Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus

doi:10.1016/S2095-3119(16)61446-6

Journal of Integrative Agriculture ›› 2017, Vol. 16 ›› Issue (01): 57-64.DOI: 10.1016/S2095-3119(16)61446-6

收稿日期:2015-12-01 出版日期:2017-01-20 发布日期:2017-01-08

Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus

GUO Xiao^1*, YANG Xiao-hui1^*, YANG Yu^1*, MAO Zhen-chuan², LIU Feng², MA Wei-qing¹, XIE Bing-yan², LI Guang-cun^{1, 2}

¹Institute 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

Received:2015-12-01 Online:2017-01-20 Published:2017-01-08
Contact: Correspondence XIE Bing-yan, Tel: +86-10-82109546, Fax: +86-10-62174123, E-mail: xiebingyan@caas.cn; LI Guang-cun, Tel: +86-10-82105955, Fax: +86-10-62174123, E-mail: liguangcun@caas.cn
Supported by:
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).

摘要/Abstract

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 CopyControl^TM 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.

Key words: pepper, bacterial artificial chromosome library (BAC), root-knot nematode, Me3 gene, contig

GUO Xiao, YANG Xiao-hui, YANG Yu, MAO Zhen-chuan, LIU Feng, MA Wei-qing, XIE Bing-yan, LI Guang-cun. [J]. Journal of Integrative Agriculture, 2017, 16(01): 57-64.

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

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Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus

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