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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (6): 869-877.doi: 10.3724/SP.J.1006.2020.94110

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

Construction and characterization of a BAC library for flue-cured tobacco line with high resistance to blank shank

DONG Qing-Yuan1,4,MA De-Qing1,4,YANG Xue2,LIU Yong1,HUANG Chang-Jun1,YUAN Cheng1,FANG Dun-Huang1,YU Hai-Qin1,TONG Zhi-Jun1,SHEN Jun-Ru3,XU Yin-Lian5,LUO Mei-Zhong2,LI Yong-Ping1,ZENG Jian-Min1,*()   

  • Received:2019-07-29 Accepted:2020-01-15 Online:2020-06-12 Published:2020-01-24
  • Contact: Jian-Min ZENG E-mail:zengjm2017@163.com
  • Supported by:
    Grants of Yunnan Province Tobacco Company(2017YN04);Grants of Yunnan Province Tobacco Company(2019530000241004)

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Abstract:

Tobacco (N. tabacum) is an important model crop in molecular biology research. In this study, a bacterial artificial chromosome (BAC) library of a flue-cured tobacco line 14-60 with high blank shank resistance and good quality was constructed. High molecular weight DNA was isolated using intact nuclei from tobacco, partially cleaved with Hind III and cloned into the pIndigoBAC536-S vector. The BAC library consisted of 414,720 clones arrayed in one thousand and eighty 384-microtite plates, with an average insert size of 123 kb ranging from 97.0-145.5 kb. No empty insert clone was found. Based on an estimated genome size of 4500 Mb for common tobacco, the BAC library was estimated to cover 11 times of genome equivalents. The utility of the library was further confirmed by screening the library with the primers of tobacco hemA, NtFT, and eIF4E-1 genes. The high capacity library will serve as a giant genomic resource for map-based cloning of quantitative trait loci or genes associated with important agronomic and smoking quality traits or resistance to blank shank, physical mapping and comparative genome analysis.

Key words: tobacco, genomic DNA, bacterial artificial chromosome (BAC), gene screening

Fig. 1

Partial digestions of tobacco DNA plugs The first lane is the λ ladder PFG marker, lanes 2-9 are enzyme digested bands of half plug. The order of enzyme concentration is 0, 0.3, 0.5, 1.0, 1.5, 2.0, 4.0, and 10.0 U μL-1. The enzyme digestion time is 15 min. "

Fig. 2

First size selection of tobacco genomic DNA DNA was separated on 1% CHEF agarose gel at 1 s-50 s linear ramp, 6 V cm-1, 14℃ in 0.5×TBE buffer for 18 h. The marker is λ ladder PFG marker. "

Fig. 3

Second size selection of tobacco genomic DNA DNA fragments were separated on 1% CHEF agarose gel at 4 s-4 s linear ramp, 6 V cm-1, 14℃ in 0.5×TBE buffer for 18 h. The marker used is λ ladder PFG marker. "

Fig. 4

Concentration of recovered DNA fragments The loading volumes for the recovered DNA fractions a1, a2, b1, b2, and the standard λDNA solutions of 1, 2, 3, 4 ng μL-1 are 1 μL each. "

Table 1

Statistics of common tobacco BAC library"

名称
Name		 指标
Index
克隆数 Total clones 414720
384孔板数 Total 384-well plates 1080
载体 BAC vector pIndigoBAC536-S
感受态细胞 Competent cell DH10B
平均插入片段大小 Average insert size (kb) 123
烟草基因组覆盖度 Tobacco genome equivalent 11×
空载率 Insert-empty BACs (%) 0

Fig. 5

Insert detection of random tobacco BAC library clones (partially) It is the insert detection image of 40 randomly selected clones. Marker is λ ladder PFG. "

Fig. 6

PCR screening of Primary Mixing Pools 132-166 in No.2 plate (hemA) The marker is Trans2K DNA marker, and the bands from top to bottom represent 2000, 1000, 750, 500, 250, and 100 bp. "

Fig. 7

PCR Screening of Primary Mixing Pools 161-190 in No. 2 plate (eIF4E-1) The marker is Trans2K DNA marker, and the bands from top to bottom represent 2000, 1000, 750, 500, 250, and 100 bp. "

Fig. 8

PCR Screening of Primary Mixing Pools 898-927 in No. 9 plate (NtFT) The Marker is Trans2K DNA marker, and the bands from top to bottom represent 2000, 1000, 750, 500, 250, and 100 bp. "

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