Huagang He

Efficient marker-assisted screening of structural changes involving Haynaldia villosa chromosome 6V using a double-distal-marker strategy

Abstract 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.

Pm21, Encoding a Typical CC-NBS-LRR Protein, Confers Broad-Spectrum Resistance to Wheat Powdery Mildew Disease

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Map-based cloning of the gene Pm21 that confers broad spectrum resistance to wheat powdery mildew

Common wheat (Triticum aestivum L.) is one of the most important cereal crops. Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a continuing threat to wheat production. The Pm21 gene, originating from Dasypyrum villosum, confers high resistance to all known Bgt races and has been widely applied in wheat breeding in China. In this research, we identify Pm21 as a typical coiled-coil, nucleotide-binding site, leucine-rich repeat gene by an integrated strategy of resistance gene analog (RGA)-based cloning via comparative genomics, physical and genetic mapping, BSMV-induced gene silencing (BSMV-VIGS), large-scale mutagenesis and genetic transformation.

Characteristics and origins of non-functional Pm21 alleles in Dasypyrum villosum and wheat genetic stocks

Most Dasypyrum villosum resources are highly resistant to wheat powdery mildew that carries Pm21 alleles. However, in the previous studies, four D. villosum lines (DvSus-1 ∼ DvSus-4) and two wheat-D. villosum addition lines (DA6V#1 and DA6V#3) were reported to be susceptible to powdery mildew. In the present study, the characteristics of non-functional Pm21 alleles in the above resources were analyzed after Sanger sequencing. The results showed that loss-of-functions of Pm21 alleles Pm21-NF1 ∼ Pm21-NF3 isolated from DvSus-1, DvSus-2/DvSus-3 and DvSus-4 were caused by two potential point mutations, a 1-bp deletion and a 1281-bp insertion, respectively. The non-functional Pm21 alleles in DA6V#1 and DA6V#3 were same to that in DvSus-4 and DvSus-2/DvSus-3, respectively, indicating that the susceptibilities of the two wheat genetic stocks came from their D. villosum donors. The origins of non-functional Pm21 alleles were also investigated in this study. Except the target variants involved, the sequences of Pm21-NF2 and Pm21-NF3 were identical to that of Pm21-F2 and Pm21-F3 in the resistant D. villosum lines DvRes-2 and DvRes-3, derived from the accessions GRA961 and GRA1114, respectively. It was suggested that the non-functional alleles Pm21-NF2 and Pm21-NF3 originated from the wild-type alleles Pm21-F2 and Pm21-F3. In summary, this study gives an insight into the sequence characteristics of non-functional Pm21 alleles and their origins in natural population of D. villosum.

Fine Physical Bin Mapping of the Powdery Mildew Resistance Gene Pm21 Based on Chromosomal Structural Variations in Wheat

Pm21, derived from wheat wild relative Dasypyrum villosum, is one of the most effective powdery mildew resistance genes and has been widely applied in wheat breeding in China. Mapping and cloning Pm21 are of importance for understanding its resistance mechanism. In the present study, physical mapping was performed using different genetic stocks involving in structural variations of chromosome 6VS carrying Pm21. The data showed that 6VS could be divided into eight distinguishable chromosomal bins, and Pm21 was mapped to the bin FLb4–b5/b6 closely flanked by the markers 6VS-08.6 and 6VS-10.2. Comparative genomic mapping indicated that the orthologous regions of FLb4–b5/b6 carrying Pm21 were narrowed to a 117.7 kb genomic region harboring 19 genes in Brachypodium and a 37.7 kb region harboring 5 genes in rice, respectively. The result was consistent with that given by recent genetic mapping in diploid D. villosum. In conclusion, this study demonstrated that physical mapping based on chromosomal structural variations is an efficient method for locating alien genes in wheat background.

Development of a novel T-vector supporting efficient green-white screening

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.