Ⅱ ） Breeding of excellent strains of Ural licorice ( Ⅱ )
甘草属五种不同来源植物遗传基础的 AFLP 分析 ——AFLP Analysis of Genetic Basis of Five Different Sources of Licorice
，庾晓红 ， 高文远 Zhou Chengming , Xi Xiaohong1 , Gao Wenyuan2
1. 北京时珍中草药技术有限公司 , ( 1. Beijing Shizhen Chinese Herbal Technology Co. , Ltd. , Beijing 天津大学药物 科学与技术学院，天津 102609; 2. School of Pharmaceutical Science and Technology, Tianjin University, Tianjin ） 300072 )
Pick To : purpose 分子标记技术在乌拉尔甘草优良栽培品系选育中的应用以及甘草属五种不同来源的植物的亲缘关系。 The genetic basis of five different sources of licorice was studied, and the application of AFLP molecular marker technology in the selection and breeding of excellent cultivars of urallic licorice and the genetic relationship of five different sources of licorice were discussed. method 扩增片段长度多态性 (AFLP) 标记技术 对乌拉尔甘草栽培新品系 “阿勒泰1号”、“乌什1号”和常规栽培品系 内蒙古乌拉尔甘草以及胀果甘草、光果甘草 进行基因组DNA多态性、指纹图谱和UPGMA聚类分析 。 Amplified fragment length polymorphism (AFLP) marker technology was used to perform genomic DNA analysis on the new cultivars "Altay 1 ", "Wushi 1 " and conventional cultivars Inner Mongolia Ural Glycyrrhiza, Glycyrrhiza uralensis and Glycyrrhiza uralensis Morphology, fingerprint and UPGMA cluster analysis . result 64 对引物组合中筛选出了 15 对引物组合进行多态性分析， 以多态位点百分率最高的 E-AGG/M-CAA 组合构建甘草属五种植物不同来源的指纹图谱。 From the 64 primer combinations, 15 primer combinations were selected for polymorphism analysis. The fingerprints of five different species of Glycyrrhiza were constructed using the E-AGG / M-CAA combination with the highest percentage of polymorphic sites . 聚类分析将五种不同来源的植物分为两组，表现出不同的亲缘关系。 UPGMA cluster analysis divided five different sources of plants into two groups, showing different genetic relationships. in conclusion 号”、“乌什1号”形成了独特的基因构成，可以作为乌拉尔甘草优良栽培新品系选育目标进行深入研究；内蒙古产乌拉尔甘草与光果甘草的亲缘关系最近。 "Altay 1 " and "Wushi 1 " formed a unique genetic composition, which can be used as an in-depth study on the selection of new cultivars of excellent cultivation of Ural licorice. Inner Mongolia ’s Ural licorice has a close relationship with Glycyrrhiza uralensis.
乌拉尔甘草，光果甘草，胀果甘草；良种选育； AFLP ；指纹图谱 Key words: Glycyrrhiza Uralensis, Glycyrrhiza glabra, Glycyrrhiza inflata; Breeding of fine varieties; AFLP ; Fingerprint
Study on Selective Breeding of Glycyrrhiza uralensis Ⅱ ) ( Ⅱ )
AFLP analysis for genus —— AFLP analysis for genus Glycyrrhiza
1 , Yu Xiao-hong 1 , GAO Wen-yuan 3 ZHOU Cheng-ming 1 , Yu Xiao-hong 1 , GAO Wen-yuan 3
(1. Beijing Shizhen herbal medicine technology Ltd., Beijing 102609, China; 2. The College of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China)
Abstract: Object Studies on genetic background of Glycyrrhiza and the application of amplified fragment length polymorphism (AFLP) to the select of breeding of Glycyrrhiza uralensis and relative relationships of different species in genus Glycyrrhiza. Methods The DNA polymorphism, fingerprinting, and UPGMA analysis of different species in genus Glycyrrhiza from Aletai, Wushi and Inner Mongolia were detected by AFLP technique. Results 15 primer combinations were screened from 64 primer combinations to analysis DNA polymorphism and the DNA fingerprinting were generated by primer combination E-AGG / M-CAA. UPGMA analysis showed that all the studied populations were clustered into two groups and have different relative. Conclusion The results showed that “Aletai No.1”, “Wushi No.1” have inimitable gene structure and should be studied more as new breed resource. On the other hand, Glycyrrhiza uralensis produced in Inner Mongolia is relative of Glycyrrhiza glabra .
Key words: Glycyrrhiza uralensis; ; Glycyrrhiza inflata ; Glycyrrhiza glabra; selective breeding; AFLP; DNA fingerprinting
1 号”的遗传基础及品系内的亲缘关系  。 The last paper mainly studied the genetic basis of the excellent cultivar "Urqin No. 1 " of Ural Glycyrrhiza uralensis and the genetic relationship within the line  . 1 号”的遗传基础及与甘草属内植物的亲缘关系。 This thesis mainly studies the genetic basis of the new cultivar "Altay 1 " of Ural licorice and its genetic relationship with the endogenous plants of the genus Glycyrrhiza. Due to the large amount of artificially excavated wild Ural licorice for decades, the source of Ural licorice in the main producing area of Inner Mongolia has decreased sharply, and the quality of seeds has decreased, which cannot meet the needs of large-scale cultivation. 2002 年开始采集内蒙古以外地区的乌拉尔甘草种源进行大面积栽培并选育优良栽培新品系，从中发现“阿勒泰 1 号”，根系发达，主根直、长，根皮淡红色，地上植株矮小紧凑，适合密植。 We have collected Ural licorice provenances from areas outside Inner Mongolia for large-scale cultivation and selection of excellent new cultivation lines since 2002. We found "Altay 1 ", which has a developed root system with straight, long main roots, reddish root bark, and short ground plants. Compact for dense planting. 3 年每公顷产量约 30000 公斤 鲜根，比内蒙古乌拉尔甘草产量每公顷产量约高 4500 公斤 ，甘草酸含量超过 2% 。 Yield in Jiuquan , Gansu Province for 3 years, yields about 30,000 kg of fresh root per hectare, which is about 4,500 kg more per hectare than that of Ural licorice in Inner Mongolia . 4000 公顷 “阿勒泰 1 号”。 At present, our company has promoted about 4,000 hectares of "Altay 1 ". 1 号”叶小，茎杆直立，抗到伏，正在选育过程中。 "Wushi No. 1 " has small leaves, erect stems and resistance to volts, and is in the process of breeding.
“阿勒泰 1 号”、“乌什 1 号”与常规内蒙古产乌拉尔甘草 在遗传本质上是否有所不同，以及不同栽培品系的乌拉尔甘草与光果甘草及胀果甘草的亲缘关系，本文应用 AFLP 技术 [2 、 3] 对甘草属以上五种不同来源的植物进行遗传基础分析。 In order to confirm whether the new Ural licorice cultivated lines "Altay 1 " and "Wushi 1 " are genetically different from conventional Inner Mongolian Ural licorice , and whether the different cultivated lines of Ural licorice are related to Glycyrrhiza uralensis and Glycyrrhiza uralensis. Genetic relationship, this paper applies AFLP technology [2 , 3] to analyze the genetic basis of plants from five different sources of Glycyrrhiza.
1 Materials and Methods
“ 阿勒泰 1 号 ” 、 “ 乌什 1 号 ” 、光果甘草和胀果甘草种子。 The materials used in the tests were conventional Inner Mongolia Ural licorice and new Ural licorice cultivation lines " Altay 1 " , " Wushi 1 " , Glycyrrhiza uralensis and Glycyrrhiza inflata. The seeds were collected and identified by Zhou Chengming of Beijing Shizhen Chinese Herbal Medicine Technology Co., Ltd. 10 株胚根，混为一份样品，提取 DNA 组织。 Dried seeds were routinely germinated. Ten radicles of each material were taken and mixed into one sample to extract DNA tissue.
组织提取 1.2 DNA Tissue Extraction
CTAB 法提取  Extraction using CTAB method  .
分析 1.3 AFLP analysis
DNA 的酶切、连接、预扩增和选择性扩增的反应液配方和反应程序由北京鼎国生物公司设计进行。 The reaction solution formulation and reaction procedure for template DNA digestion, ligation, pre-amplification and selective amplification were designed and performed by Beijing Dingguo Biological Company.
1.4 图谱分析 AFLP atlas analysis
ABI 377 测序仪进行 AFLP 多态性分析。 ABI 377 sequencer was used for AFLP polymorphism analysis.
1.5 data processing
1 ” ，同一位置没有出现的条带记为“ 0 ” ，生成由“ 1 ” 和“ 0 ” 组成的原始矩阵。 The clearly visible and reproducible bands on the electropherogram are recorded as " 1 " , and the bands that do not appear in the same position are recorded as " 0 " , and the original matrix composed of " 1 " and " 0 " is generated . (p): p=(k/n) × 100 ％，其中 k 是多态位点数目； n 为所测位点总数。 Calculate the percentage of polymorphic sites (p): p = (k / n) × 100 %, where k is the number of polymorphic sites; n is the total number of measured sites. AFLP-SURVEY With AFLP-SURVEY 1.0 (Vekemans 软件计算每个居群的多态位点数 , et al., 2002) The software calculates the number of polymorphic loci in each population . ( P ) ，并计算居群、物种水平的遗传多样性 ( He )( Nei, 1978 )  。 The percentage of polymorphic loci ( P ) , and the genetic diversity at the population and species level ( He ) ( Nei, 1978 )  . Jaccard By Jaccard NTSYSpc 2.0 (Rohlf ,1994)  对 40 个乌拉尔甘草样品用非加权配对算术平均的方法 (Unweighted pair-group method with arithmetic mean , UPGMA) The similarity coefficient is the parameter using NTSYSpc 2.0 (Rohlf, 1994)  Unweighted pair-group method with arithmetic mean (UPGMA) on 40 Ural licorice samples Perform cluster analysis.
2 Knot fruit
分析的引物筛选及其标记的多态性 2.1 Primer screening for AFLP analysis and its labeled polymorphisms
Eco Thus for Eco 标记引物 E-AAC 、 E-AAG 、 E-ACA 、 E-ACT 、 E-ACC 、 E-ACG 、 E-AGC 、 E-AGG(8 个 ) 和 Mse RI- labeled primers E-AAC , E-AAG , E-ACA , E-ACT , E-ACC , E-ACG , E-AGC , E-AGG (8 ) and Mse I M-AA 、 M-AC 、 M-AG 、 M-AT 、 M-TA 、 M-TC 、 M-TG 、 M-TT(8 个 ) 完全排列组成的 64 对引物组合进行筛选，从中选出了 15 个 谱带清晰、带型分布均匀并且多态性高的引物组合，共扩增出 2001 条带谱，平均每个引物扩增出 133 条。 The primers M-AA , M-AC , M-AG , M-AT , M-TA , M-TC , M-TG , and M-TT (8 ) are arranged in a sequence of 64 pairs of primers. A total of 15 primer combinations with clear bands, uniform band patterns and high polymorphisms were amplified. A total of 2001 bands were amplified , with an average of 133 primers per primer . , 对引物共扩增出多态性带 1030 15 , Amplification of primer polymorphic bands 1030
, The percentage of polymorphic loci is 51.47% 表 1) 。 ( Table 1) .
1 Table 1 AFLP 分析的 15 个引物组合序列及其扩增结果 Sequences of 15 primer combinations suitable for AFLP analysis of Glycyrrhiza and their amplification results
Table 1 The base sequence of 15 primer combinations for AFLP analysis; and AFLPs generated among 50 individuals using the 15 combinations
组合 Primer set
′ -3 ′ ) Prime sequence (5 ′ -3 ′ )
Total number of sites
P Polymorphic site percentage P
E-AAC / M-CAA
E-AAC / M-CAC
E-AAC / M-CAG
E-AAC / M-CAT
E-AAC / M-CTC
E-AAC / M-CTG
E-AAC / M-CTT
E-AAG / M-CAA
E-AAG / M- CAC
E-AAG / M- CAG
E-AAG / M-CTC
E-AGG / M-CAA
E-AGG / M-CAG
E-AGG / M-CTG
E-AGG / M-CTT
/ 平均 Total / Average
1030 / 67.5
2.2 Analysis of Genetic Diversity of Glycyrrhiza
65~74 之间，多态位点百分率从 46.8% （胀果甘草）到 56.6% （光果甘草）不等（表 2 ）；而乌拉尔甘草不同栽培品系的多态位点数以内蒙古对照最高，“阿勒泰 1 号”次之，“乌什 1 号”最低，多态位点百分比率分别为 55.2% 、 53.4% 、 48.3% 。 The number of polymorphic loci of five different sources of licorice is between 65 and 74. The percentage of polymorphic loci ranges from 46.8% (Glycyrrhiza inflata) to 56.6% (Glycyrrhiza glabra) (Table 2 ); and Ural The number of polymorphic loci in different cultivars of licorice was highest in Inner Mongolia control, "Altay 1 " was the second, and "Wushi 1 " was the lowest. The percentages of polymorphic loci were 55.2% , 53.4% , and 48.3% .
0.1673 到 0.1986 之间，其中光果甘草的遗传多样性最大 (0.1986) ，遗传多样性最小的是胀果甘草 (0.1673) ，属内的平均遗传多样性 Hep =0.1864 。 The genetic diversity of five different sources of licorice is between 0.1673 and 0.1986 . Among them, Glycyrrhiza glabra has the largest genetic diversity (0.1986) , and the smallest genetic diversity is Glycyrrhiza uralensis (0.1673) . Diversity Hep = 0.1864 . 0.1982 ）最高，“阿勒泰 1 号”（ 0.1893 ）次之，“乌什 1 号”（ 0.1784 ）最低（表 2 ）。 The genetic diversity of the cultivars of Glycyrrhiza uralensis was highest in the Inner Mongolia control ( 0.1982 ), followed by "Altay 1 " ( 0.1893 ), and "Wushi 1 " ( 0.1784 ) was the lowest (Table 2 ).
2 Table 2 Analysis on Genetic Diversity of Five Different Sources of Licorice
Table 2 Genetic diversity within different species of Glycyrrhiza
Number of samples
Number of sample
Percentage of polymorphic loci
(He) Genetic diversity (He)
Neimongo Neimongo, Inner Mongolia
1 号 Altay 1
1 号 Ush 1
PPB=Percentage of polymorphic sites Note: PPB = Percentage of polymorphic sites
2.2 Fingerprint construction
15 对引物组合中， E-AGG/M-CAA 组合共扩增出 140 条带谱，扩增的 DNA 片段在 50bp~450bp 之间，其中多态性带谱为 77 条，共有带 43 条，多态位点百分率达到了 55% ，为各引物中最高。 Among the 15 primer pairs screened , the E-AGG / M-CAA combination amplified a total of 140 bands, and the amplified DNA fragments ranged from 50bp to 450bp , of which 77 were polymorphic bands . With 43 bands , the percentage of polymorphic loci reached 55% , the highest among all primers. 12 条，其中“阿勒泰 1 号”分别在 82bp,90bp,116bp,295bp,330bp 五 个位点处有区别于内蒙古乌拉尔甘草的特征谱带，“乌什 1 号”具有的特异性谱带数分别为 2 条（ 310bp,430bp ），而光果甘草与内蒙古乌拉尔甘草相比较在 102bp,165bp, 291bp, 330bp,350bp 五个位点处具有特征谱带，胀果甘草则具有 3 条特征谱带（ 85bp, 98bp, 210bp ）。 There are 12 specific bands , of which "Altay 1 " has characteristic bands different from Ural licorice in Inner Mongolia at five sites of 82bp, 90bp, 116bp, 295bp, and 330bp, respectively, and the specificity of "Wushi 1 " The number of bands is 2 ( 310bp, 430bp ). Compared with Inner Mongolia Ural licorice , Guangguo Glycyrrhiza has characteristic bands at five sites: 102bp, 165bp, 291bp, 330bp, and 350bp . Characteristic bands ( 85bp, 98bp, 210bp ). ( 图 1) 。 Therefore, the primer combination has a strong ability to detect genetic variation differences between genotypes of plants of different genus Glycyrrhizae, and is used to construct a fingerprint map ( Figure 1) .
内蒙古对照 , 2: 阿勒泰 1 号 , 3: 乌什 1 号 , 4: 胀果甘草 , 5: 光果甘草 1: Inner Mongolia control , 2: Altay No. 1 , 3: Wushi No. 1 , 4: Glycyrrhiza uralensis , 5: Glycyrrhiza glabra
1: Inner Mongolia, 2: Aletai No. 1, 3: Wushi No. 1, 4: Glycyrrhiza inflata , 5: Glycyrrhiza glabra
1 Figure 1 E-AGG/M-CAA 引物组合的甘草属植物的 AFLP 指纹图谱 AFLP fingerprinting of Glycyrrhiza plants based on E-AGG / M-CAA primer combination
Fig.1 AFLP fingerprinting amplified with primer E-AGG / M-CAA
2.3 Cluster analysis
15 对引物所扩增出的甘草属的不同植物的 2001 个 DNA 带谱的数据，按照 Jaccard Based on the data of 2001 DNA bands of different licorice plants amplified by the above 15 pairs of primers , according to Jaccard UPGMA 聚类，构建供试材料间的亲缘关系树状图（图 2 ）。 The similarity coefficients were subjected to UPGMA clustering to construct a tree diagram of the relationship between the test materials (Figure 2 ).
2 可以看出，所研究的甘草属的植物被聚为两组。 It can be seen from Fig. 2 that the plants of the genus Glycyrrhiza studied are grouped into two groups. 1 号”、“阿勒泰 1 号”与胀果甘草被聚为一组，在 0.60 处胀果甘草被分离出。 Among them, Inner Mongolia control and Glycyrrhiza uralensis were grouped into one group; "Wushi 1 ", "Altay 1 " and Glycyrrhiza uralensis were grouped into one group, and Glycyrrhiza uralensis was isolated at 0.60 . The results show, 1 号”与“乌什 1 号”之间的亲缘关系也较近，而胀果甘草与其余种的亲缘关系较远。 The genetic relationship between Ural licorice and Glycyrrhiza uralensis in Inner Mongolia is the closest; the new cultivars "Altay 1 " and "Wushi 1 " of Ural licorice are also closer, and the relationship between Glycyrrhiza inflata and other species .
：内蒙古对照； c1 : Inner Mongolia control; ：阿勒泰 1 号； c2 : Altay 1 ; ：乌什 1 号； c3 : No. 1 Wushi ; ：胀果甘草； c5 ：光果甘草 c4 : Glycyrrhiza inflata; c5 : Glycyrrhiza glabra
； c2: Aletai No.1 c3:Wushi No.1 c4: c1: Inner Mongolia ; c2: Aletai No.1 c3: Wushi No.1 c4: Glycyrrhiza inflata c5: Glycyrrhiza glabra
2 Figure 2 AFLP Based on AFLP 甘草属五种不同来源植物 的 UPGMA Data of UPGMA from five different sources of licorice Cluster graph
Fig. 2 Dendrogram illustrating the genetic relationships among Glycyrrhiza accessions based on UPGMA cluster analysis of AFLP data
3 discuss s
The north and south of Xinjiang Tianshan Mountains and the surrounding areas of the Taklimakan Desert are the birthplaces of licorice plants. The region has harsh weather conditions, frequent sandstorms, large temperature differences between day and night, and rich licorice plant resources. Ural licorice, Glycyrrhiza uralensis and Glycyrrhiza uralensis have been growing in the region for a long time at the same time. There must be a certain relationship between them and a certain degree of mutation, which is exactly what we selected for the new licorice strain. basis.
1 号”、“乌什 1 号”分别采集于新疆的阿勒泰地区、乌什县的野生种群，经连续七年的大面积引种栽培，发现与常规人工栽培品系内蒙古产乌拉尔甘草相比，在单位面积产量、株型、抗性、产品品质等方面具有显著的优势，目前正大面积推广累积超过 4000 公顷 。 "Altay 1 " and "Wushi 1 " were collected from wild populations in Altay region, Xinjiang, and Wushi county, respectively. After large-scale introduction and cultivation for seven consecutive years, it was found that compared with conventional artificially cultivated strains of Inner Mongolia, Ural licorice, It has significant advantages in terms of yield per unit area, plant type, resistance, and product quality. At present, it is being promoted in a large area and accumulated more than 4000 hectares .
The analysis results show that Glycyrrhiza uralensis has the closest relationship to Ural licorice from Inner Mongolia, while Glycyrrhiza uralensis is far away from the two. From this, it seems that there is some evolutionary relationship between G. uralensis and G. uralensis and G. uralensis from Ural. Licorice is likely to evolve into Glycyrrhiza glabra after millions of years of evolution. This inference is for further study.
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