Identification and Genetic Diversity of Polygonatum cyrtonema Hua and P.filipes Merr. Based on ISSR Marker
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摘要: 利用ISSR分子标记，对福建省19份黄精属种质资源进行鉴别和遗传多样性分析。结果显示：（1）根据性状及原植物进行样品鉴别，种质1、6、7、8、9、10、11、12、13、15、16、18、19为多花黄精，种质2、3、4、5、14、17为长梗黄精；（2）从100个引物里筛选出11个能扩增出清晰具多态性的引物，共扩增出条带170条，片段大小在250~2 000 bp，其中多态性条带170条，多态性比例为100%。Popgen 32软件分析显示，供试样品种质资源遗传多样性丰富，相似系数在0.604 9~0.824 4，遗传距离在0.193 1~0.502 7，霞浦种质与建瓯种质亲缘关系最近，柘荣种质与武夷山1种质亲缘关系最远；（3）UPGMA法聚类分析显示，19份样品被分为2大类，4个亚类，多花黄精种质全部聚在第Ⅰ类，长梗黄精种质全部聚在第Ⅱ类；（4）用Popgen 32软件进行遗传多样性分析，黄精属的有效等位基因数（Ne）、期望杂合度（H）、Shannon信息指数（Ⅰ）分别为1.484 5、0.292 3、0.423 9，4个亚类种水平的基因多样性Ht=0.4132。研究表明，ISSR分子标记适用于多花黄精与长梗黄精的鉴别及遗传多样性分析，研究成果为野生黄精属植物合理引种、驯化、保护和利用提供了重要的参考依据和数据支持。Abstract: Identification and genetic diversity analysis based on ISSR marker for 19 germplasms of Polygonatum were conducted. The results were as follows. (1) Based upon the morphological characteristics of the plants, germplasms No. 1, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, and 19 were determined to be P. cyrtonema Hua; and No. 2, 3, 4, 5, 14, and 17, P. filipes Merr. (2) From the 100 primers isolated, 11 were amplified to render 170 distinctive and polymorphic bands with sizes ranging from 250 bp to 200 0 bp. (3) Using Popgen 32 software, an abundant genetic diversity was found to exist among these germplasms with similarity coefficients ranging from 0.604 9 to 0.824 4, and genetic distances from 0.193 1 to 0.502 7; and, No. 6 and 7 were closely related, while the No. 10 and 17 differed the most. (4) The UPGMA cluster analysis classified the germplasms into 2 groups and 4 subgroups with all P. cyrtonema Hua in Group Ⅰ and all P. filipes Merr. in Group Ⅱ. And, (5) as analyzed by Popgen 32, the genetic diversity of the 4 subclasses showed an effective number of alleles (Ne) to be 1.484 5, Nei's genetic diversity index (h), 0.292 3, Shannon diversity index (Ⅰ), 0.423 9, and total gene diversity (Ht), 0.413 2. It appeared that the current method based upon ISSR marker could be used to identify the individual species as well as show the genetic diversity and relationship among the species in the genus providing valuable molecular information for the introduction, acclimation, protection and utilization of the Polygonatum resources in the wild.
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图 1 样品基因组DNA电泳图谱
注：A1、B1、C1、D1、E1为新鲜样品；A2、B2、C2、D2、E2为阴干样品。
Figure 1. Genomic DNAs extracted from Polygonatum electrophoresed on 0.8% agarose TBE gel
图 2 19个样品UBC826引物扩增结果
注：M为DNA Marker，S1~S19为样品。
Figure 2. Amplified primer UBC826 in 19 Polygonatum germplasms
图 3 基于ISSR的19个受试黄精属样品Nei′s遗传相似度UPGMA聚类
Figure 3. UPGMA dendrogram of Sarcandra glabra based on Nei's genetic distance
表 1 19份供试材料
Table 1. Basic information on 19 germplasm samples
样品 原产地 叶下面短毛 总花梗长
/cmS1 福建泰宁 无 3~4 S2 福建梅列 有 5~8 S3 福建沙县 有 6~8 S4 福建光泽1 有 6~8 S5 福建光泽2 有 5~8 S6 福建霞浦 无 3~4 S7 福建建瓯 无 2~4 S8 福建建阳 无 3~4 S9 福建尤溪 无 2~4 S10 福建柘荣 无 3~4 S11 福建永泰 无 3~4 S12 福建泉港 无 2~4 S13 福建松溪 无 3~4 S14 福建闽侯 有 6~8 S15 福建新罗 无 3~4 S16 福建上杭 无 3~4 S17 福建武夷山1 有 6~8 S18 福建武夷山2 无 2~4 S19 福建武夷山3 无 3~4 
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表 2 11条ISSR引物扩增条带数与多态性比率
Table 2. Number and ratio of polymorphic bands amplified by 11 ISSR primers
引物 引物序列 退火温
度/℃扩增
条带数多态性
条带数多态性
比率/%UBC807 AGA GAG AGA GAG AGA GT 52 13 13 100 UBC811 GAG AGA GAG AGA GAG AC 54 17 17 100 UBC818 CACACACACACACACAG 54 18 18 100 UBC820 GTG TGT GTG TGT GTG TC 54 14 14 100 UBC826 ACA CAC ACA CAC ACA CC 54 18 18 100 UBC827 ACA CAC ACA CAC ACA CG 54 11 11 100 UBC842 GAG AGA GAG AGA GAG AYG 51 15 15 100 UBC849 GTG TGT GTG TGT GTG TYA 53 19 19 100 UBC854 TCT CTC TCT CTC TCT CRG 51 12 12 100 UBC855 ACA CAC ACA CAC ACA CYT 53 14 14 100 UBC889 DBD ACA CAC ACA CAC AC 52 19 19 100 统计 170
(合计)170
(合计)100
(均值)注:R为碱基A或G；B为碱基C、G或T；D为碱基A、G或T；V为碱基A、C或G；H为碱基A、C或T。
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表 3 Nei's遗传相似性(对角线上方)和遗传距离(对角线下方)
Table 3. Nei's genetic identity (above diagonal line) and distance (below diagonal line)
项目 S1 S2 S3 S4 S5 S 6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S1 0.7317 0.6976 0.6683 0.6829 0.7073 0.6683 0.6829 0.6878 0.6927 0.6780 0.6634 0.6634 0.6683 0.7073 0.7024 0.6780 0.6829 0.7366 S2 0.3124 0.7415 0.7415 0.7463 0.6829 0.6732 0.6780 0.7122 0.6683 0.6537 0.6780 0.6585 0.7024 0.6829 0.6976 0.6732 0.6390 0.6732 S3 0.3602 0.2991 0.7171 0.6634 0.6878 0.7073 0.6829 0.7073 0.7122 0.6585 0.6829 0.6634 0.6976 0.6780 0.6732 0.6390 0.6244 0.6683 S4 0.4030 0.2991 0.3326 0.8195 0.6780 0.6976 0.6732 0.6878 0.6927 0.6683 0.6927 0.6732 0.7171 0.6683 0.6732 0.6878 0.6439 0.6683 S5 0.3814 0.2926 0.4104 0.1990 0.7512 0.7024 0.6878 0.6927 0.6488 0.6634 0.6878 0.6488 0.7610 0.6732 0.6878 0.7122 0.6878 0.6829 S6 0.3643 0.3814 0.3743 0.3885 0.2861 0.8244 0.8000 0.7659 0.7512 0.7268 0.7512 0.7220 0.6976 0.7268 0.7024 0.6683 0.7122 0.6976 S7 0.4030 0.3958 0.3463 0.3602 0.3532 0.1931 0.8195 0.7951 0.7610 0.6878 0.7610 0.7610 0.6878 0.7561 0.7610 0.6293 0.6927 0.6683 S8 0.3814 0.3885 0.3814 0.3958 0.3743 0.2231 0.1990 0.8000 0.7756 0.7805 0.7268 0.7659 0.6927 0.7610 0.7463 0.6537 0.6878 0.7024 S9 0.3743 0.3394 0.3463 0.3743 0.3672 0.2668 0.2293 0.2231 0.8098 0.7073 0.6927 0.8000 0.6488 0.7756 0.6927 0.6390 0.6634 0.6683 S10 0.3672 0.4030 0.3394 0.3672 0.4327 0.2861 0.2732 0.2541 0.2110 0.7415 0.7366 0.7659 0.6244 0.7317 0.7268 0.6049 0.6293 0.6634 S11 0.3885 0.4252 0.4 177 0.4030 0.4104 0.3191 0.3743 0.2478 0.3463 0.2991 0.7902 0.7317 0.6683 0.6780 0.7122 0.7073 0.6439 0.6878 S12 0.4104 0.3885 0.3814 0.3672 0.3743 0.2861 0.2732 0.3191 0.3672 0.3057 0.2354 0.7463 0.7220 0.6829 0.7463 0.7024 0.6878 0.6829 S13 0.4104 0.4177 0.4104 0.3958 0.4327 0.3258 0.2732 0.2668 0.2231 0.2668 0.3124 0.2926 0.6927 0.7317 0.7171 0.6537 0.6878 0.7024 S14 0.4030 0.3532 0.3602 0.3326 0.2732 0.3602 0.3743 0.3672 0.4327 0.4710 0.4030 0.3258 0.3672 0.6585 0.6634 0.7366 0.6829 0.7171 S15 0.3463 0.3814 0.3885 0.4030 0.3958 0.3191 0.2796 0.2732 0.2541 0.3124 0.3885 0.3814 0.3124 0.4177 0.7707 0.6683 0.7024 0.6780 S16 0.3532 0.3602 0.3958 0.3958 0.3743 0.3532 0.2732 0.2926 0.3672 0.3191 0.3394 0.2926 0.3326 0.4104 0.2604 0.7415 0.6976 0.7122 S17 0.3885 0.3958 0.4478 0.3743 0.3394 0.4030 0.4632 0.4252 0.4478 0.5027 0.3463 0.3532 0.4252 0.3057 0.4030 0.2991 0.7024 0.6878 S18 0.3814 0.4478 0.4710 0.4402 0.3743 0.3394 0.3672 0.3743 0.4104 0.4632 0.4402 0.3743 0.3743 0.3814 0.3532 0.3602 0.3532 0.7122 S19 0.3057 0.3958 0.4030 0.4030 0.3814 0.3602 0.4030 0.3532 0.4030 0.4104 0.3743 0.3814 0.3532 0.3326 0.3885 0.3394 0.3743 0.3394
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表 4 基于ISSR的黄精属遗传多样性
Table 4. Genetic diversity of Polygonatum based on ISSR markers
亚类 有效等位基
因数(Ne)期望杂合度
(H)Shannon信息
指数(Ⅰ)Ⅰ1 1.3473 0.1930 0.2763 Ⅰ2 1.3799 0.2257 0.3391 Ⅱ1 1.3741 0.2162 0.3180 Ⅱ2 1.0 0 0 种级All 1.4845 0.2823 0.4239
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