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.

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Seven kinds of transgenic tobacco plants transformed with combinations of three FBE genes were obtained. The transgenic plants transformed with Ta1-SST + Ta6-SFT genes appeared to have the highest fructan or soluble sugar content and the strongest salt tolerance. Fructan is thought to be one of the important regulators involved in plant tolerance to various abiotic stresses. In this study, wheat-derived genes, Ta1-SST, Ta6-SFT, and Ta1-FFT, encoding fructan biosynthesis enzymes (FBE) were isolated and cloned into vectors modified pBI121 or pZP211. Seven different combinations of the three target genes were transformed into tobacco plants through an Agrobacterium-mediated approach, and transgenic tobacco plants were identified by PCR, ELISA, and Southern blotting. Compared with tobacco plants transformed with other six combinations of the three target genes and with wild-type plants, the transgenic plants transformed with Ta1-SST + Ta6-SFT genes contained the highest fructan and soluble sugar content. All seven types of transgenic tobacco plants displayed a much higher level of tolerance to drought, low temperature, and high salinity compared with the wild type. Differences of drought and low temperature tolerance between the transgenic plants containing a single FBE gene and those harboring two or three FBE genes were not significant, but the salt tolerance level of the transgenic plants with different FBE gene combinations from high to low was: Ta1-SST + Ta6-SFT > Ta1-SST + Ta6-SFT + Ta1-FFT > Ta1-SST + Ta1-FFT > Ta1-SFT + Ta1-FFT > single FBE gene. These results indicated that the tolerances of the transgenic tobacco plants to various abiotic stresses were associated with the transformed target gene combinations and the contents of fructan and soluble sugar contained in the transgenic plants.

Twenty-five Dasypyrum villosum 6V#4S-specific PCR markers were developed using transcriptome data and further assigned to comparative genomic maps of wheat chromosome 6A, 6B, and 6D and barley chromosome 6H contrasting their homologous genes in these genomes. Two Dasypyrum villosum accessions, D.v#2 and No. 1026 from England and Russia, respectively, contain Pm21 on chromosome 6V#2S and PmV on chromosome 6V#4S. Both genes confer high resistance to powdery mildew (PM) in wheat. Even though several molecular markers have been developed to detect Pm21 and PmV, only the MBH1 marker can simultaneously detect both Pm21 and PmV. In this study, we first used a high-throughput sequencing technique to obtain the transcriptome sequences of a wheat-D. villosum translocation line, Pm97033-which contains chromosome 6V#4S carrying the PmV locus, under wheat PM pathogen induction. Twenty-five 6V#4S chromosome-specific markers were developed. Three of them were able to clearly distinguish chromosomes 6V#4S and 6V#2S by product size, four amplified the product specific for chromosome 6V#4S only, and the remaining 18 markers identified chromosome 6VS in wheat backgrounds. Two different D. villosum accessions, their derived translocation lines and wheat varieties carrying different chromosome 6VS were identified using these specific markers. The 25 newly developed markers together with the known PM resistance gene Stpk-V were used to construct comparative genomic maps with the homoeologous chromosome arms of wheat and barley. The colinearity of the identified gene sequences amplified by the 25 markers among wheat chromosomes 6A, 6B, and 6D and barley chromosome 6H was not very conserved and interrupted frequently by inversion and insertion. Our markers have potential in marker assisted selection for PM resistance breeding, and for locating other potential important genes and cloning the PmV gene on chromosome 6V#4S.

Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops.

The wheat line H960642 is a homozygous wheat-Thinopyrum intermedium translocation line with resistance to BYDV by genomic in situ hybridization (GISH) and RFLP analysis. The genomic DNA of Th. intermedium was used as a probe, and common wheat genomic DNA as a blocking in GISH experiment. The results showed that the chromosome segments of Th. intermedium were transferred to the distal end of a pair of wheat chromosomes. RFLP analysis indicated that the translocation line H960642 is a T7DS-7DL-7XL translocation by using 8 probes mapped on the homoeologous group 7 in wheat. The translocation breakpoint is located between Xpsr680 and Xpsr965 about 90-99 cM from the centromere. The RFLP markers psr680 and psr687 were closely linked with the BYDV resistance gene. The gene is located on the distal end of 7XL around Xpsr680 and Xpsr687.

Background Dasypyrum villosum is an important wild species of wheat ( Triticum aestivum L.) and harbors many desirable genes that can be used to improve various traits of wheat. Compared with other D. villosum accessions, D. villosum#4 still remains less studied. In particular, chromosomes of D. villosum#4 except 6V#4 have not been introduced into wheat by addition or substitution and translocation, which is an essential step to identify and apply the alien desired genes. RNA-seq technology can generate large amounts of transcriptome sequences and accelerate the development of chromosome-specific molecular markers and assisted selection of alien chromosome line. Results We obtained the transcriptome of D. villosum#4 via a high-throughput sequencing technique, and then developed 76 markers specific to each chromosome arm of D. villosum#4 based on the bioinformatic analysis of the transcriptome data. The D. villosum#4 sequences containing the specific DNA markers were expected to be involved in different genes, among which most had functions in metabolic processes. Consequently, we mapped these newly developed molecular markers to the homologous chromosome of barley and obtained the chromosome localization of these markers on barley genome. Then we analyzed the collinearity of these markers among D. villosum , wheat, and barley. In succession, we identified six types of T. aestivum - D. villosum#4 alien chromosome lines which had one or more than one D. villosum#4 chromosome in the cross and backcross BC 3 F 5 populations between T. durum – D. villosum#4 amphidiploid TH3 and wheat cv. Wan7107 by employing the selected specific markers, some of which were further confirmed to be translocation or addition lines by genomic in situ hybridization (GISH). Conclusion Seventy-six PCR markers specific to chromosomes of D. villosum#4 based on transcriptome data were developed in the current study and their collinearity among D. villosum , wheat, and barley were carried out. Six types of Triticum aestivum - D. villosum#4 alien chromosome lines were identified by using 12 developed markers and some of which were further confirmed by GISH. These novel T. aestivum - D. villosum#4 chromosome lines have great potential to be used for the introduction of desirable genes from D. villosum#4 into wheat by chromosomal translocation to breed new wheat varieties. Electronic su...

Transcriptome data were used to develop 134 Aegilops longissima specific PCR markers and their comparative maps were constructed by contrasting with the homologous genes in the wheat B genome. Three wheat- Ae. longissima 1BL·1S S translocation lines were identified using the correspondence markers. Aegilops longissima is an important wild species of common wheat that harbors many genes that can be used to improve various traits of common wheat (Triticum aestivum L.). To efficiently transfer the traits conferred by these Ae. longissima genes into wheat, we sequenced the whole expression transcript of Ae. longissima. Using the transcriptome data, we developed 134 specific polymerase chain reaction markers located on the 14 chromosome arms of Ae. longissima. These novel molecular markers were assigned to specific chromosome locations based on a comparison with the homologous genes in the B genome of wheat. Annotation of these genes showed that most had functions related to metabolic processes, hydrolase activity, or catalytic activity. Additionally, we used these markers to identify three wheat-Ae. longissima 1BL·1SS translocation lines in somatic variation populations resulting from a cross between wheat cultivar Westonia and a wheat-Ae. longissima substitution line 1S(1B). The translocation lines had several low molecular weight glutenin subunits encoding genes beneficial to flour processing quality that came from Ae. longissima 1SS. The three translocation lines were also confirmed by genomic in situ hybridization. These translocation lines will be further evaluated for potential quality improvement of bread-making properties of wheat.