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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (11): 1640-1649.doi: 10.3724/SP.J.1006.2018.01640

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2018-11-12 Published:2018-09-04
  • Contact: Guo-Lu LIANG E-mail:lianggl@swu.edu.cn
  • Supported by:
    This study was supported by the Chongqing Science and Technology Project of China National Tobacco Corporation(NY20170401070001)

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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

Fig. 1

Origin of the materials"

Fig. 2

Primers selectionM: marker; ♂: N. plumbaginifolia; ♀: Yunyan 87 octaploid. Arrows indicate primers which can amplify different loci."

Table 1

Sequences of 29 pairs of SSR primer which can amplify different bonds from Yunyan87 genomic DNA and N. plumbaginifolia genomic DNA"

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

Fig. 3

Filial generation genomic DNA map amplified by part primers M: marker; ♂: N. plumbaginifolia; ♀: Yunyan 87 octaploid; (1): PT30138; (2): PT50178; (3): PT50090; (4): PT50117; (5): PT50030; (6): PT50132; (7): PT50199; (8): PT50200. Thin arrow: loci of N. plumbaginifolia; thick arrow: loci of Yunyan 87 octaploid."

Fig. 4

Genomic DNA amplified by primer PT50178 in Yunyan 87, N. plumbaginifolia, and some backcross plants M: marker; Arrows indicate N. plumbaginifolia amplificatory specific loci; 1-35 indicate 1-35 BC1 plants; ♂: N. plumbaginifolia; ♀: Yunyan 87 octaploid."

Table 2

Distribution of N. plumbaginifolia loci in backcross plants"

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

Supplementary fig. 1

Dendiagram of 190 hexaploid progenies"

Fig. 5

Amplification in part hybrid backcross progenies by fifth linkage group primersM: marker; A: PT30146; B: PT30167; C: PT50200; D: PT50254."

Table 3

Distribution of 29 N. plumbaginifolia loci in BC1 plants"

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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