Phylogenetic relationships among 12 cultivars of Ilex verticillata based on ISSR molecular markers
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摘要: 北美冬青Ilex verticillata作为切枝观赏和景观美化植物在中国花卉市场受到越来越多的关注。为给北美冬青新品种选育提供保障，采用简单重复序列间扩增多态性（ISSR）分子标记分析了北美冬青品种间的亲缘关系，从12个ISSR引物中筛选出4个多态性引物；对12个北美冬青品种进行了扩增，共获得63个条带，其中多态性条带占90.48%，所选引物的多态性较高，可用于鉴定北美冬青品种间的相对亲缘关系；12个北美冬青品种间的遗传相似系数为0.54~0.87，亲缘关系相对较近；品种间的遗传相似系数聚类结果表明，各品种间存在不同程度的亲缘关系，其中I. verticillata ‘Jim Dandy’和I. verticillata ‘Cacapon’间亲缘关系最近。研究结果为北美冬青品种的选育奠定了理论和实践基础。Abstract: As an important ornamental and landscape plant, Ilex verticillata has received increasing attention in flower markets of China. To foster breeding of new I. verticillata cultivars, genetic relationships between I. verticillata cultivars were analyzed by inter-simple sequence repeat (ISSR) markers. Four primers with polymorphism among I. verticillata cultivars were screened from 12 candidate ISSR primers. Results revealed a total of 63 bands with polymorphic bands accounting for 90.48% implying they could be used to identify relative relationships among different I. verticillata cultivars. The genetic similarity coefficient of the 12 I. verticillata cultivars varied from 0.54 to 0.87 indicating that the genetic relationships among I. verticillata were relatively close. Clustering of these genetic similarity coefficients showed that genetic relationships among cultivars were different with the relationship between I. verticillata 'Jim Dandy' and I. verticillata 'Cacapon' being the closest. This study will help lay an important theoretical and practical basis for genetic breeding of I. verticillata.
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Key words:
- forest tree breeding /
- Ilex verticillata /
- ISSR marker /
- molecular identification /
- genetic relationship
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图 1 12个北美冬青品种的ZBSI-002引物指纹图谱
Figure 1 Fingerprints of ISSR primer ZBSI-002 between the 12 cultivars of Ilex verticillata
图 2 12个北美冬青品种遗传相似系数聚类
Figure 2 Clustering of genetic similarity coefficients among 12 cultivars of Ilex verticillata
表 1 北美冬青品种信息
Table 1. Ilex verticillata cultivar information
编号 品种名称 来源 表型特征 R1 Ilex verticillata ‘Oosterwijk’ 荷兰 灌木，叶片8.0 cm × 4.0 cm，花期6月初，果红色，果径9~12mm R2 Ilex verticillata ‘Apollo’ 美国 灌木，雄株，叶片8.0 cm × 4.0 cm，花期5月底 R3 Ilex verticillata ‘Red Sprite’ 美国 灌木，株型紧凑，叶片5.5 cm × 2.5 cm，花期5月下旬，果径10~12mm R4 Ilex verticillata ‘Winter Red’ 美国 灌木，叶片6.0 cm × 3.0 cm，花期6月初，果小而密，果径7~9 mm R5 Ilex verticillata ‘A. Gray’ 美国 灌木，叶片6.5 cm × 3.5 cm，花期5月中旬，果红色，果径10~12mm R6 Ilex verticillata ‘Winter Gold’ 美国 灌木，叶片6.0 cm × 3.0 cm，花期6月初，果黄色，果径7~9 mm R7 Ilex verticillata ‘Jim Dandy’ 美国 灌木，雄株，花期5月中旬 R8 Ilex verticillata ‘Afterglow’ 美国 灌木，株型紧凑，叶片5.5 cm × 2.5 cm，花期5月下旬，果径7~9mm R9 Ilex verticillata × serrata ‘Sparkleberry ’ (female) 美国 灌木，雄株 R10 Ilex verticillata × serrata ‘Sparkleberry ’ (male) 美国 灌木，叶片5.0 cm × 2.5 cm，花期5月下旬，果径5~7mm R11 Ilex verticillata ‘Cacapon’ 美国 灌木，叶片3.5 cm × 5.5 cm，色浓绿，紧凑，花期5月下旬，果径7~9mm R12 Ilex verticillata ‘Southern Gentleman’ 美国 灌木，雄株，叶片6.0 cm × 4.0 cm，花期5月下旬 ck Ilex verticillata 美国 乔木，叶片5.0 cm × 2.0 cm，花期5月初，果径5~7 mm 
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表 2 青属ISSR引物
Table 2. ISSR primers for Ilex
引物编号 引物序列(5′→3′) T退火/℃ ZBSI-001 (CT)8T 51.3 ZBSI-002 (CT)8G 51.3 ZBSI-003 (TC)8G 52.5 ZBSI-004 (AC)8T 52.5 ZBSI-005 (AC)8C 52.5 ZBSI-006 (AC)8G 61.0 ZBSI-007 (GA)8YT 51.3 ZBSI-008 (CA)8RT 56.4 ZBSI-009 (AC)8YG 52.5 ZBSI-010 (TG)8RA 54.2 ZBSI-011 (ATG)5 58.9 ZBSI-012 GAT CAA GC TNN NNN NAT GTG 54.2
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表 3 北美冬青品种在4条ISSR引物位点上的扩增条带
Table 3. Statistics of bands amplified by 4 ISSR primers in the 12 cultivars of Ilex verticillata
引物编号 总条带数 多态性条带数 多态性条带比例/% ZBSI-002 16 15 93.75 ZBSI-004 15 13 86.67 ZBSI-007 18 16 88.89 ZBSI-009 14 13 92.86 合计 63 57 90.48
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表 4 北美冬青品种间在4条ISSR引物位点上的遗传相似系数
Table 4. Genetic similarity coefficients of 4 ISSR primers in Ilex verticillata cultivars
编号 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 ck R1 1 R2 0.63 1 R3 0.57 0.75 1 R4 0.71 0.70 0.67 1 R5 0.60 0.62 0.78 0.70 1 R6 0.63 0.67 0.57 0.71 0.70 1 R7 0.67 0.68 0.62 0.76 0.59 0.57 1 R8 0.70 0.65 0.78 0.79 0.81 0.70 0.68 1 R9 0.63 0.62 0.59 0.76 0.65 0.70 0.78 0.68 1 R10 0.70 0.59 0.59 0.79 0.62 0.63 0.78 0.75 0.84 1 R11 0.63 0.71 0.68 0.73 0.62 0.60 0.87 0.75 0.81 0.78 1 R12 0.71 0.67 0.57 0.78 0.54 0.62 0.86 0.63 0.76 0.76 0.83 1 ck 0.54 0.56 0.46 0.60 0.46 0.44 0.68 0.56 0.65 0.68 0.65 0.63 1
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