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He H.-G.,Jiangsu University | Bie T.-D.,Yangzhou Academy of Agricultural science | Zhou M.-X.,Jiangsu University | Wu S.-F.,Jiangsu University | And 2 more authors.
Biologia (Poland)

T-vectors play an important role in cloning of polymerase chain reaction products. In the present study, a novel pUEG-T vector was developed using enhanced green fluorescent protein (EGFP) as an indicator. To improve EGFP-based green-white screening, the lipoprotein mutant promoter, a strong constitutive promoter, was utilized to control the expression of egfp gene. Two other efficient expression elements, the ColE1 replication origin of pUC18 and the expression cassette of pET-28a, were also integrated into pUEG-T vector. Expression analysis demonstrated the efficient accumulation of active EGFPs in Escherichia coli DH5α cells carrying the T-vector precursor pUEG. In T-A cloning using pUEG-T vector, white colonies containing foreign DNA and green colonies having no insertion could be handily distinguished under normal white light, without any chemical inducer or chromogenic substrate. Furthermore, no false positive was observed in any of the tested white colonies. This proves that pUEG-T is an inexpensive, convenient and efficient T-vector. © 2013 Versita Warsaw and Springer-Verlag Wien. Source

Pu J.,Nanjing Agricultural University | Wang Q.,Nanjing Agricultural University | Shen Y.,Nanjing Agricultural University | Zhuang L.,Nanjing Agricultural University | And 5 more authors.
Theoretical and Applied Genetics

Key message: Gamma radiation induced a series of structural aberrations involvingThinopyrum bessarabicumchromosome 4J. The aberrations allowed for deletion mapping of 101 4J-specific markers and fine mapping of blue-grained geneBaThb. Abstract: Irradiation can induce translocations and deletions to assist physically locating genes and markers on chromosomes. In this study, a 12-Gy dosage of 60Co-γ was applied to pollen and eggs of a wheat (Triticum aestivum) landrace Chinese Spring (CS)–Thinopyrum bessarabicum chromosome 4J disomic addition line (DA4J), and the gametes from irradiated plants were fertilized with normal CS eggs or pollen to produce M1 seeds. Based on genomic in situ hybridization analysis of 261 M1 plants, we identified 74 lines carrying structural aberrations involving chromosome 4J with the higher aberration rate in treated pollen (31.2 %) than in the treated eggs (21.3 %). We further identified 43 (53.8 %) lines with structural aberrations on chromosome 4J by analyzing another 80 M1 plants with 74 4J-specific markers, indicating that combining molecular and cytological methods was more efficient for detecting chromosome aberrations. Marker analysis thus was performed prior to cytogenetic identification on M2–M4 seeds to detect chromosome structural aberrations. Sixty-eight M3 lines with structural aberrations on chromosome 4J and six previously obtained chromosome 4J alien lines were then analyzed using 101 chromosome 4J-specific markers. After combining marker results with chromosome aberrations in each line, chromosome 4J was physically divided into 24 segmental blocks with 7 in the short arm and 17 in the long arm. The blue-grained gene BaThb was further mapped into the region corresponding to block 4JL-11. The chromosome aberrations and the physical map developed in this research provide useful stocks and tools for introgression of genes on chromosome 4J into wheat. © 2015, Springer-Verlag Berlin Heidelberg. Source

He H.,Jiangsu University | Zhu S.,Jiangsu University | Wang W.,Jiangsu University | Bie T.,Yangzhou Academy of Agricultural science | Chen P.,Nanjing Agricultural University
African Journal of Biotechnology

A differentially expressed fragment EST145 was isolated by suppression subtractive hybridization (SSH) method. Using EST145 as the probe, a blue copper-binding protein gene designated as DvBCB was screened from Dasypyrum villosum cDNA Library. The DvBCB gene was 845 bp in length with an open reading frame (ORF) which encoded a 178-amino acid polypeptide and contained the deduced functional sites: H 66, C 107, H 112 and M 121. Northern blot analysis showed that, the expression of DvBCB gene was enhanced in leaves after inoculation with Erysiphe graminis; reached a peak level at 24 h and decreased to constitutive level at 72 h after inoculation in resistant Gh21 line. The expression level in susceptible mutant M14S line was slightly lower than that in the resistant Gh21 line at all stages after inoculation, and the peak could not appear in M14S line. The function of DvBCB gene might include lignification of cell wall or scavenging of reactive oxygen species (ROS) during powdery mildew attack. © 2011 Academic Journals. Source

Bie T.,Yangzhou Academy of Agricultural science | Zhao R.,Yangzhou Academy of Agricultural science | Jiang Z.,Yangzhou Academy of Agricultural science | Gao D.,Yangzhou Academy of Agricultural science | And 2 more authors.
Molecular Breeding

Wild relatives of wheat possess many agronomic traits important to wheat improvement. Wheat–alien translocation and deletion lines are important genetic stocks in wheat breeding or physical mapping of important alien genes. However, screening for chromosomal structural changes by conventional cytogenetic analyses is time-consuming work. It is necessary to develop an efficient method of finding alien chromosomal structural changes. In this research, two amplicon markers, 6VS-381 and 6VL-358, specific to the distal regions of the short and long arms, respectively, of Haynaldia villosa chromosome 6V, were developed based on the expressed sequence tags located on the distal regions of wheat group 6. Marker-assisted screening work was then carried out in two individual radiation-induced populations. The first contained 365 plants involving 43 M1:2 families derived from a pollen irradiation treatment of a wheat–H. villosa monosomic 6V addition line. The other was a M1 population containing 100 plants derived from an irradiation treatment of a wheat–H. villosa disomic 6V(6A) substitution line. The female parent in both treatments was a common wheat cultivar Chinese Spring. Those plants marked by a single positive distal marker were considered to be putative structural changes of 6V. After cytogenetic analysis, a total of 20 structure-changed chromosomes were identified, comprising 12 whole-arm translocations, four terminal translocations, one 6VL terminal deletion, one translocation deletion, and two intercalary translocations. The double-distal-marker strategy proposed in this study gives an efficient model for finding structural aberrations involving a specific alien chromosome. © 2015, Springer Science+Business Media Dordrecht. Source

Bie T.,Yangzhou Academy of Agricultural science | Zhao R.,Yangzhou Academy of Agricultural science | Zhu S.,Jiangsu University | Chen S.,Yangzhou Academy of Agricultural science | And 8 more authors.
Molecular Breeding

Wheat-Haynaldia villosa translocations T6V#2S.6AL and T6V#4S.6DL, carriers of Pm21 and PmV, respectively, continue to contribute powdery mildew resistance in wheat varieties in China. Based on colinearity between Brachypodium distachyon and Triticeae species, genomic sequences from gene intervals of B. distachyon physically linked to a homolog of Stpk-V, a key part of Pm21, were used to design primers and screen for codominant and stable markers. An anonymous marker, MBH1, tagging both Pm21 in 6V#2S and PmV in 6V#4S was identified. It also distinguished wheat 6AS- and 6DS-derived homoeoalleles of MBH1. Sequence length comparisons showed a decreasing order of 6AS > 6DS > 6V#2S > 6V#4S. F2 segregation analyses were conducted on crosses Yangmai 18 (T6V#2S.6AL)/Yangmai 19 and Yangmai 16/Yangmai 22 (T6V#4S.6DL). MBH1 detected homozygosity of both Pm21 and PmV in segregating populations. The results also showed that T6V#2S.6AL is more transmittable than T6V#4S.6DL. Using MBH1, we reconfirmed the identities of powdery mildew resistance genes in Yangmai 21, Zhenmai 9, and Yangmai 97G59. The resistance donor to all three was T6V#2S.6AL, indicating that the published pedigrees were not correct. Pm21 in NAU419-Y15, an intercalary translocation carrying Pm21, was also tagged by MBH1. MBH1 is a broadly applicable marker for selecting and distinguishing the translocation chromosomes carrying Pm21 and PmV, which continue to be effective in China. © 2015, Springer Science+Business Media Dordrecht. Source

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