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Polyploidy is a common mode of evolution in flowering plants. Both the natural tetraploid Thinopyrum elongatum and the diploid one from the same population show a diploid-like pairing in meiosis. However, debate on the chromosome composition and origin of the tetraploid Th. elongatum is ongoing. In the present study, we obtained the induced tetraploid Th. elongatum and found that the induced and natural tetraploids are morphologically close, except for slower development and lower seed setting. Using probes developed from single chromosome microdissection and a Fosmid library, obvious differentiations were discovered between two chromosome sets (E

Centromeres are the genomic regions that organize and regulate chromosome behaviours during cell cycle, and their variations are associated with genome instability, karyotype evolution and speciation in eukaryotes. The highly repetitive and epigenetic nature of centromeres were documented during the past half century. With the aid of rapid expansion in genomic biotechnology tools, the complete sequence and structural organization of several plant and human centromeres were revealed recently. Here, we systematically summarize the current knowledge of centromere biology with regard to the DNA compositions and the histone H3 variant (CENH3)-dependent centromere establishment and identity. We discuss the roles of centromere to ensure cell division and to maintain the three-dimensional (3D) genomic architecture in different species. We further highlight the potential applications of manipulating centromeres to generate haploids or to induce polyploids offspring in plant for breeding programs, and of targeting centromeres with CRISPR/Cas for chromosome engineering and speciation. Finally, we also assess the challenges and strategies for de novo design and synthesis of centromeres in plant artificial chromosomes. The biotechnology applications of plant centromeres will be of great potential for the genetic improvement of crops and precise synthetic breeding in the future.

Thinopyrum intermedium and barley are two close relatives of wheat and carry many genes that are potentially valuable for the improvement of various wheat traits. In this study we created wheat double substitution lines by hybridizing different wheat–Th. intermedium and wheat–barley disomic alien substitution lines, with the aim of using genes in Th. intermedium and barley for wheat breeding and investigating the genetic behavior of alien chromosomes and their wheat homoeologs. As expected, we obtained two types of wheat double substitution lines, 2D2Ai#2(2B)2H(2A) and 2A2Ai#2(2B)2H(2D), in which different group 2 wheat chromosomes were replaced by barley chromosome 2H and Th. intermedium chromosome 2Ai#2. The new materials were characterized using molecular markers, genomic in situ hybridization (GISH), and fluorescent in situ hybridization (FISH). GISH and FISH experiments revealed that the double substitution lines harbor 42 chromosomes including 38 wheat chromosomes, a pair of barley chromosomes, and a pair of Th. intermedium chromosomes. Analysis using specific DNA markers showed that two pairs of wheat homoeologous group 2 chromosomes in the new lines were substituted by a pair of 2H and a pair of 2Ai#2 chromosomes. Chromosome 2H showed a higher transmission rate than 2Ai#2, and both chromosomes were preferentially transmitted between generations via female gametes. Evaluation of botanic and agronomic traits demonstrated that, compared with their parents, the new lines showed similar growth habits and plant type but differences in plant height, flowering date, and self-fertility. Cytological observations using different probes suggested that the double substitution lines showed nearly normal genetic behavior before and during meiosis. The novel substitution lines can potentially be used in wheat meiosis research and breeding programs. Keywords: Wheat, Double substitution lines, Molecular markers, Transmission rate of alien chromosomes, Genomic in situ hybridization

The lack of resistance resources is a major bottleneck for wheat Fusarium head blight (FHB) resistance breeding. Three wheat-Th. elongatum FHB resistant translocation lines have been developed and used for wheat breeding without yield penalty. Transcriptomic analysis identified a derivative glutathione S-transferase transcript T26102, which was homologous to Fhb7 and induced dramatically by Fusarium graminearum. Unlike other studies, Fhb7 homologs were detected not only in Thinopyrum but also in Elymus, Leymus, Pseudoroegeria and Roegeria. We also found that several wheat-Th. ponticum derivatives carrying Fhb7 and its homologs were highly susceptible to FHB. Moreover, the transgenic plants expressing Fhb7 and its homolog on different backgrounds did not improve the FHB resistance.One Sentence SummaryThe GST-encoding Fhb7 candidate cannot improve Fusarium head blight resistance in wheat breeding.

Summary Previous studies revealed that the promoters for driving both Cas9 and sgRNAs are quite important for efficient genome editing by CRISPR/Cas9 in plants. Here, we report our results of targeted genome editing using the maize dmc1 gene promoter combined with the U3 promoter for Cas9 and sgRNA, respectively. Three loci in the maize genome were selected for targeting. The T0 plants regenerated were highly efficiently edited at the target sites with homozygous or bi‐allelic mutants accounting for about 66%. The mutations in T0 plants could be stably transmitted to the T1 generation, and new mutations could be generated in gametes or zygotes. Whole‐genome resequencing indicated that no off‐target mutations could be detected in the predicted loci with sequence similarity to the targeted site. Our results show that the dmc1 promoter‐controlled (DPC) CRISPR/Cas9 system is highly efficient in maize and provide further evidence that the optimization of the promoters used for the CRISPR/Cas9 system is important for enhancing the efficiency of targeted genome editing in plants. The evolutionary conservation of the dmc1 gene suggests its potential for use in other plant species.

Summary 1RS.1BL translocations are centric translocations formed by misdivision and have been used extensively in wheat breeding. However, the role that the centromere plays in the formation of 1RS.1BL translocations is still unclear. Fluorescence in situ hybridization (FISH) was applied to detect the fine structures of the centromeres in 130 1RS.1BL translocation cultivars. Immuno-FISH, chromatin immunoprecipitation (ChIP)-qPCR and RT-PCR were used to investigate the functions of the hybrid centromeres in 1RS.1BL translocations. New 1R translocations with different centromere structures were created by misdivision and pollen irradiation to elucidate the role that the centromere plays in the formation of 1RS.1BL translocations. We found that all of the 1RS.1BL translocations detected contained hybrid centromeres and that wheat-derived CENH3 bound to both the wheat and rye centromeres in the 1RS.1BL translocation chromosomes. Moreover, a rye centromere-specific retrotransposon was actively transcribed in 1RS.1BL translocations. The frequencies of new 1RS hybrid centromere translocations and group-1 chromosome translocations were higher during 1R misdivision. Our study demonstrates the hybrid nature of the centromere in 1RS.1BL translocations. New 1R translocations with different centromere structures were created to help understand the fusion centromere used for wheat breeding and for use as breeding material for the improvement of wheat.

Recently, engineered minichromosomes have been produced using a telomere-mediated truncation technique in some plants. However, the study on transferring genes to minichromosomes is very limited. Here, telomere-mediated truncation was successfully performed in common wheat (Triticum aestivum) to generate stable truncated chromosomes accompanied by a relatively high frequency of chromosomal rearrangements. After the cross between transgenic parents, a promoter-less DsRed gene in a chromosome from one parent was transferred to another chromosome from the other parent at the site behind a maize ubiquitin promoter via the Cre/lox system. DsRed transcripts and red fluorescent proteins were detected in the recombinant plants. In one such seedling, transgenic signals were detected at the centric terminus of chromosome 4D and the distal terminus of chromosome 3A. Clear translocations could be detected at the transgenic loci of these two chromosomes. Intriguingly, signals of centric-specific sequences were co-localized with the translocated D-group chromosomal segment in the terminal region of chromosome 3A. Our results indicate that the Cre/lox system induces the gene swapping to the target chromosome and non-homologous chromosomal recombination simultaneously. These approaches could offer a platform to transfer large DNA fragments or even terminal chromosomal segments to other chromosomes of the natural genome.