Identification Of Chromosomal Rearrangements In Wheat And Genes Involved In Starch Metabolism In Barley

The newly published survey sequences of bread wheat (’Chinese Spring’,’CS’) have been facilitating isolating genes related to important traits, analyzing genome sequences and constructing physical and genetic maps. Due to the highly conserved gene orders among different grass genomes, comparative information has been widely used to order and orientate contigs in wheat genome sequence assemblies. However, previous studies have shown some structural differences among chromosomes of the three bread wheat subgenomes. Previous analyses of chromosomal rearrangements based on cytology and marker gene locations had limitations. Recent developments in genome sequencing offer an excellent opportunity to characterize these translocations at the gene level. Based on the available sequence data from wheat and its relativities, we extensively analysed the chromosomal translocations and inversions in the bread wheat genotype’CS’. As the fourth major cereal crop produced in the world, barley has been utilized mainly for malting and brewing and as animal feed. Starch, the main constituent of barley grains (60-64%of kernel dry weight), is the most abundant storage reserve carbohydrate and can be used as our nutrition and as a feedstock for bioethanol production in industry. Here, we systematically identified several key genes related to starch metabolism from barley, aiming at comprehensively understanding regulation of these genes as a step toward at genetically modifying them for improving starch. Main conclusions derived from thess studies include:1. We have identified sequences bordering each of the main translocation and inversion breakpoints on chromosomes 4A,5A and 7B of the modern bread wheat genome. The locations of these breakpoints allow, for the first time, a detailed description of the evolutionary origins of these chromosomes at the gene level. Results from this study also demonstrate that, although the strategy of exploiting sorted chromosome arms has dramatically simplified the efforts of wheat genome sequencing, simultaneous analysis of sequences from homoeologous and non-homoeologous chromosomes is essential in understanding the origins of DNA sequences in polyploid species.2. Based on patterns of their homoeologous arm locations,551 genes indicated the presence of pericentric inversions in at least 10 of the 21 chromosomes. Available data from deletion bin-mapped expressed sequence tags and genetic mapping in wheat indicated that all inversions had breakpoints in the low-recombinant gene-poor pericentromeric regions. The large number of putative intrachromosomal rearrangements suggests the presence of extensive structural differences among the three subgenomes, at least some of which likely occurred during the production of the aneuploid lines of this hexaploid wheat genotype. These differences could have significant implications in wheat genome research where comparative approaches are used such as in ordering and orientating sequence contigs and in gene cloning.3. A total of 720 of genes representing interchromosomal rearrangements in wheat were identified. They were located on each of the 42 chromosome arms. About 58% of these translocated genes were those involved in the well-characterized translocations involving chromosomes 4A,5A and 7B. The other 42% of the genes represent a large numbers of putative translocations which have not yet been described. Surprisingly, when calculated by the numbers of events the distribution of these interchromosomal rearrangements was not significantly biased toward any of the three subgenomes although the times of interaction between the A and B subgenomes almost doubled that between either of them and the D subgenome. The possible existence of such a large number of interchromosomal rearrangements provide further evidence that caution should be taken when using synteny in ordering sequence contigs or in cloning genes in hexaploid wheat. The identification of these putative translocations in’CS’also provide a base for a systematic evaluation of their presence or absence in the full spectrum of bread wheat and its close relatives, which could have significant implications in a wide array of fields ranging from classification to practical breeding.4. We isolated and characterized waxy alleles of three waxy (GSHO 908, GSHO 1828 and NA 40) and two non-waxy barley accessions (PI 483237 and Clho 15773), estimated the expression patterns of waxy genes via real-time quantitative PCR (RT-qPCR), investigated promoter activity by analysing promoter-GUS expression, and examined possible effects of waxy alleles on starch granule morphology in barley accessions by scanning electron microscopy (SEM). A 193-bp insertion in introns 1, a 15-bp insertion in the coding region, and some single nucleotide polymorphic sites were detected in the waxy barley accessions. In addition, a 397-bp deletion containing the TATA box, transcription starting point, exon 1 and partial intron 1 were also identified in the waxy barley accessions. RT-qPCR analysis showed that waxy accessions had lower waxy expression levels than those of non-waxy accessions. Transient expression assays showed that GUS activity driven by the 1029-bp promoter of the non-waxy accessions was stronger than that driven by the 822-bp promoter of the waxy accessions. SEM revealed no apparent differences of starch granule morphology between waxy and non-waxy accessions. Our results showed that the 397-bp deletion identified in the waxy barley accessions is likely responsible for the reduction of waxy transcript, leading to lower concentrations of GBSS I protein thus lower amylase content.5. hi this study, we determined the amylose content, characterized granule-binding proteins, analyzed the expression of key genes involved in starch synthesis, and examined starch granule structure of both normal (Bowman and Morex) and shrunken endosperm(seg1, seg3, seg4a, seg4b, seg5, seg6, seg7, and sex1) barley accessions. Our results showed that amylose contents of shrunken endosperm mutants ranged from 8.9%(seg4a) to 25.8%(seg1). SDS-PAGE analysis revealed that an 87 kDa proteins corresponding to the starch branching enzyme Ⅱ (SBEII) and starch synthase Ⅱ (SSII) were not present in seg1, seg3, seg6, and seg1 mutants. Real-time quantitative PCR (RT-qPCR) analysis indicated that waxy expression levels of segl, seg3, seg6, and seg1 mutants decreased in varying degrees to lower leveles until 27 days after antheis (DAA) after reaching the peak at 15-21 DAA, which differed from the pattern of normal barley accessions. Further characteriztion of waxy alleles revealed 7 non-synonymous single nucleotide polymorphisms (SNPs) in the coding sequences and 16 SNPs and 8 indels in the promoter sequences of the mutants. Results from starch granule by scanning electron microscopy (SEM) indicated that, in comparison with normal barley accessions, seg4a, seg4b, and sexl had fewer starch granules per grain; seg3 and seg6 had less small B-type granules; some large A-type granules in seg1 had a hollow surface. These results improve our understanding about effects of seg and sex mutants on starch biosynthesis and granule structure during endosperm development and provide information for identification of key genes responsible for these shrunken endosperm mutants.6. We isolated starch phosphorylase genes (Phol and Pho1) from barley, characterized the gene and protein structures, predicated their promoter’s cis-elements and analysed expression patterns. Multiple alignments of these genes showed that 1) both Phol and Pho2 genes possess 15 exons and 14 introns in all but three of the species analysed, Aegilops tacushcii (for Phol which contains 16 exons and 15 introns), potato (for Pholb which contains 14 exons and 13 introns), and Triticum uraru (for Pho2 which contains 15 exons and 14 introns); 2) the exon-intron junctions of Phol and Pho2 flanking the ligand-binding sites are more conservative than the other regions. Analysis of protein sequences revealed that Phol and Pho2 were highly homologous except for two regions, the N terminal domain and the L78 insertion region. The results of real-time quantitative PCR (RT-qPCR) indicated that Pho2 is mainly expressed in germinating seeds, and the expression of Phol is similar to that of starch synthesis genes during seed development in barley. Microarray-based analysis indicated that 1) the accumulation of Phol or Pho2 transcripts exhibited uniform pattern both in various tissues and various stages of seed development among species of barley, rice, and Arabidopsis; 2) Phol of barley was significantly down-regulated under cold and drought treatments, and up-regulated under stem rust infection. Pho2 exhibited similar expression to Phol in barley. However, significant difference in expression was not detected for either Phol or Pho2 under any of the investigated abiotic stresses. In Arabidopsis, significant down-regulation was detected for Phol (PHS1) under abscisic acid (ABA) and for Pho2 (PHS2) under cold, salt, and ABA. Our results provide valuable information to genetically manipulate phosphorylase genes and to further elucidate their regulatory mechanism in the starch biosynthetic pathway.7. In this study, we isolated and chromosomally mapped barley SEX4, characterized its gene and protein structure, predicted the cis-elements of its promoter, and analysed its expression based on RT-PCR and publically available microarray data. The full length of barely SEX4 (HvSEX4) was 4,598 bp and it was mapped on the long arm of chromosome 4H (4HL). This gene contained 14 exons and 13 introns in all but two of the species analysed, Arabidopsis (13 exons and 12 introns) and Oryza brachyantha (12 exons and 11 introns). An exon-intron junction composed of intron 4 to intron 7 and exon 5 to exon 8 was highly conserved among the analysed species. SEX4 is characterized with conserved functional domains (dual specificity phosphatase domain and carbohydrate-binding module 48) and varied chloroplast transit peptide and C-terminal. Expression analyses indicated that (1) SEX4 was mainly expressed in anthers of barley, young leaf and anthers of rice, and leaf of Arabidopsis; (2) it exhibited a diurnal pattern in barley, rice and Arabidopsis; (3) significant difference in the expression of SEX4 was not detected for either barley or rice under any of the investigated stresses; and (4) it was significantly down-regulated at middle stage and up-regulated at late stage under cold treatment, down-regulated at early stage under heat treatment, and up-regulated at late stage under salt treatment in Arabidopsis. The strong relationships detected in the current study between SEX4 and glucan, water dikinases (GWD) or phosphoglucan, water dikinases (PWD) were discussed. Collectively, our results provide insights into genetic manipulation of SEX4, especially in monocotyledon and uncovering the possible roles of SEX4 in plant development.8. We identified and mapped the LSF1 and LSF2 genes in barley(HvLSF1 and HvLSF2), characterized structures of these genes and proteins, predicted the cis-elements of their promoters, and analysed their expression patterns. HvLSFl and HvLSF2 were mapped on the long arm of chromosome 1H (1HL) and 5H (5HL), respectively. Our results revealed varied exon-intron structures and conserved exon-intron junctions in both LSF1 and LSF2 from a range of species. Alignment of protein sequences indicated that cTP and CT domains are much less varied than the functional domains (PDZ, DPS and CBM48). LSF2 was mainly expressed in anthers of barley and rice, and in leaf of Arabidopsis. LSF1 was mainly expressed in endosperm of barley and leaf of Arabidopsis and rice. The expression of LSF1 exhibited a diurnal pattern in rice only and that of LSF2 in both rice and Arabidopsis. Of the investigated stresses, only cold stress significantly reduced expression level of LSF1 and LSF2 in barley and LSF2 in Arabidopsis at late stages of the treatments. While heat treatment significantly decreased expression levels of LSF1 at middle stage (4 h) of a treatment in Arabidopsis only. The strong relationships detected between LSF2 and starch excess4 (SEX4), glucan, water dikinases (GWD) or phosphoglucan, water dikinases (PWD) were identified and discussed. Taken together, these results provide information of genetic manipulation of LSF1 and LSF2, especially in monocotyledon and further elucidate their regulatory mechanism in plant development.