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Construction, characterization, and screening of a transformation-competent artificial chromosome library of peach

  • Genetic Resources
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Abstract

As a genome model of fruit trees, peach (Prunus persica [L.] Batch) has advantages for studying structural and functional genomics. Okubo, a traditional peach variety used as a parent in Asian peach breeding, displays economically valuable agronomic traits. To develop an efficient platform for peach gene cloning and genomic research, a large-insert genomic DNA library of Okubo was constructed in a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, which can accept and stably maintain large genomic DNA fragments in bothEscherichia coli andAgrobacterium tumefaciens. The TAC library contains 41,472 recombinant clones with an average insert size of approximately 42 kb, and it is equivalent to 6 haploid peach genomes. The TAC library was stored in 2 ways: one copy as frozen cultures in 108 pieces of 384-well plates and another copy as bulked pools in 36 pieces of 96-well plates, each well containing 12 individual clones. The lack of hybridization signal to chloroplast and mitochondrial genes indicated that the TAC library had no significant cytoplast organelle DNA contamination. TAC clones were stable inE. coli cells until generation 100 and stable in bothE. coli andA. tumefaciens. Twenty-one clones containing the polygalacturonase-inhibiting protein (PGIP) gene were detected by using pooled PCR in the TAC library. Positive clones can be used for peach PGIP gene cloning and functional analysis. The library is well suited for gene cloning and genetic engineering in peach.

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Abbreviations

BAC:

bacterial artificial chromosome

CIAP:

calf intestinal alkaline phosphatase

EDTA:

ethylenediaminetetraacetic acid

HB:

homogenization buffer

HMW:

high molecular weight

LMP:

low melting point

ORF:

open reading frame

PFGE:

pulsed field gel electrophoresis

PG:

polygalacturonase

PGIP:

polygalacturonase-inhibiting protein

PMSF:

phenylmethyl sulfonyl fluoride

TBE:

buffer of Tris-base, boric acid, and EDTA

TAC:

transformation-competent artificial chromosome

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Authors and Affiliations

  1. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101, Beijing, China

    Feng Shan Liang, Zhan Wang Yu, Ji Liang Yang, Jin De Min & Bin Wang

  2. Forestry and Fruit Research Institute, Academy of Agriculture and Forestry Science of Beijing City, 100093, Beijing, China

    Kai Chun Zhang & Xiao Ming Zhang

  3. College of Agronomy, Hebei Agricultural University, 071001, Baoding, China

    Feng Shan Liang

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  1. Feng Shan Liang
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  2. Kai Chun Zhang
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Liang, F.S., Zhang, K.C., Yu, Z.W. et al. Construction, characterization, and screening of a transformation-competent artificial chromosome library of peach. Plant Mol Biol Rep 22, 37–48 (2004). https://doi.org/10.1007/BF02773347

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