Abstract:In this study， the data of 22 phenotypic traits and 3 quality traits of 708 tartary buckwheat resources were used to construct the core collection of tartary buckwheat germplasm. By comparing merits of four different combination strategies including the genetic distance， sampling method， sampling proportion and clustering method， the ‘euclidean distance + multiple cluster deviation sampling + 20% sampling proportion + maximum distance method’ was determined as the optimal sampling strategy of tartary buckwheat core collection and the core collection containing 141 tartary buckwheat resources was constructed. The mean difference percentage， variance difference percentage， range coincidence rate and variation coefficient change rate of core collection and original collection were 0，84.00%， 97.60% and 115.42%， respectively. Finally， the phenotypic and quality traits of the core collection were compared with the original germplasm. The mean value of 25 traits between core collection and original germplasm had no significant difference. The variance of core collection of 25 traits was higher than or equal to that of original germplasm， and the variance of 22 traits between core collection and original germplasm had significant difference at 0.05 leve or 0.01 level. It showed that the heterogeneity of core collection was better. t-test of Shannon-Weaver index showed that there was no significant difference between core collection and original germplasm on all traits. Both the core collection and the original germplasm had 8 principal components， and the cumulative contribution rates were 77.525% and 76.191%，respectively. The core collection germplasm was proved to represent the genetic diversity of the original germplasm， which might be useful in collection， preservation and effective utilization of tartary buckwheat germplasm resources.

Abstract:In order to understand the molecular mechanism of tartary buckwheat against bacterial blight， we isolated the transcription factor gene FtEIN3 in Chuanqiao 1， a representative variety of tartary buckwheat.The FtEIN3 contains the CDS sequence length of 1623 bp， encoding 540 amino acids. The secondary structure of FtEIN3 protein was composed of α-helix （33.52%）， extended chain （6.67%）， β-fold （2.41%） and irregular coil （57.41%）. Phylogenetic tree indicated that FtEIN3 protein was closely related to GhEIN3 and DzEIN3 protein. Five different EIN3 sequence haplotypes were detected in 108 tartary buckwheat germplasm， and out of them Hap3 was an elite haplotype. qRT-PCR revealed that the expression of FtEIN3 gene in tartary buckwheat was induced by Rhizoctonia solani. The FtEIN3 gene in tartary buckwheat was localized in the nucleus under confocal laser microscopy. To further verify the function of FtEIN3 gene， FtEIN3 transgenic Arabidopsis thaliana were constructed and their resistance to blip disease was analyzed. The results showed that overexpression of FtEIN3 gene significantly improved the resistance of transgenic Arabidopsis thaliana to bacterial blight compared with the wild type. These results proved that FtEIN3 gene was involved in the defense process of tartary buckwheat against bacterial blight， and laid a foundation for further research on the molecular mechanism of FtEIN3 regulation of tartary buckwheat resistance to blight.

Abstract:The transcriptome analysis of tartary buckwheat identified a flavonol synthase gene FtFLS1. In order to further understand its structure， function and diversity in tartary buckwheat genome， we identified 104 members of FLS gene family with 10 subgroups， in which FtFLS1 was found in DF8 subgroup. Promoter analysis revealed two MeJA response elements at the upstream of 1500 bp sequence. We analyzed the expression of FtFLS1 in different organs and its response to MeJA treatments. The transcriptional level of FtFLS1 in stems and leaves was comparable but higher in roots. Expression of FtFLS1 also increased significantly with the treatment of MeJA. We subsequently cloned the CDS sequence of FtFLS1， and then generated FtFLS1 over-expressed hairy root lines of tartary buckwheat and detected their flavonoid content. Over-expression transformants over accumulated the downstream products of FLS， which including kaempferol， quercetin and rutin， while the contents of dihydrokaempferol and dihydroquercetin， the substrates of brass synthetase， decreased significantly. Furthermore， we analyzed the diversity of FtFLS1 gene in different populations of tartary buckwheat， and found that Northern landraces， Southwestern landraces and Himalayan wild accessions present obvious differentiation. The results are helpful for understanding the FtFLS1-mediated synthesis of flavonoids and the domestication process of buckwheat.

Abstract:In this study，we cloned the transcription factor gene FtMYB41 in tartary buckwheat cultivar Pinku-1. Sequence analysis showed that the MYB super gene family member FtMYB41 contained one 5′UTR and two introns，with the complete coding sequence of 705bp which encodes for 234 amino acids. Phylogenetic tree analysis showed that FtMYB41 protein had close homology with tomato NP_001234262.1 and tobacco NP_001312384.1. Transcriptional activation analysis revealed the transcriptional activation activity at FtMYB41 C-terminal. Moreover，the FtMYB41 gene had the higher expression in roots and was induced by drought and salt stresses. The expression level of FtMYB41 gene was significantly increased under drought stress for 9 h. Overexpressing FtMYB41 gene in Arabidopsis thaliana（L.）Heynh. and tartary buckwheat hair root resulted in improved drought resistance by increasing germination rate，increasing CAT and SOD activity，and decreasing MDA content.

Abstract:AP2/ERF transcription factor family members have been identified to play important roles in abiotic stress responses. In this study, the full-length coding sequence of the FtDREB6 gene was cloned with a length of 615 bp, which encodes for 204 amino acids residues, with a molecular weight of 22.7 kDa and an isoelectric point of 4.96 from tartary buckwheat (Fagopyrum tataricum) cultivar “Pinku 1”. The sequence of FtDREB6 represented higher homology with Arabidopsis thaliana AtERF043 by the sequence alignment on TAIR website. FtDREB6 gene was showed without the transcription activity by transactivation analysis. Moreover, by transforming the coding sequence of FtDREB6 into A. thaliana by Agrobacterium-mediated transformation approach, transgenic FtDREB6-overexpressing plants showed significant increase on the drought resistance. The expression vector containing FtDREB6 gene was transformed into Agrobacterium rhizogenes A4, which was subjected to infect explant for inducing the hairy roots. Under D-Mannitol treatment, the superoxide dismutase activity and catalase activity in overexpressing hairy roots were significantly higher than that of the control, and the content of malondialdehyde was significantly lower than the control. These results indicated that FtDREB6 was involved in responses to drought stress, which provided a reference for future deciphering the molecular mechanism for drought tolerance in tartary buckwheat.

Abstract:In this study, we generated a mutant population of tartary buckwheat cultivar Chuanqiao 2 treated by 50 mmol ethyl methanesulfonate (EMS) solution. We recorded the phenotypic variations of M2 to M3 lines, and also measured the content of chlorophyll and anthocyanin in the mutant plants with leaf and stem color variation in the M3 population as well as the transcription level of flavonoid synthesis and metabolite related genes (CHS, F3H, F3H, FLS and UFGT). 177 mutant plants showing alternations on stem and/or leaf morphology and/or growth stages were found in M2 population with a mutation rate of 10.56%. 665 mutant plants in M3 population were found, including leaves, growth stages, stems, and other types of mutant plants, accounting for 45.86% of the M3 population. We also observed that the shape, color and size of grains in M3 generation changed in varying degrees. We found that the change trend of chlorophyll and anthocyanin content in plants with color variation were almost the same, the content of the red plant at the base of the leaf was higher than that of the normal plant, and the content of the other types of mutant plants were lower than that of the normal plant. Among them, the yellow-green spotted leaf had the lowest content. We also found that CHS and F3H genes had the highest expression level in red stem plants, the FLS genes had the highest expression level in yellow leaf plants, and the expression of F3H was higher in the two types of plants with red leaf than the other types, and the content was the highest in the plants with the leaf edge turned red. It is speculated that leaf and stem color variations in the M3 population may be contributed by the expression of flavonoid synthesis-related genes. Collectively, these mutants gained from this study provided the basic material for future deciphering the rutin metabolism mechanism.

Abstract:NAC (NAM, ATAF1/2, CUC2) gene plays an important role in responses to plant abiotic stresses. In this study, we cloned FtNAC11 from tartary buckwheat cultivar ‘Pinku No.1’. Sequence analysis showed that the full-length coding sequence of FtNAC11 was 774bp encoding for 257 amino acids residues, with a molecular weight of 8954.58 Da and an isoelectric point of 5.91.The sequence alignment and phylogenetic tree analysis showed that FtNAC11 had higher homology with Arabidopsis thaliana AtNAC61. The expression of FtNAC11 gene was induced under PEG or NaCl treatments. Sub-localization by transient expression and yeast transactivation assay indicated that FtNAC11 encoded for a nuclear transcription activator with an activation domain at the region of C-terminal. Moreover, the coding sequence of FtNAC11 was transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation, and the transgenic plants over-expressing this gene resulted in significant enhancement under drought and salt tolerance treatments. Collectively, these results indicated that FtNAC11 is likely involved in abiotic stress adaptation, providing a basis for further investigation of its molecular mechanism responding to drought and salt tolerance in tartary buckwheat.

Abstract:Tartary buckwheat is one of minor grain crops in China, which has rich nutrition and is an important source of natural rutin. It is an important aspect of present research to break through of breeding and create new germplasm in tartary buckwheat. The mutation library was constructed in “Heifengyihao” by EMS. The half lethal treatment of EMS mutagenesis was 1.2%, the effect of plentiful phenotype was recorded in M1 generation. 102 plants with obvious phenotypic variation, including leaf color, leaf shape, plant type and grain sizes, were obtained from M3 materials and the mutation rate was 3.85%. The rutin content of 1000 plants in mutants’ library was determined by HPLC, 2 mutant lines with high (rutin content >16 mg/g) and 5 lines of low rutin (rutin content <10 mg/g) were obtained respectively. Comparison of rutin synthesis-related genes through qRT-PCR showed a relative weaker relationship between FtCHS, FtF3H, Ft4CL, FtUFGT transcripts and rutin content mutation lines. However, it also found that FtFLS showed the higher expression level in line with higher rutin content, which reached 4.55 times of control. The screening of mutants enriched the resources of tartary buckwheat, innovated the new germplasm of tartary buckwheat, and provided material guarantee and technical support for the molecular basis research of tartary buckwheat metabolism.

Abstract:R2R3-MYB transcription factors are known to play important regulation role in the flavonoid biosynthesis of plants. In this study, a gene FtMYB1 belonging to the R2R3-MYB gene family was cloned from Tartary buckwheat cultivar ‘Chuanqiao No.1’.The FtMYB1 transcription factor was found to exhibit the transcriptional activation activity in yeast, and its expression of FtMYB1 gene was induced by Methyl jasmonate (MeJA) and abscisic acid (ABA) treatment. Overexpressing the FtMYB1 gene in Tartary buckwheat hairy root showed a significant reduction in contents of total flavonoid and rutin if compared with wild type. Furthermore,the expression of FLS and RT1 genes, which encode for flavonol synthetase (FLS) and rhamnosyltransferase (RT) in Tartary buckwheat, respectively, were significantly lower in the FtMYB1 overexpressing hairy roots than that of wild type. Taken together, ourresults suggested that FtMYB1 may function in inhibition of the flavonol biosynthesis by repressing the expression of FLS and RT1 genes.

Abstract:Tartary buckwheat is widely cultivated in different areas of China and gradually evolved into genetic diversity. In order to study and utilize the tartary buckwheat, the PAL gene differences of the tartary buckwheat were analyzed on 67 samples, which were collected from different provinces of China. We amplified the PAL gene by PCR and then acquired the sequence results. Based on the sequences, genetic diversity was analyzed and cluster of analysis of 67 samples was also carried out by NJ method. Results showed that after aligning and splicing, the length the PAL sequence was 2011 bp in the matrix, the variable sites was 160, while the parsimony informative sites was 33, accounting for 7.9% and 1.64% of the total length, respectively. Variable type are main base transition and base transversions. Variable site mainly concentrated in the region of 600-1200, which was the site of the N-terminal of the exon-2. The intra-specific mean distance of the Sichuan group was 0.016, while the tartary buckwheat from Inner Mongolia group showed that the intra-specific mean distance was 0.002. And it revealed that there is a significant difference among the tartary buckwheat resources in genetics diversity from different geographic origins. We checked the 67 PAL sequences using software. And it showed that the average value of the Nucleotide polymorphism (?) and the average heterozygosity (θ) were 0.0034 and 0.0148, respectively. The Nucleotide polymorphism (?) value in samples from Sichuan is the highest (0.0148). From the phylogenetic tree, it has indicated that the 67 samples from the different province could be clustered into seven category. And the 5 sample in Tibet were clustered into another category. These results showed that the PAL sequences of the majority tartary buckwheat samples is stable and the differences between the most samples is not significant. The samples in Sichuan province had abundant genetic diversity. But only in samples from Tibet it revealed many SNPs site, and this should be the new mutation spots in the PAL coding regions.