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Barley (Hordeum vulgareL.) is one of the oldest domesticated cereal crops.It isone of the first cultivated grains and is now grown worldwide. It iswell-adapted to drought environments and can be grown normally under moisture stress and low soil fertility condition. Barley has been used as animalfodder, as a source of fermentable material forbeer and certaindistilled beverages, and as a component of varioushealth foods. It is used in soups and stews, and inbarley breadof various cultures.Currently 55-60% of the barley production is used as fodder, 30-40% in malt, 2-3% in human nutrition and about 5% is kept as seed for the newcrops.Barley is enriched with nutrients and is attractive for human food as well as for animal feed.Besides the economic importance of barley, it is also considered as a model species for cereal genetic and physiological studies.
1. QTLmapping of plant height, spike length, peduncle length and number of grains plant-1in barley (Hordeum vulgareL.) using ‘Steptoe’_‘Morex’ DH population grown in Northwest of China.
QTL analysis is of importance in the evolution of improved cultivars.The mapping of quantitative trait loci in plants is being regularly conducted by using a population resulting from a cross between two lines.In this study, 120 out of 150 double haploid lines of barley, obtained from the cross ‘Steptoe’ x ‘Morex’ along with their parents which were kindly provided by NABGMP (the North American Barley Genome Mapping Project), were screened for the traits plant height, spike length, peduncle length and number of grains plant-1, during three crop years in the dry area of Northwestern China. All the lines and parents were sown in the trial field of NWSUAF (N34.28, E108.06) under rain-fed conditions in three crop seasons 2014-15, 2015-16 and 2016-17, respectively. The DH lines were arranged in a replicates-in-sets design with three replicates and a plot size of 1m2. The plant height, peduncle length and spike length of each line was measured manually with the engineering rule and the grains plant-1were counted from 10 plants per replication. Quantitative trait loci (QTL) mapping was performed from the available genotype datasets using the QTL IciMapping Version 4.1 (http://www.isbreeding.net/software/, access by 2016-3-8) software with the inclusive composite interval mapping of additive (ICIM-ADD) and epistatic QTL (ICIM-EPI) methods. 1.0cM steps were employed in the detection of additive QTLs. The stepwise regression was adopted with the significance probability set at 0.001. 1000 permutations were used to determine significant LOD thresholds. Type I error was set at P <0.05. Epistatic QTLs were recognized by using a scanning step of 5.0cM with a probability of 0.0001 in stepwise regression with a significant LOD threshold of 3.0.“Steptoe × Morex”genetic map is the reference map of the NABGMP in which many QTLs controlling agronomic and malting traits are mapped. The genotyping data with 437 SSR and RFLP markers was downloaded from Grain gene database (http://wheat.pw.usda.gov/ggpages/SxM/) and used in constructing the genetic map and the genotyping data of the selected 120 lines extracted for QTL mapping. In total 15 QTLs were mapped on six linkage groups (1H, 2H, 3H, 4H, 5H and 7H) which explained 5.42% to 47.27% phenotypic variation. 4 QTLs were identified for plant height in three years including two major QTLs, one located on chromosome 2H in 2014-15 and 2015-16, and the other was detected only in 2016-17 on chromosome 4. 4 QTLs were identified for spike length. Six QTLs were identified underlying the trait peduncle length with three major QTLs, two localized on chromosome 2H in 2015-16 and 2016-17, and oneon chromosome 3H in 2014-15 and 2015-16. Only one major QTL was identified for number of grains plant-1in 2015-16. Our study could lay the foundation for fine mapping of these agronomic traits as well as increase the efficiency of marker assisted selection (MAS) of these traits in the barley breeding programs.
2.Prediction and Identification of RNA Editing Sites in the Chloroplast Genome of Barley (Hordeum vulgareL.)
RNA editing is one of the most important approaches to regulate the gene expression at post-transcriptional levels in the chloroplast genomes of land plants.The complete sequence of the barley chloroplast genome was analyzed in 2006 and submitted to the NCBIgene bank database(http://www.ncbi.nlm.nih.gov/)vide accession number NC_008590.Based on the annotation information in the NCBI database, 83 protein-coding genes were retrieved from the database and were used in this study. RNA editing sites were predicted by using Prep-Cp database and CUREsoftware. To ensure the accuracy of detection of RNA-editing site, the prediction parameter cutoff value was set to 0.8. Further, to validate the 83 protein coding genes, the DNA sequences obtained from barley chloroplast genome were subjected to the identification of ESTs using BLASTn tool. To ensure the high accuracy of the results, the parameters were set to match the lengthof greater than 200bp havingsimilarity of 85% or more withthe threshold value ofE-10. Besides this, the normal sequence encodedby the ndhBgene and the encoded protein after RNA editing was used for comparison study regarding domain detection, transmebrane detection and formation of secondary structure. The protein transmembrane segment prediction was performed using TMHMM ServerV.2.0 (http://genome.cbs.dtu.dk/services/TMHMM/)whileSMART tool (http://smart.embl-heidelberg.de/) was used to analyze protein-specific domains. Moreover NPS (http://npsa-pbil.ibcp.fr/cgi-bin) online tool was utilized to analyze protein secondary structure composition. At present, RNA editing sites present inchoroplast genomesofrye, rice, sugarcane, maizeand spike spp. have been reported in literature.In order to study the common characteristics, patterns and conservatism of chloroplast RNA editing, the chloroplast RNA editing sites of these species were compared with the predicted RNA editing sites of barley.Results showed that 37 editing sites were found from 16 genes, and all of them are Cto U conversion. Among them, 5 sites were located in the first position of codon, 32 sites located in the second position of codon and no RNA editing sites were found in the third position of codon. Through BLAST search in the EST sequences of barley in NCBI database, 34 sites in 16 genes were validated as the actual RNA editing sites, of which thendhBhad the most editing sites (9 sites). Furthermore, the secondary structures and the transmenbrane domains of editedndhB protein were analyzed using the bioinformatics methods. Results indicated that editing inndhB-467resulted in an increase in transmembrane protein domains and editing inndhB-149changed the secondary structure. Finally, the comparison analysis of the editing sites in barley with other 5grass chloroplast genomes was also performed. Our study analyzed the composition and distribution of RNA editing in barley chloroplast genome comprehensively, which provided the useful information for exploring the function and mechanism of RNA editing, aswell as for revealing the origin and molecular evolution of RNA editing in organelle genomes.
1. QTLmapping of plant height, spike length, peduncle length and number of grains plant-1in barley (Hordeum vulgareL.) using ‘Steptoe’_‘Morex’ DH population grown in Northwest of China.
QTL analysis is of importance in the evolution of improved cultivars.The mapping of quantitative trait loci in plants is being regularly conducted by using a population resulting from a cross between two lines.In this study, 120 out of 150 double haploid lines of barley, obtained from the cross ‘Steptoe’ x ‘Morex’ along with their parents which were kindly provided by NABGMP (the North American Barley Genome Mapping Project), were screened for the traits plant height, spike length, peduncle length and number of grains plant-1, during three crop years in the dry area of Northwestern China. All the lines and parents were sown in the trial field of NWSUAF (N34.28, E108.06) under rain-fed conditions in three crop seasons 2014-15, 2015-16 and 2016-17, respectively. The DH lines were arranged in a replicates-in-sets design with three replicates and a plot size of 1m2. The plant height, peduncle length and spike length of each line was measured manually with the engineering rule and the grains plant-1were counted from 10 plants per replication. Quantitative trait loci (QTL) mapping was performed from the available genotype datasets using the QTL IciMapping Version 4.1 (http://www.isbreeding.net/software/, access by 2016-3-8) software with the inclusive composite interval mapping of additive (ICIM-ADD) and epistatic QTL (ICIM-EPI) methods. 1.0cM steps were employed in the detection of additive QTLs. The stepwise regression was adopted with the significance probability set at 0.001. 1000 permutations were used to determine significant LOD thresholds. Type I error was set at P <0.05. Epistatic QTLs were recognized by using a scanning step of 5.0cM with a probability of 0.0001 in stepwise regression with a significant LOD threshold of 3.0.“Steptoe × Morex”genetic map is the reference map of the NABGMP in which many QTLs controlling agronomic and malting traits are mapped. The genotyping data with 437 SSR and RFLP markers was downloaded from Grain gene database (http://wheat.pw.usda.gov/ggpages/SxM/) and used in constructing the genetic map and the genotyping data of the selected 120 lines extracted for QTL mapping. In total 15 QTLs were mapped on six linkage groups (1H, 2H, 3H, 4H, 5H and 7H) which explained 5.42% to 47.27% phenotypic variation. 4 QTLs were identified for plant height in three years including two major QTLs, one located on chromosome 2H in 2014-15 and 2015-16, and the other was detected only in 2016-17 on chromosome 4. 4 QTLs were identified for spike length. Six QTLs were identified underlying the trait peduncle length with three major QTLs, two localized on chromosome 2H in 2015-16 and 2016-17, and oneon chromosome 3H in 2014-15 and 2015-16. Only one major QTL was identified for number of grains plant-1in 2015-16. Our study could lay the foundation for fine mapping of these agronomic traits as well as increase the efficiency of marker assisted selection (MAS) of these traits in the barley breeding programs.
2.Prediction and Identification of RNA Editing Sites in the Chloroplast Genome of Barley (Hordeum vulgareL.)
RNA editing is one of the most important approaches to regulate the gene expression at post-transcriptional levels in the chloroplast genomes of land plants.The complete sequence of the barley chloroplast genome was analyzed in 2006 and submitted to the NCBIgene bank database(http://www.ncbi.nlm.nih.gov/)vide accession number NC_008590.Based on the annotation information in the NCBI database, 83 protein-coding genes were retrieved from the database and were used in this study. RNA editing sites were predicted by using Prep-Cp database and CUREsoftware. To ensure the accuracy of detection of RNA-editing site, the prediction parameter cutoff value was set to 0.8. Further, to validate the 83 protein coding genes, the DNA sequences obtained from barley chloroplast genome were subjected to the identification of ESTs using BLASTn tool. To ensure the high accuracy of the results, the parameters were set to match the lengthof greater than 200bp havingsimilarity of 85% or more withthe threshold value ofE-10. Besides this, the normal sequence encodedby the ndhBgene and the encoded protein after RNA editing was used for comparison study regarding domain detection, transmebrane detection and formation of secondary structure. The protein transmembrane segment prediction was performed using TMHMM ServerV.2.0 (http://genome.cbs.dtu.dk/services/TMHMM/)whileSMART tool (http://smart.embl-heidelberg.de/) was used to analyze protein-specific domains. Moreover NPS (http://npsa-pbil.ibcp.fr/cgi-bin) online tool was utilized to analyze protein secondary structure composition. At present, RNA editing sites present inchoroplast genomesofrye, rice, sugarcane, maizeand spike spp. have been reported in literature.In order to study the common characteristics, patterns and conservatism of chloroplast RNA editing, the chloroplast RNA editing sites of these species were compared with the predicted RNA editing sites of barley.Results showed that 37 editing sites were found from 16 genes, and all of them are Cto U conversion. Among them, 5 sites were located in the first position of codon, 32 sites located in the second position of codon and no RNA editing sites were found in the third position of codon. Through BLAST search in the EST sequences of barley in NCBI database, 34 sites in 16 genes were validated as the actual RNA editing sites, of which thendhBhad the most editing sites (9 sites). Furthermore, the secondary structures and the transmenbrane domains of editedndhB protein were analyzed using the bioinformatics methods. Results indicated that editing inndhB-467resulted in an increase in transmembrane protein domains and editing inndhB-149changed the secondary structure. Finally, the comparison analysis of the editing sites in barley with other 5grass chloroplast genomes was also performed. Our study analyzed the composition and distribution of RNA editing in barley chloroplast genome comprehensively, which provided the useful information for exploring the function and mechanism of RNA editing, aswell as for revealing the origin and molecular evolution of RNA editing in organelle genomes.