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Plant Molecular Biology Reporter

, Volume 22, Issue 1, pp 37–48 | Cite as

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

  • Feng Shan Liang
  • Kai Chun Zhang
  • Zhan Wang Yu
  • Ji Liang Yang
  • Xiao Ming Zhang
  • Jin De Min
  • Bin Wang
Genetic Resources

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.

Key words

genomic library Okubo peach Prunus persica TAC transformation-competent artificial chromosome 

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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Copyright information

© International Society for Plant Molecular Biology. Printed in Canada 2004

Authors and Affiliations

  • Feng Shan Liang
    • 1
    • 3
  • Kai Chun Zhang
    • 2
  • Zhan Wang Yu
    • 1
  • Ji Liang Yang
    • 1
  • Xiao Ming Zhang
    • 2
  • Jin De Min
    • 1
  • Bin Wang
    • 1
  1. 1.Institute of Genetics and Developmental BiologyChinese Academy of SciencesBeijingChina
  2. 2.Forestry and Fruit Research InstituteAcademy of Agriculture and Forestry Science of Beijing CityBeijingChina
  3. 3.College of AgronomyHebei Agricultural UniversityBaodingChina

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