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作物学报 ›› 2018, Vol. 44 ›› Issue (11): 1640-1649.doi: 10.3724/SP.J.1006.2018.01640

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于SSR分子标记的Nicotiana tobacum-N. plumbaginifolia异源染色体植株的鉴定与筛选

尚维1,赵申清玉1,党江波1,郭启高1,梁国鲁1,*(),杨超2,张艳2,陈益银2   

  1. 1 西南大学园艺园林学院, 重庆 400716
    2 中国烟草总公司重庆市公司烟草科学研究所, 重庆 400716
  • 收稿日期:2017-12-14 接受日期:2018-08-20 出版日期:2018-11-12 网络出版日期:2018-09-04
  • 通讯作者: 梁国鲁
  • 基金资助:
    本研究由中国烟草总公司重庆市公司科技项目(NY20170401070001)

Identification and Screening of Nicotiana tobacam-N. plumbaginifolia Heterologous Chromosome Plants Based on SSR Marker

Wei SHANG1,Shen-Qing-Yu ZHAO1,Jiang-Bo DANG1,Qi-Gao GUO1,Guo-Lu LIANG1,*(),Chao YANG2,Yan ZHANG2,Yi-Yin CHEN2   

  1. 1 College of Horticulture and Landscape, Southwest University, Chongqing 400716, China
    2 Chongqing Tobacco Research Institute, Chongqing Municipal Tobacco Company, Chongqing 400716, China
  • Received:2017-12-14 Accepted:2018-08-20 Published:2018-11-12 Published online:2018-09-04
  • Contact: Guo-Lu LIANG
  • Supported by:
    This study was supported by the Chongqing Science and Technology Project of China National Tobacco Corporation(NY20170401070001)

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摘要：

以烟草(Nicotiana tabacum)品种云烟87的八倍体(2n = 8x = 96)和野生烟草N. plumbaginifolia (2n = 2x = 20)的基因组DNA为模板, 对340对烟草SSR引物进行筛选以获得能扩增多态性条带的引物。利用多态性引物对种间杂交后代及190株回交后代的基因组DNA进行扩增, 并对N. plumbaginifolia中的SSR标记的连锁情况进行简要分析。经筛选获得了多态性引物29对。结果显示, 在190株后代中, 159株的基因组DNA能扩增出N. plumbaginifolia的特异SSR位点, 可以判定该159株为N. tabacum的N. plumbaginifolia异源染色体植株, 其余31株植株可能不含有N. plumbaginifolia的染色体。经UPGMA聚类分析, 本群体中植株的遗传多样性较为丰富, 部分分子标记在后代中的出现具有完全相关性。29个标记中14个可确定来源于5条不同染色体, N. plumbaginifolia的29个位点在回交后代中的扩增效率并不相同, 且效率均较低(低于31.00%), 说明该杂种中N. plumbaginifolia基因组的垂直传递效率较低。利用SSR分子标记可以判定云烟87八倍体与N. plumbaginifolia杂交获得的后代为真杂种, 且自该远缘杂种回交后代中筛选获得大量异源染色体植株。这些结果和筛选获得异源染色体植株为进一步创制N. tabacum-N. plumbaginifolia抗黑胫病单体附加系以及易位系奠定了基础。

关键词: Nicotiana tabacum, N. plumbaginifolia, SSR分子标记, 异源染色体, 黑胫病

Abstract:

The 340 pairs SSR primers were amplified to select polymorphic primers amplifying polymorphic bands, in which the genomic DNA of the octoploid of Nicotiana tabacum Yunyan 87 (2n = 8x = 96) and N. plumbaginifolia was extracted as a template. The polymorphic primers were used to amplify the genomic DNA of interspecific hybrids and 190 backcross progenies, and the linkage of SSR markers in N. plumbaginifolia was briefly analyzed. A total of 29 pairs of polymorphic primers. We found that N. plumbaginifolia specific SSR loci were amplified from 159 of the 190 BC1 plants. It was verified that 159 plants were N. tabacum-N. plumbaginifolia alien chromosome plants. By contrast, the other 31 plants might have no chromosomes of N. plumbaginifolia. Clustering analysis based on UPGMA indicated that the genetic diversity of the plants in BC1 population was relatively high, the appearance of some of the molecular markers in the offspring had a complete correlation. Fourteen in 29 markers could be identified from five chromosomes. The 29 N. plumbaginifolia specific loci were detected in BC1 plants in different rates, which were all lower than 31.00%, showing that vertical transmission rate of N. plumbaginifolia genome in the hybrid is low. The interspecies distant hybrids of Yunyan 87 octuploid (2n = 8x = 96) and N. plumbaginifolia could be identified as a true hybrid by using SSR marker, and a large number of N. tabacum-N. plumbaginifolia alien chromosome plants were screened out. All these results lay a foundation for production of black shank-resistant N. tabacum-N. plumbaginifolia monomer alien addition lines and alien translocations lines.

Key words: Nicotiana tabacum, N. plumbaginifolia, SSR marker, alien chromosomes, black shank

图1

材料的来源"

图2

部分引物筛选M: marker; ♂: N. plumbaginifolia; ♀: 云烟87八倍体。箭头所指示为能扩增出差异条带的引物。"

表1

可在云烟87、N. plumbaginifolia基因组DNA中扩增出差异条带的29对引物"

引物编号
Primer No.		 正向引物序列
Forward primer (5°-3')		 反向引物序列
Reverse primer (5°-3')
PT30061 TCGTCCATTTCTTTCTCTCTCA CATAAATAGTTGCTCATTCAATCG
PT30372 TGCACATGCTATGACGATTATCT GGTCATTGATCGCCAAGTTT
PT30138 AGTTGCAGGATTGTTCGCTT CGACTGCAAGAGTTGGCAAT
PT50199 CTCAATCAGCCAAATCCCTC TCTCCATTGTTAGAGTGAAGAGAAA
PT20213 TGTGGAGCTCCTTTCTTTGC TCAAATCAACAACAAATCCAAT
PT30482 CTTCTCTCTCCACCGCAGAC ACAGTTGGATATGGTGGCGT
PY50282 GCCAGCGAGAATGAGTGAGT TTTGTAGCCTCGGCTGATGT
PT50178 GATTGCAAAGGCGGTTACAT ACCGGACGGTCCAAACTAAG
PT50200 TTCTTCCGGTGATGTTCCTC TCTACACGTGTTCCTGTATCTGTG
PT50088 CAACAAATTGGAGTGGAGGG TGCACATCCTCAGCCTACAG
PT50022 AAACTTGCTTTCTTTGGACATC TTCATGTTGCAACGAATCCT
PT50090 CCTATATGATGAGTTGAATATTGCG TGCTCTATAATCATAACGTTGAAGAAG
PT50030 GGGCCAATCCGTGTAGAAAG CCCGATTTAGCACTTGATTGA
PT50254 TATAATCCCTCCCTGTGCCA TCGCGGTGAGATACAAAGAA
PT30412 CATTTAGCCGGGAACATTCA CATGGGATACACACGCAAAG
PT50051 AGATCGATCGGGTGAGTGAG TCACTCCACGCACAGAGAGT
PT40035 GAGGTGGAAGTCATCGGAAA CGTCTGTCATACACGCGAAA
PT50117 GCAGAATCGCAGATCCAGA GGGAGACAGTGGAGGTAGAGG
PT30167 TGATACAGAATATGGCGAACTTT CCGCTTCATCATTGAGGTTT
PT50225 TGTGTCCTCGTCAACTGTGG TTTGTTATGCACGCATTGCT
PT50243 CCCACTCTCTCTTGCCCATA TCAGAATAGCATGTGGGTGTG
PT30272 GAACCTAACCTCGCTCCACA AAATGGTAGCTGCGAGGAGA
PT50132 CATTCAGCACGCACCTAAGA ATGCTTCCAAACCTTTCTCG
PT30259 CAGCCAAGAGAACCCTTCAG GATTACCCTTCAAATGCCGA
PT30241 AAGTCTCGTGTGGTTGCTTT AAAGGGCAATGTGTCTAGCTC
PT50081 GTGACCGGAAGACGGTGAT CTTCCGTTGAAACTTCGCAC
PT50029 GATAAACTTGGTAGGTCCGGC AGGATGAGCAGGAGCATGAC

图3

部分引物扩增种间远缘杂种基因组DNA图谱M: marker; ♂: N. plumbaginifolia; ♀: 云烟87八倍体; (1): PT30138; (2): PT50178; (3): PT50090; (4): PT50117; (5): PT50030; (6): PT50132; (7): PT50199; (8): PT50200。细箭头为N. plumbaginifolia的 特异条带; 粗箭头为云烟87八倍体的特异条带。 "

图4

引物PT50178在云烟87、N. plumbaginifolia基因组DNA和部分回交后代中的扩增情况 M: marker; 箭头所指为N. plumbaginifolia扩增出的特异位点; 1~35为1~35号回交后代; ♂为N. plumbaginifolia; ♀为云烟87八倍体。"

表2

杂种回交后代N. plumbaginifolia位点的分布情况"

序号
No.		 扩增位点
Amount of loci		 植株数量
Number of plants		 比例
Rate (%)
1 0 31 16.32
2 1 26 13.68
3 2 18 9.47
4 3 36 18.95
5 4 23 12.11
7 6 14 7.37
8 7 8 4.21
9 8 5 2.63
10 9 5 2.63
11 10 2 1.05
12 11 1 0.53
13 12 2 1.05
14 13 1 0.53
15 14 1 0.53
合计Total 91 190 100.00

附图1

190株六倍体后代的聚类图。"

图5

第5个相关标记群的引物在部分后代中的扩增情况"

表3

29个N. plumbaginifolia位点在回交后代植株中的分布情况"

引物/位点
Primer/locus		 扩增植株数量
Plants number		 所占比例
Proportion (%)		 引物/位点
Primer/locus		 扩增植株数量
Plants number		 所占比例
Proportion (%)
PT30061 2 1.05 PT30241 19 10.00
PT20213 2 1.05 PT50029 24 12.63
PT30412 5 2.63 PT50199 24 12.63
PT50022 6 3.16 PT50243 28 14.74
PT50051 7 3.68 PT40035 34 17.89
PT50225 12 6.32 PT30272 34 17.89
PT30259 12 6.32 PT50081 34 17.89
PT30361 15 7.89 PT30138 34 17.89
PT50200 16 8.42 PT50178 34 17.89
PT50254 16 8.42 PT50030 38 20.00
PT30167 16 8.42 PT50117 38 20.00
PT30146 16 8.42 PT50132 38 20.00
PT50088 18 9.47 PY50282 39 20.53
PT30482 19 10.00 PT50090 58 30.53
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