Identification of TP-M13-SSR molecular markers and genetic relationship analysis of seven new ornamental peach germplasms
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摘要:
目的 以7份选育出的观赏桃新种质和15份市面上常见桃品种的叶片为试验材料，通过简单重复序列（SSR）分子标记对其遗传多样性及亲缘关系进行分析鉴定，旨在探讨新种质与市面上常见桃品种的种间遗传距离与亲缘关系，为观赏桃起源演化、开发利用和亲本选配提供参考。 方法 利用36对引物进行TP-M13-SSR PCR扩增以及荧光毛细管电泳检测，并对扩增产物的扩增效率及引物多态性进行分析。对7份观赏桃新种质‘T20’、‘T22’、‘T28’、‘T10’、‘T13’、‘T9-1’、‘早花’与15份已有常见桃品种的亲缘关系进行鉴定，利用6个SSR位点构建22份观赏桃种质资源的指纹图谱，进行Neighbor-Joining聚类并结合表型性状开展综合分析。 结果 36对引物中筛选得到25对高多态性引物，共检测到183个多态性等位基因和98.396个有效等位基因，观察杂合度平均值为0.341，预期杂合度平均值为0.739，Shannon’s信息指数平均值为1.546，引物多态性信息指数平均值为0.683，介于0.510 ~ 0.841之间。新种质‘T20’和‘T10’与已有‘台阶’品种相似系数最大，分别为0.95和0.92，‘T20’和‘台阶’的枝型、花瓣颜色及花瓣类型等表型性状都相同；新种质‘T13’与已有‘五宝桃’品种相似系数最大，为0.86；新种质‘T22’与已有各品种相似系数很小，其表型性状与其余21份种质相比有较大不同；新种质‘T9-1’和‘早花’与已有‘朱粉垂枝’品种相似系数最大，为0.94。 结论 本研究通过构建SSR指纹图谱，更加直观和快捷的对观赏桃种质资源进行鉴定，为后期观赏桃种质资源的创新、种质资源库的建立及种质资源的保护与利用提供技术与理论支撑。 Abstract:Objective Genetic diversity and genetic relationship of the leaves from 15 commercially available common types and 7 new ornamental peach germplasms were studied and identified by simple sequence repeat (SSR). The purpose of this study was to explore the genetic distance between the new germplasm and commercially available common varieties, and to provide reference for the origin, evolution, development and utilization of ornamental peach and parental selection. Method TP-M13-SSR PCR amplification and fluorescent capillary electrophoresis detection were carried out using 36 pairs of primers, and the amplification efficiency and polymorphism were analyzed. Genetic relationship of new ornamental peach germplasm ‘T13’, ‘T9-1’, ‘T10’, ‘T20’, ‘T22’ and ‘Zaohua’ was identified, and fingerprints of 22 ornamental peach germplasm resources were constructed using 6 SSR loci, and Neighbor-Joining clustering and comprehensive analysis combined with phenotypic traits was performed. Result 36 pairs of highly polymorphic primers were screened from 29 pairs of primers. A total of 183 polymorphic alleles and 98.396 effective alleles were detected. Average of observed heterozygosity was 0.341, and average of expected heterozygosity was 0.739. Average of Shannon’s information index was 1.546, and average of polymorphism information content of primer was 0.683, ranging from 0.510 to 0.841. The new germplasm ‘T20’ and ‘T10’ had the largest similarity coefficients with the existing variety ‘Taijie’, which were 0.95 and 0.92, respectively. ‘T20’ and ‘Taijie’ had the same phenotypic traits, including branch shape, petal color and petal type. The new germplasm ‘T13’ and the existing variety ‘Wubao tao’ had the highest similarity coefficient, which was 0.86. Similarity coefficient between the new germplasm ‘T22’ and other existing varieties was very small, and its phenotypic characters were quite different from the other 21 germplasms. Similarity coefficient of the new germplasm ‘T9-1’ and ‘Zaohua’ with the existing variety ‘Zhufen chuizhi’ was the largest, which was 0.94. Conclusion In this study, through the construction of SSR fingerprint map, the ornamental peach germplasm resources can be identified more intuitively and quickly, providing technical and theoretical support for the later innovation of ornamental peach germplasm resources, the establishment of germplasm resource banks, and the protection and utilization of germplasm resources. -
Key words:
- ornamental peach /
- SSR /
- genetic relationship analysis /
- new germplasm identification
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图 1 引物BPPCT017在新种质资源‘T13’、‘T9-1’、‘T10’、‘T20’、‘T22’及‘早花’中的扩增产物毛细管电泳检测结果
Figure 1. Results of capillary electrophoresis detection of amplified products of primer BPPCT017 in new germplasm resources ‘T13’, ‘T9-1’, ‘T10’, ‘T20’, ‘T22’ and ‘Zaohua’
图 2 22份观赏桃种质资源的Neighbor-Joining聚类分析图
Figure 2. Neighbor-Joining cluster analysis diagram of 22 ornamental peaches
表 1 供试的观赏桃种质
Table 1. Ornamental peach germplasm for test
编号
No.种质
Germplasm枝型或树形
Branch type or tree type花瓣颜色
Petal color花瓣类型
Petal type花型
Flower pattern1 ‘T20’ 直枝型
Orthocladous group粉色淡粉色混合
Mix pink and light pink重瓣
Double petal牡丹型
Peony flower shape2 ‘T22’ 直枝型
Orthocladous group白粉色
White pink重瓣
Double petal菊花型
Chrysanthemum shape3 ‘T28’ 直枝型
Orthocladous group红色白色混合
Mix red and white重瓣
Double petal海棠花型
Crabapple flower shape4 ‘T10’ 直枝型
Orthocladous group粉色
Pink单瓣
Single-lobe海棠花型
Crabapple flower shape5 ‘T13’ 直枝型
Orthocladous group粉色
Pink单瓣
Single-lobe海棠花型
Crabapple flower shape6 ‘T9-1’ 直枝型
Orthocladous group玫红色
Rose red重瓣
Double petal牡丹型
Peony flower shape7 ‘早花’
‘Zaohua’直枝型
Orthocladous group淡玫红色
Pale rose red单瓣
Single-lobe海棠花型
Crabapple flower shape8 ‘二色’
‘Erse’直枝型
Orthocladous group粉红色或深红色
Pink or dark red重瓣
Double petal蔷薇型
Rose flower shape9 ‘寒红’
‘Hanhong’直枝型
Orthocladous group红色
Red重瓣
Double petal梅花型
Plum blossom shape10 ‘京舞子’
‘Kyou-maiko’直枝型
Orthocladous group红色
Red重瓣
Double petal菊花型
Chrysanthemum shape11 ‘撒金’
‘Sajin’直枝型
Orthocladous group粉色或红色
Pink or red重瓣
Double petal蔷薇型
Rose flower shape12 ‘菊花桃’
‘Kikoumomo’直枝型
Orthocladous group粉色
Pink重瓣
Double petal菊花型
Chrysanthemum shape13 ‘科林斯粉’
‘Corinthian pink’帚型
Pillar group粉红色
Shocking pink重瓣
Double petal月季型
China rose flower shape14 ‘绿萼子’
‘Lü e’垂枝型
Weeping group白色
White重瓣
Double petal梅花型
Plum blossom shape15 ‘品霞’
‘Pinxia’直枝型
Orthocladous group淡粉色
Light pink重瓣
Double petal梅花型
Plum blossom shape16 ‘陕甘山桃’
‘Shangan mountain peach’直枝型
Orthocladous group粉红色
Shocking pink单瓣
Single-lobe海棠花型
Crabapple flower shape17 ‘寿红’
‘Shouhong’寿星型
Dwarf group红色
Red重瓣
Double petal梅花型
Plum blossom shape18 ‘台阶’
‘Taijie’直枝型
Orthocladous group粉色淡粉色混合
Mix pink and light pink重瓣
Double petal蔷薇型
Rose flower shape19 ‘五宝垂’
‘Wubao chui’垂枝型
Weeping group粉色
Pink重瓣
Double petal牡丹型
Peony flower shape20 ‘五宝桃’
‘Wubao tao’直枝型
Orthocladous group红色或粉色
Red or pink重瓣
Double petal牡丹型
Peony flower shape21 ‘云龙’
‘Unriumomo’直枝型
Orthocladous group淡粉色
Light pink单瓣
Single-lobe海棠花型
Crabapple flower shape22 ‘朱粉垂枝’
‘Zhufen chuizhi’垂枝型
Weeping group粉红色
Shocking pink重瓣
Double petal梅花型
Plum blossom shape注：1 ~ 7号材料为选育出的新种质，8 ~ 22号材料为市面上常见观赏桃品种。Notes: materials No.1 to No.7 are selected as new germplasm, and materials No.8 to No.22 are common ornamental peach varieties in the market. 
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表 2 36对SSR引物信息
Table 2. Information of 36 pairs of SSR primers
引物编号
Primer No.连锁群
Linkage group引物序列（5′—3′）
Primer sequence (5′−3′)扩增长度
Amplification length/bp核心序列重复单元
Core sequence repeat motif参考文献
ReferenceBPPCT020 1 F: CGTGGATGGTCAAGATGC 196 ~ 208 (AG)14 GG(AG)7 AT(AG)8 [7] R: ATTGACGTGGACTTACAGGTG BPPCT022 1 F: TTGCGTCTCGCAGGTTATA 132 (AG)22 [7] R: CTACCCCTGCCACAAGCT BPPCT028 1 F: TCAAGTTAGCTGAGGATCGC 151 ~ 191 (TC)15 [7] R: GAGCTTGCCTATGAGAAGACC BPPCT042 1 F: AACCCTACTGGTTCCTCAGC 243 (CT)25 [7] R: GACCAGTCCTTTAGTTGGAGC CPPCT026 1 F: AGACGCAGCACCCAAACTAC 150 ~ 220 (CT)22 [22] R: CATTACATCACCGCCAACAA SSR152 1 F: GTTCTCGACTCCCATATCCAA 250 (TC)31 [23] R: CTCCAAAGTACAGAGCCTATCG SSR169 1 F: TTCTTATTCTGGAAATGCATCG 235 (TC)11 [23] R: ACATTTGCCCAAAATATGGTG UDP96-018 1 F: TTCTAATCTGGGCTATGGCG 253 (AC)21 [24] R: GAAGTTCACATTTACGACAGGG BPPCT001 2 F: AATTCCCAAAGGATGTGTATGAG 128 ~ 170 (GA)27 [7] R: CAGGTGAATGAGCCAAAGC BPPCT034 2 F: CTACCTGAAATAAGCAGAGCCAT 216 ~ 258 (GA)19 [7] R: CAATGGAGAATGGGGTGC SSR96 2 F: AACCTCAATCATTCTTTACACAAGC 146 (AG)33 [23] R: CTGCTTAAGGAGGAACCTCAAAT SSR107 2 F: TGCAGACTAGGGTTTTACAGACAA 155 (GA)8 [23] R: GATCTCCAAGTCATCTCCATCTG SSR179 2 F: ATCACGTCGGAAAGTTCCTAGA 222 (AG)8 [23] R: CGCCCTCCTCCCTCAGTA BPPCT007 3 F: TCATTGCTCGTCATCAGC 111 ~ 151 (AG)22 (CG)2 (AG)4 [7] R: CAGATTTCTGAAGTTAGCGGTA BPPCT033 3 F: GTAGCCGGAGCCGTGTAT 180 (AG)32 [7] R: CTAGAACCCTATAAACACATGGC CPPCT002 3 F: GGAGCTGCAATATTGCTG 100 (GA)10 [22] R: GTTAGGGAAGCATCTCAC SSR184 3 F: TGAATGTTCTTCCTGCTCCTG 290 (GA)33 [23] R: ATGAACATGAACCAGTCAAGGA BPPCT015 4 F: ATGGAAGGGAAGAGAAATCG 150 (AG)13 [7] R: GTCATCTCAGTCAACTTTTCCG BPPCT023 4 F: TGCAGCTCATTACCTTTTGC 183 ~ 237 (CT)21 [7] R: AGATGTGCTCGTAGTTCGGAC CPPCT005 4 F: CATGAACTCTACTCTCCA 120 ~ 185 (CT)25 [22] R: TGGTATGGACTCACCAAC SSR73 4 F: TTGCTGCTGAAAAATAATGAACA 160 (CAA)6 [23] R: GGGTGGCCTGTTGAGAATATAA BPPCT014 5 F: TTGTCTGCCTCTCATCTTAACC 190 ~ 226 (AG)23 [7] R: CATCGCAGAGAACTGAGAGC BPPCT017 5 F: TTAAGAGTTTGTGATGGGAACC 139 ~ 182 (GA)28 [7] R: AAGCATAATTTAGCATAACCAAGC BPPCT026 5 F: ATACCTTTGCCACTTGCG 134 (AG)8 GG(AG)6 [7] R: TGAGTTGGA AGAAAACGTAACA BPPCT037 5 F: CATGGAAGAGGATCAAGTGC 155 (GA)25 [7] R: CTTGAAGGTAGTGCCAAAGC BPPCT038 5 F: TATATTGTTGGCTTCTTGCATG 135 (GA)25 [7] R: TGAAAGTGAAACAATGGAAGC BPPCT008 6 F: ATGGTGTGTATGGACATGATGA 93 ~ 161 (GA)36 [7] R: CCTCAACCTAAGACACCTTCACT BPPCT025 6 F: TCC TGC GTA GAA GAA GGT AGC 147 ~ 199 (GA)29 [7] R: CGA CAT AAA GTC CAA ATG GC CPPCT015 6 F: TGGAGTGCCAATACTATTTA 200 (CT)31 [22] R: CATATGCATGGTTATGGT SSR181 6 F: AGAATGCAGGCCTTCCTTCT 224 (CT)36 [23] R: GCACCTTGCTTATCATCCGA SSR125 6 F: TAGCGCCATTGTTCACACAC 156 (GA)39 [23] R: GCTGGGAGAGAAAGATGACTGT CPPCT022 7 F: CAATTAGCTAGAGAGAATTATTG 240 (CT)28 CAA (CT)20 [22] R: GACAAGAAGCAAGTAGTTTG CPPCT033 7 F: TCAGCAAACTAGAAACAAACC 151 (CT)16 [22] R: TTGCAATCTGGTTGATGTT BPPCT041 8 F: CAATAAGGCATTTGGAGGC 220 (AG)21 [7] R: CAGCCGAACCAAGGAGAC CPPCT006 8 F: AATTAACTCCAACAGCTCCA 188 ~ 219 (CT)16 [22] R: ATGGTTGCTTAATTCAATGG SSR93 8 F: AACTGCCTTAGCTTAGACTGGCT 160 (AG)10 [23] R: AAGACGAGAAACCACCTTGAATC
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表 3 29对SSR引物扩增效率及多态性分析
Table 3. Amplification efficiency and polymorphism analysis of 29 pairs of SSR primers
引物编号
Primer No.多态性等位基因观测数
Number of polymorphic allele observation
(Na)有效等位基因数
Effective number of alleles (Ne)Shannon’s信息指数
Shannon’s information index (I)观察杂合度
Observed heterozygosity (Ho)预期杂合度
Expected heterozygosity (He)多态性信息指数
Polymorphism information
index (PIC)BPPCT017 11 6.964 2.120 0.318 0.876 0.841 CPPCT015 11 6.676 2.126 0.227 0.870 0.835 BPPCT001 9 5.661 1.938 0.409 0.843 0.803 SSR96 11 5.261 1.994 0.500 0.829 0.792 BPPCT022 10 5.042 1.884 0.227 0.820 0.778 SSR152 10 4.676 1.859 0.546 0.804 0.764 BPPCT034 8 4.820 1.762 0.381 0.812 0.764 SSR125 10 4.302 1.811 0.333 0.786 0.743 SSR169 9 4.420 1.732 0.400 0.794 0.742 SSR107 7 4.127 1.628 0.333 0.779 0.725 CPPCT026 7 4.067 1.588 0.455 0.772 0.717 BPPCT026 5 3.660 1.432 0.191 0.745 0.683 BPPCT037 6 3.646 1.451 0.250 0.757 0.679 BPPCT025 9 3.350 1.602 0.409 0.718 0.676 CPPCT006 6 3.505 1.374 0.263 0.734 0.660 BPPCT028 6 3.421 1.403 0.227 0.724 0.659 CPPCT002 5 3.238 1.289 0.618 0.707 0.634 BPPCT023 4 3.133 1.213 0.227 0.697 0.618 SSR179 5 2.892 1.291 0.476 0.670 0.612 BPPCT007 7 2.985 1.348 0.250 0.682 0.609 SSR181 4 2.788 1.140 0.100 0.658 0.571 BPPCT042 4 2.749 1.146 0.450 0.653 0.570 BPPCT020 5 2.499 1.149 0.476 0.614 0.550 UDP96-018 6 2.361 1.183 0.318 0.590 0.540 SSR93 8 2.156 1.195 0.136 0.549 0.510 BPPCT015 3 2.399 0.951 0.053 0.599 0.496 SSR73 3 2.072 0.794 0.118 0.533 0.411 CPPCT005 4 1.296 0.507 0.188 0.236 0.220 BPPCT014 4 1.278 0.478 0.191 0.223 0.208 平均值
Average6.793 3.636 0.675 1.427 0.313 0.692
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表 4 22份观赏桃新种质在6个SSR位点的指纹图谱
Table 4. Fingerprinting of 22 ornamental peach germplasm at 6 SSR loci
bp 种质
GermplasmBPPCT017 BPPCT025 BPPCT034 CPPCT015 SSR125 SSR152 ‘T20’ 191/191 193/193 245/245 213/213 163/165 256/258 ‘T22’ 191/193 193/193 245/245 213/213 163/165 256/258 ‘T28’ 171/171 193/211 243/245 215/215 167/169 270/270 ‘T10’ 179/189 193/211 231/243 211/215 147/147 268/270 ‘T13’ 169/169 207/207 245/245 223/223 147/157 246/268 ‘T9-1’ 179/179 211/211 231/243 211/215 181/181 268/268 ‘早花’
‘Zaohua’179/187 193/193 231/243 215/215 167/169 256/258 ‘二色’
‘Erse’193/193 193/193 245/245 213/213 163/165 266/272 ‘寒红’
‘Hanhong’193/199 193/199 247/247 221/221 167/169 270/270 ‘京舞子’
‘Kyou-maiko’187/187 191/191 247/247 257/257 169/169 274/274 ‘撒金’
‘Sajin’171/171 209/209 241/243 213/213 169/169 256/256 ‘菊花桃’
‘Kikoumomo’189/189 191/191 247/253 257/257 169/169 270/274 ‘科林斯粉’
‘Corinthian pink’181/189 193/193 247/247 215/215 167/167 270/270 ‘绿萼子’
‘Lü e’187/187 193/207 247/247 247/261 167/167 246/270 ‘品霞’
‘Pinxia’173/189 193/193 245/253 257/257 169/169 270/270 ‘陕甘山桃’
‘Shangan mountain peach’161/173 171/197 223/223 223/223 133/149 232/242 ‘寿红’
‘Shouhong’171/171 193/193 243/243 255/255 169/169 270/270 ‘台阶’
‘Taijie’193/193 193/193 245/245 213/213 165/165 256/258 ‘五宝垂’
‘Wubao chui’187/187 193/207 247/247 247/263 169/169 246/270 ‘五宝桃’
‘Wubao tao’171/171 207/209 241/243 213/213 169/169 256/256 ‘云龙’
‘Unriumomo’171/171 205/207 231/231 249/249 177/177 270/270 ‘朱粉垂枝’
‘Zhufen chuizhi’187/187 193/207 227/247 247/261 167/167 246/270
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