BLAST is an extensively used local similarity search tool for identifying homologous sequences. When a gene sequence (either protein sequence or nucleotide sequence) is used as a query to search for homologous sequences in a genome, the search results, represented as a list of high-scoring pairs (HSPs), are fragments of candidate genes rather than full-length candidate genes. Relevant HSPs (“signals”), which represent candidate genes in the target genome sequences, are buried within a report that contains also hundreds to thousands of random HSPs (“noises”). Consequently, BLAST results are often overwhelming and confusing even to experienced users. For effective use of BLAST, a program is needed for extracting relevant HSPs that represent candidate homologous genes from the entire HSP report. To achieve this goal, we have designed a graph-based algorithm, genBlastA, which automatically filters HSPs into well-defined groups, each representing a candidate gene in the target genome. The novelty of genBlastA is an edge length metric that reflects a set of biologically motivated requirements so that each shortest path corresponds to an HSP group representing a homologous gene. We have demonstrated that this novel algorithm is both efficient and accurate for identifying homologous sequences, and that it outperforms existing approaches with similar functionalities.

SummaryGenotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker‐free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium‐mediated method, with efficiency of up to 37.7%, as confirmed by the use of Quickstix strips, histochemical staining, PCR analysis and Southern blotting. Of particular interest, marker‐free transgenic wheat plants from various commercial Chinese varieties and their F1 hybrids were successfully obtained for the first time, with a frequency of 4.3%, using a plasmid harbouring two independent T‐DNA regions. The average co‐integration frequency of the gus and the bar genes located on the two independent T‐DNA regions was 49.0% in T0 plants. We further found that the efficiency of generating marker‐free plants was related to the number of bar gene copies integrated in the genome. Marker‐free transgenic wheat plants were identified in the progeny of three transgenic lines that had only one or two bar gene copies. Moreover, silencing of the bar gene was detected in 30.7% of T1 positive plants, but the gus gene was never found to be silenced in T1 plants. Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in the transgenic plants.

Summary Kernel size is an important trait determining cereal yields. In this study, we cloned and characterized TaDA1, a conserved negative regulator of kernel size in wheat (Triticum aestivum). The overexpression of TaDA1 decreased the size and weight of wheat kernels, while its down‐regulation using RNA interference (RNAi) had the opposite effect. Three TaDA1‐A haplotypes were identified in Chinese wheat core collections, and a haplotype association analysis showed that TaDA1‐A‐HapI was significantly correlated with the production of larger kernels and higher kernel weights in modern Chinese cultivars. The haplotype effect resulted from a difference in TaDA1‐A expression levels between genotypes, with TaDA1‐A‐HapI resulting in lower TaDA1‐A expression levels. This favourable haplotype was found having been positively selected during wheat breeding over the last century. Furthermore, we demonstrated that TaDA1‐A physically interacts with TaGW2‐B. The additive effects of TaDA1‐A and TaGW2‐B on kernel weight were confirmed not only by the phenotypic enhancement arising from the simultaneous down‐regulation of TaDA1 and TaGW2 expression, but also by the combinational haplotype effects estimated from multi‐environment field data from 348 wheat cultivars. A comparative proteome analysis of developing transgenic and wild‐type grains indicated that TaDA1 and TaGW2 are involved in partially overlapping but relatively independent protein regulatory networks. Thus, we have identified an important gene controlling kernel size in wheat and determined its interaction with other genes regulating kernel weight, which could have beneficial applications in wheat breeding.

http://genome.sfu.ca/genblast/download.html

Genotype dependency is the most important factor in wheat genetic transformation, which further limits wheat improvement by transgenic integration and genome editing approaches. The application of regeneration related genes during in vitro culture could potentially contribute to enhancement of plant transformation e ciency. In the present study, a wheat gene TaCB1 in the WUSCHEL family was identi ed to dramatically increase the transformation e ciencies of many wheat varieties without genotype dependency after its over-expression. The expression of TaCB1 in wheat calli did not prohibit shoot differentiation and root development. The application of TaCB1 can lighten the requirement to wheat immature embryo for plant regeneration. Transgenic wheat plants can be clearly recognized by the visible phenotype of wide ag leaves. The promise function of TaCB1 on improving transformation e ciency was also tested in T. monococcum, triticale, rye, barley, and maize.

The use of CRISPR/LbCpf1 and CRISPR/xCas9 systems in wheat have not yet been reported. In this study, we compared the efficiencies of three CRISPR editing systems (SpCas9, LbCpf1, and xCas9), and three different promoters (OsU6a, TaU3, and TaU6) that drive single-guide (sg)RNA, which were introduced into wheat via Agrobacterium-mediated transformation. The results indicated that TaU3 was a better choice than OsU6a or TaU6. The editing efficiency was higher using two sgRNAs than one sgRNA, and mutants with a large fragment deletion between the two sgRNAs were produced. The LbCpf1 and xCas9 systems could both be used successfully. Two endogenous genes, TaWaxy and TaMTL, were edited with high efficiency by the optimized SpCas9 system, with the highest efficiency (80.5%) being achieved when using TaU3 and two sgRNAs to target TaWaxy. Rates of seed set in the TaMTL-edited T0 transgenic plants were much lower than that of the wild-type. A haploid induction rate of 18.9% was found in the TaMTL-edited T1 plants using the CRISPR/SpCas9 system. Mutants with reverse insertion of the deleted sequences of TaMTL and TaWaxy between the two sgRNAs were identified in the edited T0 plants. In addition, wheat grains lacking embryos or endosperms were observed in the TaMTL-edited T1 generation.

The objective of this study was to evaluate the safety and efficacy of allogeneic bone marrow mesenchymal stromal/stem cell transplantation (BM-MSCT) for patients with ursodeoxycholic acid (UDCA)-resistant primary biliary cirrhosis (PBC). Ten patients were enrolled in this trial of BM-MSCT. All patients were permitted to concurrently continue their previous UDCA treatment. The efficacy of BM-MSCT in UDCA-resistant PBC was assessed at various time points throughout the 12-month follow up. No transplantation-related side effects were observed. The life quality of the patients was improved after BM-MSCT as demonstrated by responses to the PBC-40 questionnaire. Serum levels of ALT, AST, γ-GT, and IgM significantly decreased from baseline after BM-MSCT. In addition, the percentage of CD8+ T cells was reduced, while that of CD4+CD25+Foxp3+ T cells was increased in peripheral lymphocytic subsets. Serum levels of IL-10 were also elevated. Notably, the optimal therapeutic outcome was acquired in 3 to 6 months and could be maintained for 12 months after BM-MSCT. In conclusion, allogeneic BM-MSCT in UDCA-resistant PBC is safe and appears to be effective.

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