| Tibetan hulless barley(Hordeum vulgare L.var.nudum,2n=14)is a species of Gramineous Hordeum crop.It is also called naked barley because of its grain is separated from the glume.Tibetan hulless barley has constituted a staple food for the Tibetan people in China.It is the only crop that can mature normally in the high-altitude regions over 4200 meters above sea level.At present,a growing number of studies have shown that Tibetan hulless barley has “three high and two low”(high protein,high fiber,high vitamin,low fat,low sugar).It is a nutritional health-care cereal.Compared with white grain Tibetan hulless barley,black one is rich in anthocyanins,phenolic compounds,human essential amino acids and trace elements.Therefore,as an important quality index of high quality highland barley,black grain has attracted more and more attention.In this study,the black seed coat color gene of Tibetan hulless barley was mapped by a genetic linkage map constructing through specific locus amplified fragment sequencing(SLAF-seq).Then the black seed coat color candidate genes were predicted,cloned and functionally predicted according to the annotations of the comparison with the barley reference genome and the transcriptome sequencing results.In addition,the expression patterns of Hvt GT and Hvt HID in the seed coats of different grain-colored hulless barley at the late maturation stage and the expression patterns of the black-grained hulless barley “Kun Lun 17” at the early,middle and late maturation stage were studied.The competitive relationship was revealed,which between the expression of Hvt GT and Hvt HID in the pigment deposition during the formation of grain color.It provided basis for the study of the formation and regulation mechanism of barley seed coat color.The main results are as follows:1.Mapping of the black seed color gene in Tibetan hulless barley and Mining candidate genes.The phenotypic statistics were performed on the F2 population constructed by the female black-grain parent “Kun Lun 17” and the father white grain parent “Bai 91-97-3”.It was found that the ratio of black grain,intermediate type,and white grain barley of orthogonal and reverse hybridization was about 1: 2: 1,which indicated that the black grain trait was controlled by a single incomplete dominant gene.The F2 population was sequenced by SLAF-seq,the black seed color gene of Tibetan hulless barley was mapped.The black seed color trait was located on chromosome 1 at 547.69Mb-548.79 Mb,548.80Mb-548.90 Mb and 554.56Mb-555.85 Mb.Comparison with the reference genome revealed that the three candidate segments contained a total of 58 candidate genes.According to the results of database annotations such as Swissprot,NR,COG,GO,KEGG,etc.combineding with transcriptome sequencing results of different stages of barley black grain formation and known anthocyanin pathways,GT1(Gene ID: HORVU1Hr1G092650)was identified as a candidate gene for controlling the formation of barley black grains It was named Hvt GT(HORVU1Hr1G092650)that located on chromosome 1(554.56Mb-555.85Mb).2.Hvt GT gene was isolated in “Kun Lun 17” and “Bai 91-97-3” grains by RT-PCR.Allele analysis revealed that the CDS of the two genes were 1470 bp,encoding a total of 489 amino acids.What's interesting was that we found there were 5 amino acid differences between black grain Hvt GT and white grain Hvtgt due to 5 A and G alternations in the gene sequences.Bioinformatics analysis showed that Hvt GT encoded GT-B UDP glycosyltransferase,and it was a structural gene.Phylogenetic tree analysis showed that Hvt GT was closely related to Hordeum vulgare,but far away from indica rice and Oryza brachy Antha.The expression vector PYPQ203-Hvt GT was obtained by Gateway technology.Twenty-five tobacco-positive seedlings transferred Hvt GT gene were obtained by Agrobacterium mediated transformation of tobacco.But anthocyanin deposition was not found in transgenic tobacco.3.Hvt HID gene was isolated in “Kun Lun 17” grains by RT-PCR.The CDS was 1080 bp,encoding a total of 327 amino acids.The protein of Hvt HID gene was composed of random coil,?-helix,extended chain,?-turn,and random coil is the main one,with the smallest ?-turn.Phylogenetic tree analysis showed that Hvt HID was closely related to Hordeum vulgare,but far away from Setaria italica and Setaria viridis.Bioinformatics analysis showed Hvt HID encoded Abhydrolase?3(? / ? hydrolase)superfamily carboxylesterase,and it was the structural gene.The expression vector PYPQ203-Hvt HID was obtained by Gateway technology.Nine tobacco-positive seedlings transferred Hvt HID gene were obtained by Agrobacterium mediated transformation of tobacco.But anthocyanin deposition was not found in transgenic tobacco.4.In the late maturity stage of the barley with different grain colors,the expression level of Hvt GT gene was the highest in “Kun Lun 17”(black grain),while the expression level of Hvt HID gene was the highest in “Ne Rumuza”(purple grain).The expression level of Hvt GT gene was “Kun Lun 17”(black grain)> “Ne Rumuza”(purple grain)> “Da Zhangzi”(purple grain)> “Hei Laoya”(black grain)> “Kun Lun 12”(white grain)> “INB0N-7”(blue grain).The expression level of Hvt HID gene was “Ne Rumuza”(purple grain)>“Kun Lun 17”(black grain)>“Hei Laoya”(black grain)>“Kun Lun 12”(white grain)> “Da Zhangzi”(purple grain)> “INB0N-7”(blue grain).So,it is speculated that Hvt GT and Hvt HID participate in grain color deposition during the late grain filling stage of Tibetan hulless barley.During the grain formation of “Kun Lun 17”,the expression level of Hvt GT gradually increased,and reached the highest peak at 14 weeks after sowing.The expression level of Hvt HID has gradually decreased,and reached the lowest at 14 weeks after sowing.The contrary expression patterns of Hvt GT and Hvt HID in the grain formation of “Kun Lun 17” in black-grained hulless barley.It is suggested that there may be some competition between between Hvt GT and Hvt HID genes in the synthesis pathway during the formation of black hulless barley grains. |
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