PCR-based screening of BAC clones of different chromosomes in Chinese cabbage

doi:10.1007/s11703-011-1122-2

Front Agric Chin

2011, Vol. 5

Issue (4) : 524-528 https://doi.org/10.1007/s11703-011-1122-2

RESEARCH ARTICLE

PCR-based screening of BAC clones of different chromosomes in Chinese cabbage

Daling FENG^1,2, Shuxin XUAN¹, Aixia GU¹, Airu MA¹, Jiuhuan LI¹, Shuxing SHEN¹(

)

1. College of Horticulture, Agricultural University of Hebei, Baoding 071001, China; 2. College of Life Science, Agricultural University of Hebei, Baoding 071001, China

Download: PDF(118 KB) HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

Abstract

In this paper, taking SSR and functional gene sequence as the primers and the plasmid of first- and second-level pools of bacterial artificial chromosome (BAC) library as templates, the PCR method was used for specific clones of different chromosomes in Chinese cabbage. The results showed that the number of positive clones was 1–11 per primer and the average number of clone was 3.9 by screening 19200 clones of BAC library using 12 pairs of SSR primers from 10 linkage groups individually, which were nearly consistent with about 3.4 times of genome coverage. Positive clones were acquired in chromosome Nos. 2 to 5 and 8 to 10 without screening with the positive clones in chromosome Nos. 1, 6, and 7. In addition, the primer of FLC1 functional gene of chromosome No. 10 was used for PCR screening, and two BAC clones containing FLC1 gene were acquired. Therefore, different specific BAC clones of chromosomes were taken by using SSR primer and functional gene primer. Specific clone screening of chromosomes could provide a probe for identifying the chromosome accurately. Meanwhile, the BAC library screening method was optimized, serving as an effective technical means for quick BAC clone screening.

Keywords Chinese cabbage chromosome SSR primer mixing pool PCR screening

Corresponding Author(s): SHEN Shuxing,Email:shensx@hebau.edu.cn

Issue Date: 05 December 2011

Cite this article:

Daling FENG,Shuxin XUAN,Aixia GU, et al. PCR-based screening of BAC clones of different chromosomes in Chinese cabbage[J]. Front Agric Chin, 2011, 5(4): 524-528.

URL:

https://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1122-2
https://academic.hep.com.cn/fag/EN/Y2011/V5/I4/524

Tab.1 SSR primer

Fig.1 PCR-based screening of chromosome 2. M: Markers are 2000, 1000, 750, 500, 250, and 100 bp from top to bottom. A is first-level pool screening, B is second-level pool screening, and C is monoclonal screening.

Tab.2 BAC clones screening by different linkages of SSR primers

Fig.2 Positive BAC clone screening of chromosome 10. M are markers of 2000, 1000, 750, 500, 250, and 100 bp from top to bottom. A is first-level pool screening, B is second-level pool screening, and C is monoclonal screening.

1	Achenbach U C, Tang X, Ballvora A, de Jong H, Gebhardt C (2010). Comparison of the chromosome maps around a resistance hot spot on chromosome 5 of potato and tomato using BAC-FISH painting. Genome , 53(2): 103-110 doi: 10.1139/G09-086 pmid:20140028
2	Febrer M, Cheung F, Town C D, Cannon S B, Young N D, Abberton M T, Jenkins G, Milbourne D (2007). Construction, characterization, and preliminary BAC-end sequencing analysis of a bacterial artificial chromosome library of white clover (Trifolium repens L.). Genome , 50(4): 412-421 doi: 10.1139/G07-013 pmid:17546099
3	Giraudat J, Hauge B M, Valon C, Smalle J, Parcy F, Goodman H M (1992). Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell , 4(10): 1251-1261 pmid:1359917
4	Guan Y, Chen Q, Pan J S, Li Z, He H L, Wu A Z, Song R T, Cai R (2008). Isolation of linkage special clones and construction of BAC library in cucumber (Cucumis sativus L.). Progress in Natural Science , 18(2): 211-215 (in Chinese)
5	Guan Y, Chen Q, Pan J S, Li Z, He H L, Wu A Z, Song R T, Cai R (2008). BAC library building and screening of linkage specific clones on the study of Cucumis sativus L.. Prog Nat Sci , 18(2): 211-215 (in Chinese)
6	Huan P, Zhang X J, Li F H, Zhang Y, Zhao C, Liu B Z, Xiang J H (2009). Chromosomal localization and development of SNP markers of a serine protease gene in Farrer’s scallop (Chlamys farreri). Hereditas , 31(12): 1241-1247 (in Chinese) doi: 10.3724/SP.J.1005.2009.01241 pmid:20042392
7	Li X J (2010). Establishment and SSR identification of a few cabbage-Chinese cabbage alien addition lines. Dissertation for the Master Degree . Baoding: Agricultural University of Hebei, 24
8	Liu H B, Liu K, Wang J F, Ma R Z (2006). A BAC clone-based physical map of ovine major histocompatibility complex. Genomics , 88(1): 88-95 doi: 10.1016/j.ygeno.2006.02.006 pmid:16595171
9	Luo R, Wu W L, Zhang Y, Li Y H (2010). SSR Marker and its application to crop genetics and breeding. Genomics and Applied Biology , 29(1): 137-143 (in Chinese)
10	Mun J H, Kwon S J, Yang T J, Kim H S, Choi B S, Baek S, Kim J S, Jin M, Kim J A, Lim M H, Lee S I, Kim H I, Kim H, Lim Y P, Park B S (2008). The first generation of a BAC-based physical map of Brassica rapa. BMC Genomics , 9: 280 doi: 10.1186/1471-2164-9-280 pmid:18549474
11	Osoegawa K, Woon P Y, Zhao B, Frengen E, Tateno M, Catanese J J, de Jong P J (1998). An improved approach for construction of bacterial artificial chromosome libraries. Genomics , 52(1): 1-8 doi: 10.1006/geno.1998.5423 pmid:9740665
12	Schranz M E, Quijada P, Sung S B, Lukens L, Amasino R, Osborn T C (2002). Characterization and effects of the replicated flowering time gene FLC in Brassica rapa. Genetics , 162(3): 1457-1468 pmid:12454088
13	Shizuya H, Birren B, Kim U J, Mancino V, Slepak T, Tachiiri Y, Simon M (1992). Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci USA , 89(18): 8794-8797 doi: 10.1073/pnas.89.18.8794 pmid:1528894
14	Szinay D, Chang S B, Khrustaleva L, Peters S, Schijlen E, Bai Y, Stiekema W J, van Ham R C, de Jong H, Klein Lankhorst R M (2008). High-resolution chromosome mapping of BACs using multi-colour FISH and pooled-BAC FISH as a backbone for sequencing tomato chromosome 6. Plant J , 56(4): 627-637 doi: 10.1111/j.1365-313X.2008.03626.x pmid:18643986
15	Terol J, Naranjo M A, Ollitrault P, Talon M (2008). Development of genomic resources for Citrus clementina: Characterization of three deep-coverage BAC libraries and analysis of 46,000 BAC end sequences. BioMed Central Genomics, 9: 423
16	Xiong Z Y, Tan G X, You A Q, He G Y, She C W, Li L J, Song Y C (2004). Comparative physical positioning of BAC cloning linked with Glh, Bph-3 and xa-5 in cultivated rice and Oryza granulate. Chin Sci Bull , 49(3): 252-257 (in Chinese)

[1]	Chengjin GUO, Jincai LI, Wensuo CHANG, Lijun ZHANG, Xirong CUI, Shuwen LI, Kai XIAO. Effects of chromosome substitution on the utilization efficiency of nitrogen, phosphorus, and potassium in wheat[J]. Front Agric Chin, 2011, 5(3): 253-261.
[2]	Erqiao SHEN, Xiaofeng LI, Chenghe ZHANG, Xine WANG, Shuxin XUAN. Generation and identification of four new monosomic addition lines of flowering Chinese cabbage-Chinese kale[J]. Front Agric Chin, 2011, 5(2): 215-220.
[3]	WU Yuxiang, CHEN Chongqian, GAO Yanhui, ZHU Shuijin, JI Daofan. Effect of plant preservation on the fertility and chromosome configuration of a quadri-specific hybrid derived from 4 cultivated cotton species[J]. Front. Agric. China, 2008, 2(4): 380-385.
[4]	NIU Na, ZHANG Gaisheng, CAO Yue, ZHANG Yu, WEI Fang. Directional transduction of male sterile gene of NIAN type in wheat[J]. Front. Agric. China, 2008, 2(4): 386-390.
[5]	LIAO Wenhua, LIU Jianling, Wang Xinjun, JIA Ke, MENG Na. Effects of phosphate fertilizer and manure on Chinese cabbage yield and soil phosphorus accumulation[J]. Front. Agric. China, 2008, 2(3): 301-306.
[6]	SUN Feifei, HOU Xilin, LI Ying, CUI Xiumin. Molecular cloning and characterization of nitrate reductase gene cDNA from non-heading Chinese cabbage[J]. Front. Agric. China, 2007, 1(2): 188-192.
[7]	Jianyu YUAN,Xilin HOU,Changwei ZHANG,Fan YE. Active oxygen metabolism in the floral buds and leaves of the new cytoplasm male sterile (CMS) line and its maintainer line of non-heading Chinese cabbage[J]. Front. Agric. China, 2007, 1(1): 47-51.

Viewed

Full text

Abstract

Cited

Shared

Discussed