Please wait a minute...
Journal of Integrative Agriculture  2017, Vol. 16 Issue (01): 57-64    DOI: 10.1016/S2095-3119(16)61446-6
Crop Genetics · Breeding · Germplasm Resources Advanced Online Publication | Current Issue | Archive | Adv Search |
Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus
GUO Xiao1*, YANG Xiao-hui1*, YANG Yu1*, MAO Zhen-chuan2, LIU Feng2, MA Wei-qing1, XIE Bing-yan2, LI Guang-cun1, 2
1 Institute of Vegetables and Flowers, Shandong Academy of Agricultural Sciences, Molecular Biology Key Laboratory of Shandong Facility Vegetable, Jinan 250100, P.R.China
2 Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China
Export:  BibTeX | EndNote (RIS)      
Abstract  Pepper (Capsicum annuum. L.) is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China.  Pepper root-knot nematode (Meloidogyne spp.) is one of the most serious pests of pepper, which caused huge losses every year.  Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita.  HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chromosome library (BAC) via the vector of CopyControlTM pCC1 in this study.  The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes.  The average insert size is 95 kb, and most of them are 90–120 kb; but the empty clones are less than 3%.  In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study.  Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained.  Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively.  Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.
Keywords:  pepper      bacterial artificial chromosome library (BAC)      root-knot nematode      Me3 gene      contig  
Received: 01 December 2015   Accepted: 08 January 2017

This work was supported by the National High-Tech R&D Program in China (2013AA102603), the Natural Science Foundation of Shandong Province, China (ZR2014YL014), the Youth Scientific Research Foundation of Shandong Academy of Agricultural Sciences, China (2014QNZ03), the Taishan Scholars Program of Shandong Province, China (2016-2020), and the National Natural Science Foundation of China (31101425).

Corresponding Authors:  Correspondence XIE Bing-yan, Tel: +86-10-82109546, Fax: +86-10-62174123, E-mail:; LI Guang-cun, Tel: +86-10-82105955, Fax: +86-10-62174123, E-mail:    

Cite this article: 

GUO Xiao, YANG Xiao-hui, YANG Yu, MAO Zhen-chuan, LIU Feng, MA Wei-qing, XIE Bing-yan, LI Guang-cun. 2017. Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus. Journal of Integrative Agriculture, 16(01): 57-64.

Ariyadasa R, Stein N. 2012. Advances in BAC-based physical mapping and map integration strategies in plants. Journal of Biomedicine and Biotechnology, 23, 1-11.

Biradar S S, Nie X, Feng K, Weining S. 2014. Preparation of high molecular weight gDNA and bacterial artificial chromosome (BAC) libraries in plants. Methods in Molecular Biology, 1099, 41–63.

Bleve-Zacheo T, Bongiovanni M, Melillo MT, Castagnone-Sereno P. 1998. The pepper resistance genes Me1 and Me3 induce differential penetration rates and temporal sequences of root cell ultrastructural changes upon nematode infection. Plant Science, 133, 79–90.

Chalhoub B, Belcram H, Caboche M. 2004. Efficient cloning of plant genomes intobacterial artificial chromosome (BAC) libraries with larger and more uniform insert size. Plant Biotechnology Journal, 2, 181–188.

Djian-Caporalino C, Fazari A, Arguel M J, Vernie T, Vande Casteele C, Faure I, Brunoud G, Pijarowski L, Palloix A. 2007. Root-knot nematode (Meloidogyne spp.) Me resistance genes in pepper (Capsicumannuum L.) are clustered on the P9 chromosome. Theoretical and Applied Genetics, 114, 473–486.

Djian-Caporalino C, Pijarowski L, Fazari A, Samson M, Gaveau L, Byrne C O, Lefebvre V, Caranta C, Palloix A, Abad P. 2001. High-resolution genetic mapping of the pepper (Capsicum annuum L.) resistance loci Me3 and Me4 conferring heat-stable resistance to root-knot nematodes (Meloidogyne spp.). Theoretical and Applied Genetics, 103, 592–600.

Djian-Caporalino C, Pijarowski L, Januel A, Lefebvre V, Daubèze A, Palloix A, Dalmasso A, Abad P. 1999. Spectrum of resistance to root-knot nematodes and inheritance of heat-stable resistance in pepper (Capsicum annuum L.). Theoretical and Applied Genetics, 99, 496-502.

Hendy H, Dalmasso A, Cardin M C. 1985. Differences in resistant Capsicum annuum attacked by different Meloidogyne species. Nematologica, 31, 72–78.

Kim H J, Nahm S H, Lee H R, Yoon G B, Kim K T, Kang B C, Choi D, Kweon O Y, Cho M C, Kwon J K, Han J H, Kim J H, Park M K, Ahn J H, Choi S H, Her N H, Sung J H, Kim B D. 2008. BAC-derived markers converted from RFLP linked to Phytophthoracapsici resistance in pepper (Capsicum annuum L.). Theoretical and Applied Genetics, 118, 15–27.

Kruijt M, Brandwagt B F, de Wit P J G M. 2004. Rearrangements in the Cf-9 disease resistance gene cluster of wild tomato have resulted in three genes that mediate Avr9 responsiveness. Genetics, 168, 1655-1663.

Ma Z, Weining S, Sharp P J, Liu C. 2000. Non-gridded library: a new approach for BAC (bacterial artificial chromosome) exploitation in hexaploid wheat (Triticumae stivum). Nucleic Acids Research, 28, E106.

Mao Z C, Xie B Y, Yang Y H, Feng D X, Feng L X, Yang Z W. 2007. Analysis of pepper Me3 gene resistance to root knot nematodes by SSH. Acta Horticulturae Sinica, 34, 1453–1458. (in Chinese)

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, 1–8.

Pegard A, Brizzard G, Fazari A, Soucaze O, Abad P, Djian-Caporalino C. 2005. Histological characterization of resistance to different root-knot nematode species related to phenolics accumulation in Capsicum annuum. Phytopathology, 95, 158–165.

 Qin C, Yu C, Shen Y, Fang X, Chen L, Min J, Cheng J,

Zhao S, Xu M, Luo Y, Yang Y, Wu Z, Mao L, Wu H, Ling-Hu C, Zhou H, Lin H, González-Morales S, Trejo-Saavedra DL, Tian H, et al. 2014. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization. Proceedings of the National Academy of Sciences of the United States of America, 111, 5135-5140.

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. Proceedings of the National Academy of Sciences of the United States of America, 89, 8794–8797.

Sierro N, van Oeveren J, van Eijk M J, Martin F, Stormo K E, Peitsch M C, Ivanov N V. 2013. Whole genome profiling physical map and ancestral annotation of tobacco Hicks Broadleaf. The Plant Journal, 75, 880–889.

Teresa B Z, Michel B, Maria T M, Philippe C S. 1998. The pepper resistance genes Me1 and Me3 induce differential penetration rates and temporal sequences of root cell ultrastructural changes upon nematode infection. Plant Science, 133, 79–90.

Xu X, Liu F, Kang H, Zhang Z, Zou X, Xie B. 2011. Fine mapping of the root-knot nematode resistance gene Me3 in pepper. Acta Horticulturae Sinica, 38, 288–294. (in Chinese)

Yang Y, Yang X H, Li C H, Guo X, Shan W W, Ma W Q, Huang S W, Li G C. 2015. Construction and characterization of a bacterial artificial chromosome library of potato cultivar C88. Acta Horticulturae Sinica, 42, 361–366. (in Chinese)

Yoo E Y, Kim S, Kim J Y, Kim B D. 2001. Construction and characterization of a bacterial artificial chromosome library from chili pepper. Molecular Cells, 12, 117–120.

Yoo E Y, Kim S, Kim Y H, Lee C J, Kim B D. 2003. Construction of a deep coverage BAC library from Capsicum annuum ‘CM334’. Theoretical and Applied Genetics, 107, 540–543.

Zhang C Z, Liu L, Wang X X, Vossen J, Li G C, Li T, Zheng Z, Gao J C, Guo Y M, Visser R, Li J M, Bai Y L, Du Y C. 2014. The Ph-3 gene from Solanum pimpinellifolium encodes CC-NBS-LRR protein conferring resistance to Phytophthora infestans. Theoretical and Applied Genetics, 127, 1353–1364.

Zhang M, Zhang Y, Scheuring C F, Wu C C, Dong J J, Zhang H B. 1995. Preparation of megabase-sized DNA from plant nuclei. The Plant Journal, 7, 175–184.
[1] YIN Yue-yan, HUA Meng-ying, ZHAO Kuang-jie, WAN Qiong-lian, BU Shan, LU Yu-wen, ZHENG Hong-ying, RAO Shao-fei, YAN Fei, PENG Jie-jun, CHEN Hai-ru, CHEN Jian-ping. Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre/loxP system[J]. >Journal of Integrative Agriculture, 2022, 21(8): 2456-2463.
[2] CHENG Wan-li, ZENG Li, YANG Xue, HUANG Dian, YU Hao, CHEN Wen, CAI Min-min, ZHENG Long-yu, YU Zi-niu, ZHANG Ji-bin. Preparation and efficacy evaluation of Paenibacillus polymyxa KM2501-1 microbial organic fertilizer against root-knot nematodes[J]. >Journal of Integrative Agriculture, 2022, 21(2): 542-551.
[3] Hakan FIDAN, Pelin SARIKAYA, Kubra YILDIZ, Bengi TOPKAYA, Gozde ERKIS, Ozer CALIS. Robust molecular detection of the new Tomato brown rugose fruit virus in infected tomato and pepper plants from Turkey[J]. >Journal of Integrative Agriculture, 2021, 20(8): 2170-2179.
[4] WU Li-hong, ZHOU Cao, LONG Gui-yun, YANG Xi-bin, WEI Zhi-yan, LIAO Ying-jiang, YANG Hong, HU Chao-xing . Fitness of fall armyworm, Spodoptera frugiperda to three solanaceous vegetables[J]. >Journal of Integrative Agriculture, 2021, 20(3): 755-763.
[5] Pornthip RUANPANUN, Prakit SOMTA. Identification and resistant characterization of legumes sources against Meloidogyne incognita #br#[J]. >Journal of Integrative Agriculture, 2021, 20(1): 168-177.
[6] HUANG Bin, WANG Qian, GUO Mei-xia, FANG Wen-sheng, WANG Xiao-ning, WANG Qiu-xia, YAN Dong-dong, OUYANG Can-bin, LI Yuan, CAO Ao-cheng. The synergistic advantage of combining chloropicrin or dazomet with fosthiazate nematicide to control root-knot nematode in cucumber production[J]. >Journal of Integrative Agriculture, 2019, 18(9): 2093-2106.
[7] TU Ke-ling, LI Lin-juan, YANG Li-ming, WANG Jian-hua, SUN Qun. Selection for high quality pepper seeds by machine vision and classifiers[J]. >Journal of Integrative Agriculture, 2018, 17(09): 1999-2006.
[8] SUI Xiao-lei, MAO Sheng-li, WANG Li-hao, ZHANG Bao-xi, ZHANG Zhen-xian. Effect of Low Light on the Characteristics of Photosynthesis and Chlorophyll a Fluorescence During Leaf Development of Sweet Pepper[J]. >Journal of Integrative Agriculture, 2012, 12(10): 1633-1643.
[9] GUO Shuang, MA Ning, YANG Wen-cai, SUN Yu-jie, SHEN Huo-lin. Expression Analysis of Restorer Alleles-Induced Genes in Pepper[J]. >Journal of Integrative Agriculture, 2011, 10(7): 1010-1015.
[10] HUANG Huan-huan, ZHANG Zhong-hua, ZHANG Zheng-hai, MAO Sheng-li, WANG Li-hao , ZHANG Bao-xi. Analysis of SSRs Information in Capsicum spp. from EST Database[J]. >Journal of Integrative Agriculture, 2011, 10(10): 1532-1536.
No Suggested Reading articles found!