[Construction, characterization and screening of a transformation-competent artificial chromosome (TAC) library of wheat-Thinopyrum intermedium translocation line with resistance to barley yellow dwarf virus].
A transformation-competent artificial chromosome (TAC) library was constructed from the genomic DNA of wheat-Th. intermidium translocation line HW642 that harbor the barley yellow dwarf virus (BYDV) resistance gene derived from Th. intermidium. The library consists of 2.3 x 10(6) clones with an average insert size of 22kb, representing approximately 2.5 haploid genome equivalents and is able to give a greater than 95.77% probability of isolating single-copy DNA sequences from this library. The library was stored as frozen cultures in 24 96-well formats, each well containing approximately 1000 different clones. TAC clones containing interest gene could be identified by the pooled PCR technique. A sequence characterized amplified region (SCAR) marker cosegregated with BYDV resistance gene, derived from a simple sequence repeat (SSR) or microsatellite marker wms37 of wheat, was applied to screen the TAC library. Twelve clones were successfully selected by the pooled PCR method. PCR products were identified by hybridizing with the SCAR marker band of Th. intermidium. Out of 12 clones, 10 positive clones restricted by Hind III were shown to hybridize with genomic DNA of Th. intermidium. These results showed evidences that the 10 clones could be used as candidate clones for isolation of BYDV resistance and its related genes, and the TAC library is a useful resource for isolating genes.
Key Laboratory of Crop Genetics and Breeding of Ministry of Agriculture, Institute of Crop Breeding and Cultivation, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Xiao-Ping
XP
Wang
Zeng-Yan
ZY
Zhang
Qun-Yu
QY
Zhang
Yao-Guang
YG
Liu
Zhi-Yong
ZY
Xin
D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D015698:Genomic Library; D007113:Immunity, Innate / Q000235:genetics; D017793:Luteovirus / Q000254:growth & development; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000235:genetics* / Q000821:virology; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic / Q000235:genetics; D014908:Triticum / Q000235:genetics* / Q000821:virology
D018744:DNA, Plant; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII