嫩芽>种子>果实>枝芯>叶。
5、厚朴随着树龄的增长、厚朴枝皮和干皮中厚朴酚与和厚朴酚含量表现为升高-下降-升高的趋势。厚朴在中幼林时随着树龄增大,酚含量增多,大致在25年左右达到最大,以后呈下降趋势,百年老树有效成含量又有增高的现象。厚朴叶中酚类含量随树龄增大呈下降趋势,幼龄树叶中酚类含量较高。
6、同一群体内,胸径大小相近的厚朴植株酚类含量个体间存在着较大的变异,个体间各部位酚类含量差异显著。厚朴优良种源选择时,树高、冠幅、树皮厚、枝条数之间都呈极显著或显著的正相关;通直度、圆满度、健康度相关性不大;枝皮中酚含量与各树体性状之间都呈极显著或显著的正相关。
7、按特征根大于或等于1原则提取四个主成分,对24个种源各主成分与综合评分,24个种源中综合排名较前的为柘荣、尤溪、连城、福州。其次是武平、永泰、寿宁、清流、沙县、永安、浦城、农大、屏南、明溪、宁化、漳平,最少是泰宁、南平、顺昌、建阳、大田、建瓯、福安、光泽。
8、在优树选择时各性状间相关性分析,胸径与冠幅、树高、酚含量及树高与冠幅、枝下高、枝条数,冠幅与枝下高之间有极显著正相关,通直度与树皮厚显著的关系,其它指标相关性不大。
9、按主成分分析法中累计贡献率大于或等于85%原则提取了六个主成分,对54株优树各主成分与综合评分,优树中得分较高为11号、6号、131号、146号、16号家系,其次为123号、33号、49号、114号、2号家系综合排名较前。聚类分析结果分为2类种源,第一类是2、14、99、107、113、8、141、114、37、75、44、117、135、109、16、33、49、123、6、11、131、146,第二类13、102、111、43、116、145、50、70、93、136、76、90、132、147、38、115、119、45、101、20、110、142、125、21、46、120、138、129、97、94、29、112。
10、根据6个引物对厚朴资源扩增出的96条可重现RAPD谱带获得的数据矩阵,构建了89份厚朴资源的DNA指纹图谱。结果表明,S60与S69、S60与S91、S60与S1412、S60与S330、S60与S1422、S69与S91、S69与S1412、S69与S1422、S91与S1412、S1412与S1422、S1412与S330这11个组合中的每个组合形成的扩增数据矩阵都可以成为一张指纹图谱,比较容易地把89份资源相互区分鉴别开来。
11、全国89个样本的来源分析,第一类为福建建瓯,遗传结构具有特殊性。第二类包括四川江邮、湖南道县,福建明溪种源和安徽歙县,福建建阳部分种源个体。第三类中的第一亚类主要来源有湖北恩施、浙江景宁、广西资源、广西全州、江西庐山、安徽毫州、安徽合肥、四川雅安、陕西长安和安徽歙县、四川江邮、湖南永州部分个体。第二亚类为湖南张家界、重庆江津区、福建顺昌、福建浦城、福建南平、福建柘荣、福建永泰、福州森林公园、福建连城、福建光泽、福建泰宁、福建寿宁和湖南道县、福建建阳的部分个体。
Chinese medicinal materials Magnolia officinalis mainly originated from Magnolia officinalis and M.officinalis var biloba. In history, according to origination of Magnolia officinalis (produced in Hubei and Sichuan, and M.officinalis var biloba (produced in Zhejiang and Fujian).Authenticity is one of the important criterions for quality evaluation of varieties, Identification techniques is also the base of solving the problem about rights and interests protection of new varieties of seeds (seedlings) as well as its economic disputes. Selecting fine varieties, establishing evaluation system with good and comprehensive traits for Magnolia officinalis, collecting elite germplasm resources, improving high-yield cultivation technique is the prerequisite to develop chinese medicinal materials.
In this paper plus tree with high comprehensive evaluation from high-quality provenance area and high effective ingredient plus good traits were selected on the base of studying investigation and morphological index from different provenances of Fujian Province Magnolia officinalis. In addition different provenance of the country Magnolia officinalis for RAPD molecular markers and genetic diversity were analyzed.The major results were as follows:
1. Magnolia phenol extraction was experimentally studied through orthogonal design L9(34). The result showed that ethanol concentration is a key factor of extraction, followed by ultrasonic time. The optimal method for extraction was: ethanol concentration 90%, water-curing treatment time 50min, water-curing treatment temperature 60℃, ultrasonic time for 20min.
2. The investigation showed that the distribution of Magnolia officinalis was generally in dispersed state, mainly in the west and north of Fujian Province. 147 plus trees primarily selected in Magnolia officinalis from 24 town and citieswere depleted analysis. The result indicated that the content of Magnolia phenol varied greatly with different provenances, with the minimum at 0.0907 mg.g-1 but the maximum at 5.4759 mg.g-1.The impact of basic climatic factors existed in sampling land on the content of Magnolia phenol was analyzed. The result showed that annual temperature and frostless season were the greatest impact factors.
3. Seasonal changes have an impact on the content of secondary metabolites during the period of plant growth. The content of phenol in shoot cortex of Magnolia was highest in January. Afterward with the passage of time, the content would decrease at first then increased but in downward trend on September. In the Magnolia leaves honokiol content is always higher than that magnolol content,the trend of which is more obvious in tender leaves.Finally the total phenol content distribution from tender leaves to old leaves was in Increasing trend until it reached the maximum before the deciduous period.
4.There are some differences of Magnolia content among the different parts of Magnolia but the differences would be minimal among the same organs. Polyphenol content in different organs ranked in the order of root bark ,dry hide ,branch bark, tender bud , seed, fruit, stem wood and needle.
5. With the increase of Magnolia trees age, magnolol and honokiol content trend in both branch bark and stem bark were shown as increasing - decreasing– increasing. while in both middle-aged and young growth plantations, the phenol content would increased with their ages increasing. The value reached the maximum approximately at their ages of 25 but afterward it would be shown as downtrend. However, in hundred years of aged trees, the active component content would show increasing trend in the last stage. The phenol content of Magnolia leaves decreased with age increasing, which reached higher level in their young growth tree leaves.
6. The variation of phenol contents between Magnolia individuals in the same population with the diameter of the similar size were quite large and the difference of phenol contents in various parts between Magnolia individuals were significantly.When excellent magnolia provenance was selected, there exited very significant or significant positive correlation with tree height, crown, bark thick, and branches rather than straight degrees, round degree and health degree. Meanwhile, the positive correlation between phenol content of branch bark and traits of trees was very significant or significant.
7. Four main components were extracted, according to the principle in which characteristic root is equal to or greater than 1. Then the principal components were comprehensive evaluated from 24 different provenance. The evaluation result by ranking order was as follow: top front group was Zherong,Youxi, Licheng, Fuzhou, which is followed by the Wuping,Yongtai, Shouning,Qingliu.Shaxian,Yongan,Pucheng, campus of Fujian Agricultural and Forestry University,Pingnan, Mingxi,Ninghua, Zhangping, and at least was Taining,Nanping,Shunchang,Jianyang,Datian, Jianou, Fuan, Guangze.
8. Correlation between various traits in plus tree selection was analyzed. The results were shown as follow: there exited very significant positive correlation between DBH and crown width, tree height and phenol content, very significant positive correlation between tree height and crown width, clear bole height, and branches, very significant positive correlation between crown width and clear bole height, significant positive correlation between stem straightness and bark thickness, rather than other indexes.
9. Six principal components extracted by principal component analysis of cumulative contribution rate of greater than or equal to 85% were comprehensively scored from 54 plus tree. The result was shown as follow: the higher scores of plus tree family was the number 11,6,131,146,16,followed by 123,33,49,114, 2. Cluster analysis results were divided into two categories Provenance, the first category was the number 2,14,99,107,113,8,141,114,37,75,44,117,135,109,16,33,49,123, 6,11,131,146, the second category was the number 13,102,111,43,116,145,50,70,93,136,76,90,132,147,38,115,119,45,101,20,110,142,125,21,46,120, 138,129,97,94,29,112.
10. DNA fingerprinting of 89 copies of Magnolia resources was constructed based on the data matrix obtained out of 96 reproducible RAPD bands of Magnolia resources amplified by six primers. The results indicated that 11 combinations were respectively S60 and S69, S60 and S91, S60 and S1412, S60 and S330, S60 and S1422, S69 and S91, S69 and S1412, S69 and S1422, S91 and S1412, S1412 and S1422, S1412 and S330.In each combination , data matrix formed by amplification can be relatively easy for multiple authentication to distinguish from the 89 resources.
11.Source of 89 samples from the whole nation was analyzed. The analysis result can be concluded to three categories. The first category was Jiangyou,Fujian in which genetic structure possessed particularity while the second category included Jiangyou Sichuan,Daoxian Hunan,Mingxi Fujian, Shexian Anhui and part of Provenance individual of Jianyang Fujian. In addition, the third category can be divided into two sub-categories.In the first sub-category, main source region included Enshi Hubei, Jingning Zhejiang, Ziyuan Guangxi, Quanzhou Guangxi, Lushan Jiangxi, Haozhou Anhui, Hefei Anhui, Yaan Sichuan, Changan Shaanxi, Shexian Anhui,Jiangyou Sichuan and part of the Provenance individual of Yongzhou Hunan and the the second sub-category included Zhangjiajie Hunan, Jiangjin district of Chongqing, Shunchang Fujian, Pucheng Fujian, Nanping Fujian, Zherong Fujian, Yongtai Fujian, Forest Park of Fuzhou, Liancheng Fujian, Guangze Fujian,Taining Fujian, Shouning Fujian,Daoxian Hunan and part of the Provenance individualof Fujian Jianyang.
本文对福建省不同种源地厚朴调查及形态指标研究的基础上,筛选出综合评价高的优良种源区及有效成分含量高、性状优良的优树,并对全国不同种源区厚朴进行RAPD分子标记及遗传多样性等进行分析研究。主要研究结果如下:
1、厚朴酚类提取按正交设计L9(34)进行试验研究,表明乙醇浓度是厚朴酚类提取测定的关键因素,其次是超声处理时间。其提取最优方法为:乙醇浓度为90%,浸泡时间为50min,浸泡温度为60℃,超声时间为20min。
2、调查显示厚朴在福建省基本上呈零星分布状态,主要分布于福建省西部和北部。对24个县市的147株初选厚朴优树进行聚类分析,不同地理种源的厚朴酚含量差异较大,147株厚朴初选优树中酚含量最低为0.0907 mg.g-1,最高5.4759 mg.g-1。以采样地的基本气候因子对厚朴酚类含量影响分析表明,年均温和无霜期对厚朴酚类物质含量影响最大。
3、季节的变化对植物生长次生代谢物质的含量具有影响,枝皮中厚朴酚与和厚朴酚含量在1月份达到最高,后降低再升高,到9月份又下降的趋势。在厚朴叶中和厚朴酚含量始终高于厚朴酚含量,且嫩叶期趋势更明显;总的酚含量由嫩叶到老叶呈增加趋势,直到落叶前含量达到最高。
4、不同器官部位厚朴酚类物质含量存在一定的差异,但同一器官中厚朴酚含量与和厚朴酚含量相差不大。不同器官中酚类物质含量大小顺序依次是:根皮>干皮>枝皮>嫩芽>种子>果实>枝芯>叶。
5、厚朴随着树龄的增长、厚朴枝皮和干皮中厚朴酚与和厚朴酚含量表现为升高-下降-升高的趋势。厚朴在中幼林时随着树龄增大,酚含量增多,大致在25年左右达到最大,以后呈下降趋势,百年老树有效成含量又有增高的现象。厚朴叶中酚类含量随树龄增大呈下降趋势,幼龄树叶中酚类含量较高。
6、同一群体内,胸径大小相近的厚朴植株酚类含量个体间存在着较大的变异,个体间各部位酚类含量差异显著。厚朴优良种源选择时,树高、冠幅、树皮厚、枝条数之间都呈极显著或显著的正相关;通直度、圆满度、健康度相关性不大;枝皮中酚含量与各树体性状之间都呈极显著或显著的正相关。
7、按特征根大于或等于1原则提取四个主成分,对24个种源各主成分与综合评分,24个种源中综合排名较前的为柘荣、尤溪、连城、福州。其次是武平、永泰、寿宁、清流、沙县、永安、浦城、农大、屏南、明溪、宁化、漳平,最少是泰宁、南平、顺昌、建阳、大田、建瓯、福安、光泽。
8、在优树选择时各性状间相关性分析,胸径与冠幅、树高、酚含量及树高与冠幅、枝下高、枝条数,冠幅与枝下高之间有极显著正相关,通直度与树皮厚显著的关系,其它指标相关性不大。
9、按主成分分析法中累计贡献率大于或等于85%原则提取了六个主成分,对54株优树各主成分与综合评分,优树中得分较高为11号、6号、131号、146号、16号家系,其次为123号、33号、49号、114号、2号家系综合排名较前。聚类分析结果分为2类种源,第一类是2、14、99、107、113、8、141、114、37、75、44、117、135、109、16、33、49、123、6、11、131、146,第二类13、102、111、43、116、145、50、70、93、136、76、90、132、147、38、115、119、45、101、20、110、142、125、21、46、120、138、129、97、94、29、112。
10、根据6个引物对厚朴资源扩增出的96条可重现RAPD谱带获得的数据矩阵,构建了89份厚朴资源的DNA指纹图谱。结果表明,S60与S69、S60与S91、S60与S1412、S60与S330、S60与S1422、S69与S91、S69与S1412、S69与S1422、S91与S1412、S1412与S1422、S1412与S330这11个组合中的每个组合形成的扩增数据矩阵都可以成为一张指纹图谱,比较容易地把89份资源相互区分鉴别开来。
11、全国89个样本的来源分析,第一类为福建建瓯,遗传结构具有特殊性。第二类包括四川江邮、湖南道县,福建明溪种源和安徽歙县,福建建阳部分种源个体。第三类中的第一亚类主要来源有湖北恩施、浙江景宁、广西资源、广西全州、江西庐山、安徽毫州、安徽合肥、四川雅安、陕西长安和安徽歙县、四川江邮、湖南永州部分个体。第二亚类为湖南张家界、重庆江津区、福建顺昌、福建浦城、福建南平、福建柘荣、福建永泰、福州森林公园、福建连城、福建光泽、福建泰宁、福建寿宁和湖南道县、福建建阳的部分个体。
Chinese medicinal materials Magnolia officinalis mainly originated from Magnolia officinalis and M.officinalis var biloba. In history, according to origination of Magnolia officinalis (produced in Hubei and Sichuan, and M.officinalis var biloba (produced in Zhejiang and Fujian).Authenticity is one of the important criterions for quality evaluation of varieties, Identification techniques is also the base of solving the problem about rights and interests protection of new varieties of seeds (seedlings) as well as its economic disputes. Selecting fine varieties, establishing evaluation system with good and comprehensive traits for Magnolia officinalis, collecting elite germplasm resources, improving high-yield cultivation technique is the prerequisite to develop chinese medicinal materials.
In this paper plus tree with high comprehensive evaluation from high-quality provenance area and high effective ingredient plus good traits were selected on the base of studying investigation and morphological index from different provenances of Fujian Province Magnolia officinalis. In addition different provenance of the country Magnolia officinalis for RAPD molecular markers and genetic diversity were analyzed.The major results were as follows:
1. Magnolia phenol extraction was experimentally studied through orthogonal design L9(34). The result showed that ethanol concentration is a key factor of extraction, followed by ultrasonic time. The optimal method for extraction was: ethanol concentration 90%, water-curing treatment time 50min, water-curing treatment temperature 60℃, ultrasonic time for 20min.
2. The investigation showed that the distribution of Magnolia officinalis was generally in dispersed state, mainly in the west and north of Fujian Province. 147 plus trees primarily selected in Magnolia officinalis from 24 town and citieswere depleted analysis. The result indicated that the content of Magnolia phenol varied greatly with different provenances, with the minimum at 0.0907 mg.g-1 but the maximum at 5.4759 mg.g-1.The impact of basic climatic factors existed in sampling land on the content of Magnolia phenol was analyzed. The result showed that annual temperature and frostless season were the greatest impact factors.
3. Seasonal changes have an impact on the content of secondary metabolites during the period of plant growth. The content of phenol in shoot cortex of Magnolia was highest in January. Afterward with the passage of time, the content would decrease at first then increased but in downward trend on September. In the Magnolia leaves honokiol content is always higher than that magnolol content,the trend of which is more obvious in tender leaves.Finally the total phenol content distribution from tender leaves to old leaves was in Increasing trend until it reached the maximum before the deciduous period.
4.There are some differences of Magnolia content among the different parts of Magnolia but the differences would be minimal among the same organs. Polyphenol content in different organs ranked in the order of root bark ,dry hide ,branch bark, tender bud , seed, fruit, stem wood and needle.
5. With the increase of Magnolia trees age, magnolol and honokiol content trend in both branch bark and stem bark were shown as increasing - decreasing– increasing. while in both middle-aged and young growth plantations, the phenol content would increased with their ages increasing. The value reached the maximum approximately at their ages of 25 but afterward it would be shown as downtrend. However, in hundred years of aged trees, the active component content would show increasing trend in the last stage. The phenol content of Magnolia leaves decreased with age increasing, which reached higher level in their young growth tree leaves.
6. The variation of phenol contents between Magnolia individuals in the same population with the diameter of the similar size were quite large and the difference of phenol contents in various parts between Magnolia individuals were significantly.When excellent magnolia provenance was selected, there exited very significant or significant positive correlation with tree height, crown, bark thick, and branches rather than straight degrees, round degree and health degree. Meanwhile, the positive correlation between phenol content of branch bark and traits of trees was very significant or significant.
7. Four main components were extracted, according to the principle in which characteristic root is equal to or greater than 1. Then the principal components were comprehensive evaluated from 24 different provenance. The evaluation result by ranking order was as follow: top front group was Zherong,Youxi, Licheng, Fuzhou, which is followed by the Wuping,Yongtai, Shouning,Qingliu.Shaxian,Yongan,Pucheng, campus of Fujian Agricultural and Forestry University,Pingnan, Mingxi,Ninghua, Zhangping, and at least was Taining,Nanping,Shunchang,Jianyang,Datian, Jianou, Fuan, Guangze.
8. Correlation between various traits in plus tree selection was analyzed. The results were shown as follow: there exited very significant positive correlation between DBH and crown width, tree height and phenol content, very significant positive correlation between tree height and crown width, clear bole height, and branches, very significant positive correlation between crown width and clear bole height, significant positive correlation between stem straightness and bark thickness, rather than other indexes.
9. Six principal components extracted by principal component analysis of cumulative contribution rate of greater than or equal to 85% were comprehensively scored from 54 plus tree. The result was shown as follow: the higher scores of plus tree family was the number 11,6,131,146,16,followed by 123,33,49,114, 2. Cluster analysis results were divided into two categories Provenance, the first category was the number 2,14,99,107,113,8,141,114,37,75,44,117,135,109,16,33,49,123, 6,11,131,146, the second category was the number 13,102,111,43,116,145,50,70,93,136,76,90,132,147,38,115,119,45,101,20,110,142,125,21,46,120, 138,129,97,94,29,112.
10. DNA fingerprinting of 89 copies of Magnolia resources was constructed based on the data matrix obtained out of 96 reproducible RAPD bands of Magnolia resources amplified by six primers. The results indicated that 11 combinations were respectively S60 and S69, S60 and S91, S60 and S1412, S60 and S330, S60 and S1422, S69 and S91, S69 and S1412, S69 and S1422, S91 and S1412, S1412 and S1422, S1412 and S330.In each combination , data matrix formed by amplification can be relatively easy for multiple authentication to distinguish from the 89 resources.
11.Source of 89 samples from the whole nation was analyzed. The analysis result can be concluded to three categories. The first category was Jiangyou,Fujian in which genetic structure possessed particularity while the second category included Jiangyou Sichuan,Daoxian Hunan,Mingxi Fujian, Shexian Anhui and part of Provenance individual of Jianyang Fujian. In addition, the third category can be divided into two sub-categories.In the first sub-category, main source region included Enshi Hubei, Jingning Zhejiang, Ziyuan Guangxi, Quanzhou Guangxi, Lushan Jiangxi, Haozhou Anhui, Hefei Anhui, Yaan Sichuan, Changan Shaanxi, Shexian Anhui,Jiangyou Sichuan and part of the Provenance individual of Yongzhou Hunan and the the second sub-category included Zhangjiajie Hunan, Jiangjin district of Chongqing, Shunchang Fujian, Pucheng Fujian, Nanping Fujian, Zherong Fujian, Yongtai Fujian, Forest Park of Fuzhou, Liancheng Fujian, Guangze Fujian,Taining Fujian, Shouning Fujian,Daoxian Hunan and part of the Provenance individualof Fujian Jianyang.
引文
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