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1.  Relationship between homoeologous regulatory and structural genes in allopolyploid genome – A case study in bread wheat 
BMC Plant Biology  2008;8:88.
Background
The patterns of expression of homoeologous genes in hexaploid bread wheat have been intensively studied in recent years, but the interaction between structural genes and their homoeologous regulatory genes remained unclear. The question was as to whether, in an allopolyploid, this interaction is genome-specific, or whether regulation cuts across genomes. The aim of the present study was cloning, sequence analysis, mapping and expression analysis of F3H (flavanone 3-hydroxylase – one of the key enzymes in the plant flavonoid biosynthesis pathway) homoeologues in bread wheat and study of the interaction between F3H and their regulatory genes homoeologues – Rc (red coleoptiles).
Results
PCR-based cloning of F3H sequences from hexaploid bread wheat (Triticum aestivum L.), a wild tetraploid wheat (T. timopheevii) and their putative diploid progenitors was employed to localize, physically map and analyse the expression of four distinct bread wheat F3H copies. Three of these form a homoeologous set, mapping to the chromosomes of homoeologous group 2; they are highly similar to one another at the structural and functional levels. However, the fourth copy is less homologous, and was not expressed in anthocyanin pigmented coleoptiles. The presence of dominant alleles at the Rc-1 homoeologous loci, which are responsible for anthocyanin pigmentation in the coleoptile, was correlated with F3H expression in pigmented coleoptiles. Each dominant Rc-1 allele affected the expression of the three F3H homoeologues equally, but the level of F3H expression was dependent on the identity of the dominant Rc-1 allele present. Thus, the homoeologous Rc-1 genes contribute more to functional divergence than do the structural F3H genes.
Conclusion
The lack of any genome-specific relationship between F3H-1 and Rc-1 implies an integrative evolutionary process among the three diploid genomes, following the formation of hexaploid wheat. Regulatory genes probably contribute more to the functional divergence between the wheat genomes than do the structural genes themselves. This is in line with the growing consensus which suggests that although heritable morphological traits are determined by the expression of structural genes, it is the regulatory genes which are the prime determinants of allelic identity.
doi:10.1186/1471-2229-8-88
PMCID: PMC2538534  PMID: 18700978
2.  Molecular analysis of phosphomannomutase (PMM) genes reveals a unique PMM duplication event in diverse Triticeae species and the main PMM isozymes in bread wheat tissues 
BMC Plant Biology  2010;10:214.
Background
Phosphomannomutase (PMM) is an essential enzyme in eukaryotes. However, little is known about PMM gene and function in crop plants. Here, we report molecular evolutionary and biochemical analysis of PMM genes in bread wheat and related Triticeae species.
Results
Two sets of homoeologous PMM genes (TaPMM-1 and 2) were found in bread wheat, and two corresponding PMM genes were identified in the diploid progenitors of bread wheat and many other diploid Triticeae species. The duplication event yielding PMM-1 and 2 occurred before the radiation of diploid Triticeae genomes. The PMM gene family in wheat and relatives may evolve largely under purifying selection. Among the six TaPMM genes, the transcript levels of PMM-1 members were comparatively high and their recombinant proteins were all enzymatically active. However, PMM-2 homoeologs exhibited lower transcript levels, two of which were also inactive. TaPMM-A1, B1 and D1 were probably the main active isozymes in bread wheat tissues. The three isozymes differed from their counterparts in barley and Brachypodium distachyon in being more tolerant to elevated test temperatures.
Conclusion
Our work identified the genes encoding PMM isozymes in bread wheat and relatives, uncovered a unique PMM duplication event in diverse Triticeae species, and revealed the main active PMM isozymes in bread wheat tissues. The knowledge obtained here improves the understanding of PMM evolution in eukaryotic organisms, and may facilitate further investigations of PMM function in the temperature adaptability of bread wheat.
doi:10.1186/1471-2229-10-214
PMCID: PMC3017832  PMID: 20920368
3.  Sequencing of Chloroplast Genomes from Wheat, Barley, Rye and Their Relatives Provides a Detailed Insight into the Evolution of the Triticeae Tribe 
PLoS ONE  2014;9(3):e85761.
Using Roche/454 technology, we sequenced the chloroplast genomes of 12 Triticeae species, including bread wheat, barley and rye, as well as the diploid progenitors and relatives of bread wheat Triticum urartu, Aegilops speltoides and Ae. tauschii. Two wild tetraploid taxa, Ae. cylindrica and Ae. geniculata, were also included. Additionally, we incorporated wild Einkorn wheat Triticum boeoticum and its domesticated form T. monococcum and two Hordeum spontaneum (wild barley) genotypes. Chloroplast genomes were used for overall sequence comparison, phylogenetic analysis and dating of divergence times. We estimate that barley diverged from rye and wheat approximately 8–9 million years ago (MYA). The genome donors of hexaploid wheat diverged between 2.1–2.9 MYA, while rye diverged from Triticum aestivum approximately 3–4 MYA, more recently than previously estimated. Interestingly, the A genome taxa T. boeoticum and T. urartu were estimated to have diverged approximately 570,000 years ago. As these two have a reproductive barrier, the divergence time estimate also provides an upper limit for the time required for the formation of a species boundary between the two. Furthermore, we conclusively show that the chloroplast genome of hexaploid wheat was contributed by the B genome donor and that this unknown species diverged from Ae. speltoides about 980,000 years ago. Additionally, sequence alignments identified a translocation of a chloroplast segment to the nuclear genome which is specific to the rye/wheat lineage. We propose the presented phylogeny and divergence time estimates as a reference framework for future studies on Triticeae.
doi:10.1371/journal.pone.0085761
PMCID: PMC3948623  PMID: 24614886
4.  Genome-wide analysis of the rice and arabidopsis non-specific lipid transfer protein (nsLtp) gene families and identification of wheat nsLtp genes by EST data mining 
BMC Genomics  2008;9:86.
Background
Plant non-specific lipid transfer proteins (nsLTPs) are encoded by multigene families and possess physiological functions that remain unclear. Our objective was to characterize the complete nsLtp gene family in rice and arabidopsis and to perform wheat EST database mining for nsLtp gene discovery.
Results
In this study, we carried out a genome-wide analysis of nsLtp gene families in Oryza sativa and Arabidopsis thaliana and identified 52 rice nsLtp genes and 49 arabidopsis nsLtp genes. Here we present a complete overview of the genes and deduced protein features. Tandem duplication repeats, which represent 26 out of the 52 rice nsLtp genes and 18 out of the 49 arabidopsis nsLtp genes identified, support the complexity of the nsLtp gene families in these species. Phylogenetic analysis revealed that rice and arabidopsis nsLTPs are clustered in nine different clades. In addition, we performed comparative analysis of rice nsLtp genes and wheat (Triticum aestivum) EST sequences indexed in the UniGene database. We identified 156 putative wheat nsLtp genes, among which 91 were found in the 'Chinese Spring' cultivar. The 122 wheat non-redundant nsLTPs were organized in eight types and 33 subfamilies. Based on the observation that seven of these clades were present in arabidopsis, rice and wheat, we conclude that the major functional diversification within the nsLTP family predated the monocot/dicot divergence. In contrast, there is no type VII nsLTPs in arabidopsis and type IX nsLTPs were only identified in arabidopsis. The reason for the larger number of nsLtp genes in wheat may simply be due to the hexaploid state of wheat but may also reflect extensive duplication of gene clusters as observed on rice chromosomes 11 and 12 and arabidopsis chromosome 5.
Conclusion
Our current study provides fundamental information on the organization of the rice, arabidopsis and wheat nsLtp gene families. The multiplicity of nsLTP types provide new insights on arabidopsis, rice and wheat nsLtp gene families and will strongly support further transcript profiling or functional analyses of nsLtp genes. Until such time as specific physiological functions are defined, it seems relevant to categorize plant nsLTPs on the basis of sequence similarity and/or phylogenetic clustering.
doi:10.1186/1471-2164-9-86
PMCID: PMC2277411  PMID: 18291034
5.  A high-throughput method for the detection of homoeologous gene deletions in hexaploid wheat 
BMC Plant Biology  2010;10:264.
Background
Mutational inactivation of plant genes is an essential tool in gene function studies. Plants with inactivated or deleted genes may also be exploited for crop improvement if such mutations/deletions produce a desirable agronomical and/or quality phenotype. However, the use of mutational gene inactivation/deletion has been impeded in polyploid plant species by genetic redundancy, as polyploids contain multiple copies of the same genes (homoeologous genes) encoded by each of the ancestral genomes. Similar to many other crop plants, bread wheat (Triticum aestivum L.) is polyploid; specifically allohexaploid possessing three progenitor genomes designated as 'A', 'B', and 'D'. Recently modified TILLING protocols have been developed specifically for mutation detection in wheat. Whilst extremely powerful in detecting single nucleotide changes and small deletions, these methods are not suitable for detecting whole gene deletions. Therefore, high-throughput methods for screening of candidate homoeologous gene deletions are needed for application to wheat populations generated by the use of certain mutagenic agents (e.g. heavy ion irradiation) that frequently generate whole-gene deletions.
Results
To facilitate the screening for specific homoeologous gene deletions in hexaploid wheat, we have developed a TaqMan qPCR-based method that allows high-throughput detection of deletions in homoeologous copies of any gene of interest, provided that sufficient polymorphism (as little as a single nucleotide difference) amongst homoeologues exists for specific probe design. We used this method to identify deletions of individual TaPFT1 homoeologues, a wheat orthologue of the disease susceptibility and flowering regulatory gene PFT1 in Arabidopsis. This method was applied to wheat nullisomic-tetrasomic lines as well as other chromosomal deletion lines to locate the TaPFT1 gene to the long arm of chromosome 5. By screening of individual DNA samples from 4500 M2 mutant wheat lines generated by heavy ion irradiation, we detected multiple mutants with deletions of each TaPFT1 homoeologue, and confirmed these deletions using a CAPS method. We have subsequently designed, optimized, and applied this method for the screening of homoeologous deletions of three additional wheat genes putatively involved in plant disease resistance.
Conclusions
We have developed a method for automated, high-throughput screening to identify deletions of individual homoeologues of a wheat gene. This method is also potentially applicable to other polyploidy plants.
doi:10.1186/1471-2229-10-264
PMCID: PMC3017838  PMID: 21114819
6.  Molecular cloning, phylogenetic analysis, and expression profiling of endoplasmic reticulum molecular chaperone BiP genes from bread wheat (Triticum aestivum L.) 
BMC Plant Biology  2014;14(1):260.
Background
The endoplasmic reticulum chaperone binding protein (BiP) is an important functional protein, which is involved in protein synthesis, folding assembly, and secretion. In order to study the role of BiP in the process of wheat seed development, we cloned three BiP homologous cDNA sequences in bread wheat (Triticum aestivum), completed by rapid amplification of cDNA ends (RACE), and examined the expression of wheat BiP in wheat tissues, particularly the relationship between BiP expression and the subunit types of HMW-GS using near-isogenic lines (NILs) of HMW-GS silencing, and under abiotic stress.
Results
Sequence analysis demonstrated that all BiPs contained three highly conserved domains present in plants, animals, and microorganisms, indicating their evolutionary conservation among different biological species. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that TaBiP (Triticum aestivum BiP) expression was not organ-specific, but was predominantly localized to seed endosperm. Furthermore, immunolocalization confirmed that TaBiP was primarily located within the protein bodies (PBs) in wheat endosperm. Three TaBiP genes exhibited significantly down-regulated expression following high molecular weight-glutenin subunit (HMW-GS) silencing. Drought stress induced significantly up-regulated expression of TaBiPs in wheat roots, leaves, and developing grains.
Conclusions
The high conservation of BiP sequences suggests that BiP plays the same role, or has common mechanisms, in the folding and assembly of nascent polypeptides and protein synthesis across species. The expression of TaBiPs in different wheat tissue and under abiotic stress indicated that TaBiP is most abundant in tissues with high secretory activity and with high proportions of cells undergoing division, and that the expression level of BiP is associated with the subunit types of HMW-GS and synthesis. The expression of TaBiPs is developmentally regulated during seed development and early seedling growth, and under various abiotic stresses.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0260-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-014-0260-0
PMCID: PMC4189733  PMID: 25273817
Wheat; BiP; Cloning; Expression; HMW-GS silencing; Drought stress
7.  Modes of Gene Duplication Contribute Differently to Genetic Novelty and Redundancy, but Show Parallels across Divergent Angiosperms 
PLoS ONE  2011;6(12):e28150.
Background
Both single gene and whole genome duplications (WGD) have recurred in angiosperm evolution. However, the evolutionary effects of different modes of gene duplication, especially regarding their contributions to genetic novelty or redundancy, have been inadequately explored.
Results
In Arabidopsis thaliana and Oryza sativa (rice), species that deeply sample botanical diversity and for which expression data are available from a wide range of tissues and physiological conditions, we have compared expression divergence between genes duplicated by six different mechanisms (WGD, tandem, proximal, DNA based transposed, retrotransposed and dispersed), and between positional orthologs. Both neo-functionalization and genetic redundancy appear to contribute to retention of duplicate genes. Genes resulting from WGD and tandem duplications diverge slowest in both coding sequences and gene expression, and contribute most to genetic redundancy, while other duplication modes contribute more to evolutionary novelty. WGD duplicates may more frequently be retained due to dosage amplification, while inferred transposon mediated gene duplications tend to reduce gene expression levels. The extent of expression divergence between duplicates is discernibly related to duplication modes, different WGD events, amino acid divergence, and putatively neutral divergence (time), but the contribution of each factor is heterogeneous among duplication modes. Gene loss may retard inter-species expression divergence. Members of different gene families may have non-random patterns of origin that are similar in Arabidopsis and rice, suggesting the action of pan-taxon principles of molecular evolution.
Conclusion
Gene duplication modes differ in contribution to genetic novelty and redundancy, but show some parallels in taxa separated by hundreds of millions of years of evolution.
doi:10.1371/journal.pone.0028150
PMCID: PMC3229532  PMID: 22164235
8.  Evolutionary history of Methyltransferase 1 genes in hexaploid wheat 
BMC Genomics  2014;15(1):922.
Background
Plant and animal methyltransferases are key enzymes involved in DNA methylation at cytosine residues, required for gene expression control and genome stability. Taking advantage of the new sequence surveys of the wheat genome recently released by the International Wheat Genome Sequencing Consortium, we identified and characterized MET1 genes in the hexaploid wheat Triticum aestivum (TaMET1).
Results
Nine TaMET1 genes were identified and mapped on homoeologous chromosome groups 2A/2B/2D, 5A/5B/5D and 7A/7B/7D. Synteny analysis and evolution rates suggest that the genome organization of TaMET1 genes results from a whole genome duplication shared within the grass family, and a second gene duplication, which occurred specifically in the Triticeae tribe prior to the speciation of diploid wheat. Higher expression levels were observed for TaMET1 homoeologous group 2 genes compared to group 5 and 7, indicating that group 2 homoeologous genes are predominant at the transcriptional level, while group 5 evolved into pseudogenes. We show the connection between low expression levels, elevated evolution rates and unexpected enrichment in CG-dinucleotides (CG-rich isochores) at putative promoter regions of homoeologous group 5 and 7, but not of group 2 TaMET1 genes. Bisulfite sequencing reveals that these CG-rich isochores are highly methylated in a CG context, which is the expected target of TaMET1.
Conclusions
We retraced the evolutionary history of MET1 genes in wheat, explaining the predominance of group 2 homoeologous genes and suggest CG-DNA methylation as one of the mechanisms involved in wheat genome dynamics.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-922) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-922
PMCID: PMC4223845  PMID: 25342325
DNA methylation; Evolution; Genome dynamics; CG-rich isochores
9.  Intraspecific sequence comparisons reveal similar rates of non-collinear gene insertion in the B and D genomes of bread wheat 
BMC Plant Biology  2012;12:155.
Background
Polyploidization is considered one of the main mechanisms of plant genome evolution. The presence of multiple copies of the same gene reduces selection pressure and permits sub-functionalization and neo-functionalization leading to plant diversification, adaptation and speciation. In bread wheat, polyploidization and the prevalence of transposable elements resulted in massive gene duplication and movement. As a result, the number of genes which are non-collinear to genomes of related species seems markedly increased in wheat.
Results
We used new-generation sequencing (NGS) to generate sequence of a Mb-sized region from wheat chromosome arm 3DS. Sequence assembly of 24 BAC clones resulted in two scaffolds of 1,264,820 and 333,768 bases. The sequence was annotated and compared to the homoeologous region on wheat chromosome 3B and orthologous loci of Brachypodium distachyon and rice. Among 39 coding sequences in the 3DS scaffolds, 32 have a homoeolog on chromosome 3B. In contrast, only fifteen and fourteen orthologs were identified in the corresponding regions in rice and Brachypodium, respectively. Interestingly, five pseudogenes were identified among the non-collinear coding sequences at the 3B locus, while none was found at the 3DS locus.
Conclusion
Direct comparison of two Mb-sized regions of the B and D genomes of bread wheat revealed similar rates of non-collinear gene insertion in both genomes with a majority of gene duplications occurring before their divergence. Relatively low proportion of pseudogenes was identified among non-collinear coding sequences. Our data suggest that the pseudogenes did not originate from insertion of non-functional copies, but were formed later during the evolution of hexaploid wheat. Some evidence was found for gene erosion along the B genome locus.
doi:10.1186/1471-2229-12-155
PMCID: PMC3445842  PMID: 22935214
Wheat; BAC sequencing; Homoeologous genomes; Gene duplication; Non-collinear genes; Allopolyploidy
10.  The Protein Disulfide Isomerase gene family in bread wheat (T. aestivum L.) 
BMC Plant Biology  2010;10:101.
Background
The Protein Disulfide Isomerase (PDI) gene family encodes several PDI and PDI-like proteins containing thioredoxin domains and controlling diversified metabolic functions, including disulfide bond formation and isomerisation during protein folding. Genomic, cDNA and promoter sequences of the three homoeologous wheat genes encoding the "typical" PDI had been cloned and characterized in a previous work. The purpose of present research was the cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species.
Results
Eight new non-homoeologous wheat genes were cloned and characterized. The nine PDI and PDI-like sequences of wheat were located in chromosome regions syntenic to those in rice and assigned to eight plant phylogenetic groups. The nine wheat genes differed in their sequences, genomic organization as well as in the domain composition and architecture of their deduced proteins; conversely each of them showed high structural conservation with genes from other plant species in the same phylogenetic group. The extensive quantitative RT-PCR analysis of the nine genes in a set of 23 wheat samples, including tissues and developmental stages, showed their constitutive, even though highly variable expression.
Conclusions
The nine wheat genes showed high diversity, while the members of each phylogenetic group were highly conserved even between taxonomically distant plant species like the moss Physcomitrella patens. Although constitutively expressed the nine wheat genes were characterized by different expression profiles reflecting their different genomic organization, protein domain architecture and probably promoter sequences; the high conservation among species indicated the ancient origin and diversification of the still evolving gene family. The comprehensive structural and expression characterization of the complete set of PDI and PDI-like wheat genes represents a basis for the functional characterization of this gene family in the hexaploid context of bread wheat.
doi:10.1186/1471-2229-10-101
PMCID: PMC3017771  PMID: 20525253
11.  Physical mapping of a large plant genome using global high-information-content-fingerprinting: the distal region of the wheat ancestor Aegilops tauschii chromosome 3DS 
BMC Genomics  2010;11:382.
Background
Physical maps employing libraries of bacterial artificial chromosome (BAC) clones are essential for comparative genomics and sequencing of large and repetitive genomes such as those of the hexaploid bread wheat. The diploid ancestor of the D-genome of hexaploid wheat (Triticum aestivum), Aegilops tauschii, is used as a resource for wheat genomics. The barley diploid genome also provides a good model for the Triticeae and T. aestivum since it is only slightly larger than the ancestor wheat D genome. Gene co-linearity between the grasses can be exploited by extrapolating from rice and Brachypodium distachyon to Ae. tauschii or barley, and then to wheat.
Results
We report the use of Ae. tauschii for the construction of the physical map of a large distal region of chromosome arm 3DS. A physical map of 25.4 Mb was constructed by anchoring BAC clones of Ae. tauschii with 85 EST on the Ae. tauschii and barley genetic maps. The 24 contigs were aligned to the rice and B. distachyon genomic sequences and a high density SNP genetic map of barley. As expected, the mapped region is highly collinear to the orthologous chromosome 1 in rice, chromosome 2 in B. distachyon and chromosome 3H in barley. However, the chromosome scale of the comparative maps presented provides new insights into grass genome organization. The disruptions of the Ae. tauschii-rice and Ae. tauschii-Brachypodium syntenies were identical. We observed chromosomal rearrangements between Ae. tauschii and barley. The comparison of Ae. tauschii physical and genetic maps showed that the recombination rate across the region dropped from 2.19 cM/Mb in the distal region to 0.09 cM/Mb in the proximal region. The size of the gaps between contigs was evaluated by comparing the recombination rate along the map with the local recombination rates calculated on single contigs.
Conclusions
The physical map reported here is the first physical map using fingerprinting of a complete Triticeae genome. This study demonstrates that global fingerprinting of the large plant genomes is a viable strategy for generating physical maps. Physical maps allow the description of the co-linearity between wheat and grass genomes and provide a powerful tool for positional cloning of new genes.
doi:10.1186/1471-2164-11-382
PMCID: PMC2900270  PMID: 20553621
12.  Genetic interactions reveal the evolutionary trajectories of duplicate genes 
Duplicate genes show significantly fewer interactions than singleton genes, and functionally similar duplicates can exhibit dissimilar profiles because common interactions are ‘hidden' due to buffering.Genetic interaction profiles provide insights into evolutionary mechanisms of duplicate retention by distinguishing duplicates under dosage selection from those retained because of some divergence in function.The genetic interactions of duplicate genes evolve in an extremely asymmetric way and the directionality of this asymmetry correlates well with other evolutionary properties of duplicate genes.Genetic interaction profiles can be used to elucidate the divergent function of specific duplicate pairs.
Gene duplication and divergence serves as a primary source for new genes and new functions, and as such has broad implications on the evolutionary process. Duplicate genes within S. cerevisiae have been shown to retain a high degree of similarity with regard to many of their functional properties (Papp et al, 2004; Guan et al, 2007; Wapinski et al, 2007; Musso et al, 2008), and perturbation of duplicate genes has been shown to result in smaller fitness defects than singleton genes (Gu et al, 2003; DeLuna et al, 2008; Dean et al, 2008; Musso et al, 2008). Individual genetic interactions between pairs of genes and profiles of such interactions across the entire genome provide a new context in which to examine the properties of duplicate compensation.
In this study we use the most recent and comprehensive set of genetic interactions in yeast produced to date (Costanzo et al, 2010) to address questions of duplicate retention and redundancy. We show that the ability for duplicate genes to buffer the deletion of a partner has three main consequences. First it agrees with previous work demonstrating that a high proportion of duplicate pairs are synthetic lethal, a classic indication of the ability to buffer one another functionally (DeLuna et al, 2008; Dean et al, 2008; Musso et al, 2008). Second, it reduces the number of genetic interactions observed between duplicate genes and the rest of the genome by masking interactions relating to common function from experimental detection. Third, this buffering of common interactions serves to reduce profile similarity in spite of common function (Figure 1). The compensatory ability of functionally similar duplicates buffers genetic interactions related to their common function (reducing the number of genetic interactions overall), while allowing the measurement of interactions related to any divergent function. Thus, even functionally similar duplicates may have dissimilar genetic interaction profiles. As previously surmised (Ihmels et al, 2007), duplicate genes under selection for dosage amplification have differing profile characteristics. We show that dosage-mediated duplicates have much higher genetic interaction profile similarity than do other duplicate pairs. Furthermore, we show in a comparison with local neighbors on a protein–protein interaction (PPI) network, that although dosage-mediated duplicates more often have higher similarity to each other than they do to their neighbors, the reverse is true for duplicates in general. That is, slightly divergent duplicate genes more often exhibit a higher similarity with a common neighbor on the PPI network than they do with each other, and that observation is consistent with the idea that common interactions are buffered while interactions corresponding to divergent functions are observed.
We then asked whether duplicates' genetic interactions that are not buffered appear in a symmetric or an asymmetric fashion. Previous work has established asymmetric patterns with regard to PPI degree (Wagner, 2002; He and Zhang, 2005), sequence divergence (Conant and Wagner, 2003; Zhang et al, 2003; Kellis et al, 2004; Scannell and Wolfe, 2008) and expression patterns (Gu et al, 2002b; Tirosh and Barkai, 2007). Although genetic interactions are further removed from mechanism than protein–protein interactions, for example, they do offer a more direct measurement of functional consequence and, thus, may give a better indication of the functional differences between a duplicate pair. We found that duplicates exhibit a strikingly asymmetric pattern of genetic interactions, with the ratio of interactions between sisters commonly exceeding 7:1 (Figure 4A). The observations differ significantly from random simulations in which genetic interactions were redistributed between sisters with equal probability (Figure 4A). Moreover, the directionality of this interaction asymmetry agrees with other physiological properties of duplicate pairs. For example, the sister with more genetic interactions also tends to have more protein–protein interactions and also tends to evolve at a slower rate (Figure 4B).
Genetic interaction degree and profiles can be used to understand the functional divergence of particular duplicates pairs. As a case example, we consider the whole-genome-duplication pair CIK1–VIK1. Each of these genes encode proteins that form distinct heterodimeric complexes with the microtubule motor protein Kar3 (Manning et al, 1999). Although each of these proteins depend on a direct physical interaction with Kar3, Cik1 has a much higher profile similarity to Kar3 than does Vik1 (r=0.5 and r=0.3, respectively). Consistent with its higher similarity, Δcik1 and Δkar3 exhibit several similar phenotypes, including abnormally short spindles, chromosome loss and delayed cell cycle progression (Page et al, 1994; Manning et al, 1999). In contrast, a Δvik1 mutant strain exhibits no overt phenotype (Manning et al, 1999).
The characterization of functional redundancy and divergence between duplicate genes is an important step in understanding the evolution of genetic systems. Large-scale genetic network analysis in Saccharomyces cerevisiae provides a powerful perspective for addressing these questions through quantitative measurements of genetic interactions between pairs of duplicated genes, and more generally, through the study of genome-wide genetic interaction profiles associated with duplicated genes. We show that duplicate genes exhibit fewer genetic interactions than other genes because they tend to buffer one another functionally, whereas observed interactions are non-overlapping and reflect their divergent roles. We also show that duplicate gene pairs are highly imbalanced in their number of genetic interactions with other genes, a pattern that appears to result from asymmetric evolution, such that one duplicate evolves or degrades faster than the other and often becomes functionally or conditionally specialized. The differences in genetic interactions are predictive of differences in several other evolutionary and physiological properties of duplicate pairs.
doi:10.1038/msb.2010.82
PMCID: PMC3010121  PMID: 21081923
duplicate genes; functional divergence; genetic interactions; paralogs; Saccharomyces cerevisiae
13.  Multiple Mechanisms Promote the Retained Expression of Gene Duplicates in the Tetraploid Frog Xenopus laevis 
PLoS Genetics  2006;2(4):e56.
Gene duplication provides a window of opportunity for biological variants to persist under the protection of a co-expressed copy with similar or redundant function. Duplication catalyzes innovation (neofunctionalization), subfunction degeneration (subfunctionalization), and genetic buffering (redundancy), and the genetic survival of each paralog is triggered by mechanisms that add, compromise, or do not alter protein function. We tested the applicability of three types of mechanisms for promoting the retained expression of duplicated genes in 290 expressed paralogs of the tetraploid clawed frog, Xenopus laevis. Tests were based on explicit expectations concerning the ka/ks ratio, and the number and location of nonsynonymous substitutions after duplication. Functional constraints on the majority of paralogs are not significantly different from a singleton ortholog. However, we recover strong support that some of them have an asymmetric rate of nonsynonymous substitution: 6% match predictions of the neofunctionalization hypothesis in that (1) each paralog accumulated nonsynonymous substitutions at a significantly different rate and (2) the one that evolves faster has a higher ka/ks ratio than the other paralog and than a singleton ortholog. Fewer paralogs (3%) exhibit a complementary pattern of substitution at the protein level that is predicted by enhancement or degradation of different functional domains, and the remaining 13% have a higher average ka/ks ratio in both paralogs that is consistent with altered functional constraints, diversifying selection, or activity-reducing mutations after duplication. We estimate that these paralogs have been retained since they originated by genome duplication between 21 and 41 million years ago. Multiple mechanisms operate to promote the retained expression of duplicates in the same genome, in genes in the same functional class, over the same period of time following duplication, and sometimes in the same pair of paralogs. None of these paralogs are superfluous; degradation or enhancement of different protein subfunctions and neofunctionalization are plausible hypotheses for the retained expression of some of them. Evolution of most X. laevis paralogs, however, is consistent with retained expression via mechanisms that do not radically alter functional constraints, such as selection to preserve post-duplication stoichiometry or temporal, quantitative, or spatial subfunctionalization.
Synopsis
Gene duplication plays a fundamental role in biological innovation but it is not clear how both copies of a duplicated gene manage to circumvent degradation by mutation if neither is unique. This study explores genetic mechanisms that could make each copy of a duplicate gene different, and therefore distinguishable and potentially preserved by natural selection. It is based on DNA sequences of the protein-coding region of 290 expressed duplicated genes in a frog, Xenopus laevis, that underwent complete duplication of its entire genome. Results provide evidence for multiple mechanisms acting within the same genome, within the same functional classes of genes, within the same period of time following duplication, and even on the same set of duplicated genes. Each copy of a duplicate gene may be subject to distinct evolutionary constraints, and this could be associated with degradation or enhancement of function. Functional constraints of most of these duplicates, however, are not substantially different from a single copy gene; their persistence in the first dozens of millions of years after duplication may more frequently be explained by mechanisms acting on their expression rather than their function.
doi:10.1371/journal.pgen.0020056
PMCID: PMC1449897  PMID: 16683033
14.  Pervasive and Persistent Redundancy among Duplicated Genes in Yeast 
PLoS Genetics  2008;4(7):e1000113.
The loss of functional redundancy is the key process in the evolution of duplicated genes. Here we systematically assess the extent of functional redundancy among a large set of duplicated genes in Saccharomyces cerevisiae. We quantify growth rate in rich medium for a large number of S. cerevisiae strains that carry single and double deletions of duplicated and singleton genes. We demonstrate that duplicated genes can maintain substantial redundancy for extensive periods of time following duplication (∼100 million years). We find high levels of redundancy among genes duplicated both via the whole genome duplication and via smaller scale duplications. Further, we see no evidence that two duplicated genes together contribute to fitness in rich medium substantially beyond that of their ancestral progenitor gene. We argue that duplicate genes do not often evolve to behave like singleton genes even after very long periods of time.
Author Summary
Gene duplication is the primary source of new genes. To persist, duplicated genes must lose some of the original redundancy either by partitioning the ancestral function (subfunctionalization) or by gaining new non-redundant functions (neofunctionalization). The extent to which these processes shape the evolution of duplicated genes over long periods of time is unknown. We investigate these questions experimentally by building strains carrying single and double gene deletions of duplicated genes and measuring their growth rates in rich medium. Using these data, we determine that many duplicated genes are functionally redundant to a substantial degree. We also investigate how often duplicated genes gain new functionality. We demonstrate that the fitness effects of double deletions of duplicate genes are indistinguishable from our best estimate of the fitness effects of deletions of their ancestral singleton genes. We therefore argue that many duplicate genes do not gain substantial new functionality at least in the rich medium. Our results suggest that subfunctionalization does not generally proceed to completion, even after very long periods of time, and that neofunctionalization is either rare or of little consequence, at least under some growth conditions.
doi:10.1371/journal.pgen.1000113
PMCID: PMC2440806  PMID: 18604285
15.  Structural Analysis of the Wheat Genes Encoding NADH-Dependent Glutamine-2-oxoglutarate Amidotransferases and Correlation with Grain Protein Content 
PLoS ONE  2013;8(9):e73751.
Background
Nitrogen uptake and the efficient absorption and metabolism of nitrogen are essential elements in attempts to breed improved cereal cultivars for grain or silage production. One of the enzymes related to nitrogen metabolism is glutamine-2-oxoglutarate amidotransferase (GOGAT). Together with glutamine synthetase (GS), GOGAT maintains the flow of nitrogen from NH4+ into glutamine and glutamate, which are then used for several aminotransferase reactions during amino acid synthesis.
Results
The aim of the present work was to identify and analyse the structure of wheat NADH-GOGAT genomic sequences, and study the expression in two durum wheat cultivars characterized by low and high kernel protein content. The genomic sequences of the three homoeologous A, B and D NADH-GOGAT genes were obtained for hexaploid Triticum aestivum and the tetraploid A and B genes of Triticum turgidum ssp. durum. Analysis of the gene sequences indicates that all wheat NADH-GOGAT genes are composed of 22 exons and 21 introns. The three hexaploid wheat homoeologous genes have high conservation of sequence except intron 13 which shows differences in both length and sequence. A comparative analysis of sequences among di- and mono-cotyledonous plants shows both regions of high conservation and of divergence. qRT-PCR performed with the two durum wheat cvs Svevo and Ciccio (characterized by high and low protein content, respectively) indicates different expression levels of the two NADH-GOGAT-3A and NADH-GOGAT-3B genes.
Conclusion
The three hexaploid wheat homoeologous NADH-GOGAT gene sequences are highly conserved – consistent with the key metabolic role of this gene. However, the dicot and monocot amino acid sequences show distinctive patterns, particularly in the transit peptide, the exon 16–17 junction, and the C-terminus. The lack of conservation in the transit peptide may indicate subcellular differences between the two plant divisions - while the sequence conservation within enzyme functional domains remains high. Higher expression levels of NADH-GOGAT are associated with higher grain protein content in two durum wheats.
doi:10.1371/journal.pone.0073751
PMCID: PMC3775782  PMID: 24069228
16.  iTRAQ-based quantitative proteome and phosphoprotein characterization reveals the central metabolism changes involved in wheat grain development 
BMC Genomics  2014;15(1):1029.
Background
Wheat (Triticum aestivum L.) is an economically important grain crop. Two-dimensional gel-based approaches are limited by the low identification rate of proteins and lack of accurate protein quantitation. The recently developed isobaric tag for relative and absolute quantitation (iTRAQ) method allows sensitive and accurate protein quantification. Here, we performed the first iTRAQ-based quantitative proteome and phosphorylated proteins analyses during wheat grain development.
Results
The proteome profiles and phosphoprotein characterization of the metabolic proteins during grain development of the elite Chinese bread wheat cultivar Yanyou 361 were studied using the iTRAQ-based quantitative proteome approach, TiO2 microcolumns, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among 1,146 non-redundant proteins identified, 421 showed at least 2-fold differences in abundance, and they were identified as differentially expressed proteins (DEPs), including 256 upregulated and 165 downregulated proteins. Of the 421 DEPs, six protein expression patterns were identified, most of which were up, down, and up-down expression patterns. The 421 DEPs were classified into nine functional categories mainly involved in different metabolic processes and located in the membrane and cytoplasm. Hierarchical clustering analysis indicated that the DEPs involved in starch biosynthesis, storage proteins, and defense/stress-related proteins significantly accumulated at the late grain development stages, while those related to protein synthesis/assembly/degradation and photosynthesis showed an opposite expression model during grain development. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 12 representative genes encoding different metabolic proteins showed certain transcriptional and translational expression differences during grain development. Phosphorylated proteins analyses demonstrated that 23 DEPs such as AGPase, sucrose synthase, Hsp90, and serpins were phosphorylated in the developing grains and were mainly involved in starch biosynthesis and stress/defense.
Conclusions
Our results revealed a complex quantitative proteome and phosphorylation profile during wheat grain development. Numerous DEPs are involved in grain starch and protein syntheses as well as adverse defense, which set an important basis for wheat yield and quality. Particularly, some key DEPs involved in starch biosynthesis and stress/defense were phosphorylated, suggesting their roles in wheat grain development.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1029) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-1029
PMCID: PMC4301063  PMID: 25427527
Wheat; Grain proteome; iTRAQ; Phosphoproteins; qRT-PCR
17.  Genome-wide transcriptome study in wheat identified candidate genes related to processing quality, majority of them showing interaction (quality x development) and having temporal and spatial distributions 
BMC Genomics  2014;15:29.
Background
The cultivated bread wheat (Triticum aestivum L.) possesses unique flour quality, which can be processed into many end-use food products such as bread, pasta, chapatti (unleavened flat bread), biscuit, etc. The present wheat varieties require improvement in processing quality to meet the increasing demand of better quality food products. However, processing quality is very complex and controlled by many genes, which have not been completely explored. To identify the candidate genes whose expressions changed due to variation in processing quality and interaction (quality x development), genome-wide transcriptome studies were performed in two sets of diverse Indian wheat varieties differing for chapatti quality. It is also important to understand the temporal and spatial distributions of their expressions for designing tissue and growth specific functional genomics experiments.
Results
Gene-specific two-way ANOVA analysis of expression of about 55 K transcripts in two diverse sets of Indian wheat varieties for chapatti quality at three seed developmental stages identified 236 differentially expressed probe sets (10-fold). Out of 236, 110 probe sets were identified for chapatti quality. Many processing quality related key genes such as glutenin and gliadins, puroindolines, grain softness protein, alpha and beta amylases, proteases, were identified, and many other candidate genes related to cellular and molecular functions were also identified. The ANOVA analysis revealed that the expression of 56 of 110 probe sets was involved in interaction (quality x development). Majority of the probe sets showed differential expression at early stage of seed development i.e. temporal expression. Meta-analysis revealed that the majority of the genes expressed in one or a few growth stages indicating spatial distribution of their expressions. The differential expressions of a few candidate genes such as pre-alpha/beta-gliadin and gamma gliadin were validated by RT-PCR. Therefore, this study identified several quality related key genes including many other genes, their interactions (quality x development) and temporal and spatial distributions.
Conclusions
The candidate genes identified for processing quality and information on temporal and spatial distributions of their expressions would be useful for designing wheat improvement programs for processing quality either by changing their expression or development of single nucleotide polymorphisms (SNPs) markers.
doi:10.1186/1471-2164-15-29
PMCID: PMC3897974  PMID: 24433256
Wheat; Chapatti; Processing quality; Development; Interaction; Transcriptome; Gene expression
18.  Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae 
Key message
A cytogenetic map of wheat was constructed using FISH with cDNA probes. FISH markers detected homoeology and chromosomal rearrangements of wild relatives, an important source of genes for wheat improvement.
Abstract
To transfer agronomically important genes from wild relatives to bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) by induced homoeologous recombination, it is important to know the chromosomal relationships of the species involved. Fluorescence in situ hybridization (FISH) can be used to study chromosome structure. The genomes of allohexaploid bread wheat and other species from the Triticeae tribe are colinear to some extent, i.e., composed of homoeoloci at similar positions along the chromosomes, and with genic regions being highly conserved. To develop cytogenetic markers specific for genic regions of wheat homoeologs, we selected more than 60 full-length wheat cDNAs using BLAST against mapped expressed sequence tags and used them as FISH probes. Most probes produced signals on all three homoeologous chromosomes at the expected positions. We developed a wheat physical map with several cDNA markers located on each of the 14 homoeologous chromosome arms. The FISH markers confirmed chromosome rearrangements within wheat genomes and were successfully used to study chromosome structure and homoeology in wild Triticeae species. FISH analysis detected 1U-6U chromosome translocation in the genome of Aegilops umbellulata, showed colinearity between chromosome A of Ae. caudata and group-1 wheat chromosomes, and between chromosome arm 7S#3L of Thinopyrum intermedium and the long arm of the group-7 wheat chromosomes.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-013-2253-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s00122-013-2253-z
PMCID: PMC3931928  PMID: 24408375
19.  Global transgenerational gene expression dynamics in two newly synthesized allohexaploid wheat (Triticum aestivum) lines 
BMC Biology  2012;10:3.
Background
Alteration in gene expression resulting from allopolyploidization is a prominent feature in plants, but its spectrum and extent are not fully known. Common wheat (Triticum aestivum) was formed via allohexaploidization about 10,000 years ago, and became the most important crop plant. To gain further insights into the genome-wide transcriptional dynamics associated with the onset of common wheat formation, we conducted microarray-based genome-wide gene expression analysis on two newly synthesized allohexaploid wheat lines with chromosomal stability and a genome constitution analogous to that of the present-day common wheat.
Results
Multi-color GISH (genomic in situ hybridization) was used to identify individual plants from two nascent allohexaploid wheat lines between Triticum turgidum (2n = 4x = 28; genome BBAA) and Aegilops tauschii (2n = 2x = 14; genome DD), which had a stable chromosomal constitution analogous to that of common wheat (2n = 6x = 42; genome BBAADD). Genome-wide analysis of gene expression was performed for these allohexaploid lines along with their parental plants from T. turgidum and Ae. tauschii, using the Affymetrix Gene Chip Wheat Genome-Array. Comparison with the parental plants coupled with inclusion of empirical mid-parent values (MPVs) revealed that whereas the great majority of genes showed the expected parental additivity, two major patterns of alteration in gene expression in the allohexaploid lines were identified: parental dominance expression and non-additive expression. Genes involved in each of the two altered expression patterns could be classified into three distinct groups, stochastic, heritable and persistent, based on their transgenerational heritability and inter-line conservation. Strikingly, whereas both altered patterns of gene expression showed a propensity of inheritance, identity of the involved genes was highly stochastic, consistent with the involvement of diverse Gene Ontology (GO) terms. Nonetheless, those genes showing non-additive expression exhibited a significant enrichment for vesicle-function.
Conclusions
Our results show that two patterns of global alteration in gene expression are conditioned by allohexaploidization in wheat, that is, parental dominance expression and non-additive expression. Both altered patterns of gene expression but not the identity of the genes involved are likely to play functional roles in stabilization and establishment of the newly formed allohexaploid plants, and hence, relevant to speciation and evolution of T. aestivum.
doi:10.1186/1741-7007-10-3
PMCID: PMC3313882  PMID: 22277161
20.  Patterns of homoeologous gene expression shown by RNA sequencing in hexaploid bread wheat 
BMC Genomics  2014;15:276.
Background
Bread wheat (Triticum aestivum) has a large, complex and hexaploid genome consisting of A, B and D homoeologous chromosome sets. Therefore each wheat gene potentially exists as a trio of A, B and D homoeoloci, each of which may contribute differentially to wheat phenotypes. We describe a novel approach combining wheat cytogenetic resources (chromosome substitution ‘nullisomic-tetrasomic’ lines) with next generation deep sequencing of gene transcripts (RNA-Seq), to directly and accurately identify homoeologue-specific single nucleotide variants and quantify the relative contribution of individual homoeoloci to gene expression.
Results
We discover, based on a sample comprising ~5-10% of the total wheat gene content, that at least 45% of wheat genes are expressed from all three distinct homoeoloci. Most of these genes show strikingly biased expression patterns in which expression is dominated by a single homoeolocus. The remaining ~55% of wheat genes are expressed from either one or two homoeoloci only, through a combination of extensive transcriptional silencing and homoeolocus loss.
Conclusions
We conclude that wheat is tending towards functional diploidy, through a variety of mechanisms causing single homoeoloci to become the predominant source of gene transcripts. This discovery has profound consequences for wheat breeding and our understanding of wheat evolution.
doi:10.1186/1471-2164-15-276
PMCID: PMC4023595  PMID: 24726045
Wheat; Wheat transcriptome; mRNA-Seq; Diploidization; Homoeologues; Polyploidy
21.  Next-generation sequencing of flow-sorted wheat chromosome 5D reveals lineage-specific translocations and widespread gene duplications 
BMC Genomics  2014;15(1):1080.
Background
The ~17 Gb hexaploid bread wheat genome is a high priority and a major technical challenge for genomic studies. In particular, the D sub-genome is relatively lacking in genetic diversity, making it both difficult to map genetically, and a target for introgression of agriculturally useful traits. Elucidating its sequence and structure will therefore facilitate wheat breeding and crop improvement.
Results
We generated shotgun sequences from each arm of flow-sorted Triticum aestivum chromosome 5D using 454 FLX Titanium technology, giving 1.34× and 1.61× coverage of the short (5DS) and long (5DL) arms of the chromosome respectively. By a combination of sequence similarity and assembly-based methods, ~74% of the sequence reads were classified as repetitive elements, and coding sequence models of 1314 (5DS) and 2975 (5DL) genes were generated. The order of conserved genes in syntenic regions of previously sequenced grass genomes were integrated with physical and genetic map positions of 518 wheat markers to establish a virtual gene order for chromosome 5D.
Conclusions
The virtual gene order revealed a large-scale chromosomal rearrangement in the peri-centromeric region of 5DL, and a concentration of non-syntenic genes in the telomeric region of 5DS. Although our data support the large-scale conservation of Triticeae chromosome structure, they also suggest that some regions are evolving rapidly through frequent gene duplications and translocations.
Sequence accessions
EBI European Nucleotide Archive, Study no. ERP002330
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1080) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-1080
PMCID: PMC4298962  PMID: 25487001
Wheat genome; Chromosome sorting; Triticum aestivum; Genome zipper; Triticeae genome; Chromosome arm shotgun; Comparative grass genomics
22.  Wheat beta-expansin (EXPB11) genes: Identification of the expressed gene on chromosome 3BS carrying a pollen allergen domain 
BMC Plant Biology  2010;10:99.
Background
Expansins form a large multi-gene family found in wheat and other cereal genomes that are involved in the expansion of cell walls as a tissue grows. The expansin family can be divided up into two main groups, namely, alpha-expansin (EXPA) and beta-expansin proteins (EXPB), with the EXPB group being of particular interest as group 1-pollen allergens.
Results
In this study, three beta-expansin genes were identified and characterized from a newly sequenced region of the Triticum aestivum cv. Chinese Spring chromosome 3B physical map at the Sr2 locus (FPC contig ctg11). The analysis of a 357 kb sub-sequence of FPC contig ctg11 identified one beta-expansin genes to be TaEXPB11, originally identified as a cDNA from the wheat cv Wyuna. Through the analysis of intron sequences of the three wheat cv. Chinese Spring genes, we propose that two of these beta-expansin genes are duplications of the TaEXPB11 gene. Comparative sequence analysis with two other wheat cultivars (cv. Westonia and cv. Hope) and a Triticum aestivum var. spelta line validated the identification of the Chinese Spring variant of TaEXPB11. The expression in maternal and grain tissues was confirmed by examining EST databases and carrying out RT-PCR experiments. Detailed examination of the position of TaEXPB11 relative to the locus encoding Sr2 disease resistance ruled out the possibility of this gene directly contributing to the resistance phenotype.
Conclusions
Through 3-D structural protein comparisons with Zea mays EXPB1, we proposed that variations within the coding sequence of TaEXPB11 in wheats may produce a functional change within features such as domain 1 related to possible involvement in cell wall structure and domain 2 defining the pollen allergen domain and binding to IgE protein. The variation established in this gene suggests it is a clearly identifiable member of a gene family and reflects the dynamic features of the wheat genome as it adapted to a range of different environments and uses.
Accession Numbers: ctg11 =FN564426
Survey sequences of TaEXPB11ws and TsEXPB11 are provided request.
doi:10.1186/1471-2229-10-99
PMCID: PMC2887456  PMID: 20507562
23.  Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of celiac disease epitopes 
BMC Genomics  2006;7:1.
Background
Bread wheat (Triticum aestivum) is an important staple food. However, wheat gluten proteins cause celiac disease (CD) in 0.5 to 1% of the general population. Among these proteins, the α-gliadins contain several peptides that are associated to the disease.
Results
We obtained 230 distinct α-gliadin gene sequences from severaldiploid wheat species representing the ancestral A, B, and D genomes of the hexaploid bread wheat. The large majority of these sequences (87%) contained an internal stop codon. All α-gliadin sequences could be distinguished according to the genome of origin on the basis of sequence similarity, of the average length of the polyglutamine repeats, and of the differences in the presence of four peptides that have been identified as T cell stimulatory epitopes in CD patients through binding to HLA-DQ2/8. By sequence similarity, α-gliadins from the public database of hexaploid T. aestivum could be assigned directly to chromosome 6A, 6B, or 6D. T. monococcum (A genome) sequences, as well as those from chromosome 6A of bread wheat, almost invariably contained epitope glia-α9 and glia-α20, but never the intact epitopes glia-α and glia-α2. A number of sequences from T. speltoides, as well as a number of sequences fromchromosome 6B of bread wheat, did not contain any of the four T cell epitopes screened for. The sequences from T. tauschii (D genome), as well as those from chromosome 6D of bread wheat, were found to contain all of these T cell epitopes in variable combinations per gene. The differences in epitope composition resulted mainly from point mutations. These substitutions appeared to be genome specific.
Conclusion
Our analysis shows that α-gliadin sequences from the three genomes of bread wheat form distinct groups. The four known T cell stimulatory epitopes are distributed non-randomly across the sequences, indicating that the three genomes contribute differently to epitope content. A systematic analysis of all known epitopes in gliadins and glutenins will lead to better understanding of the differences in toxicity among wheat varieties. On the basis of such insight, breeding strategies can be designed to generate less toxic varieties of wheat which may be tolerated by at least part of the CD patient population.
doi:10.1186/1471-2164-7-1
PMCID: PMC1368968  PMID: 16403227
24.  Unlocking Triticeae genomics to sustainably feed the future 
Plant and Cell Physiology  2013;54(12):1931-1950.
The tribe Triticeae includes the major crops wheat and barley. Within the last few years, the whole genomes of four Triticeae species—barley, wheat, Tausch’s goatgrass (Aegilops tauschii) and wild einkorn wheat (Triticum urartu)—have been sequenced. The availability of these genomic resources for Triticeae plants and innovative analytical applications using next-generation sequencing technologies are helping to revitalize our approaches in genetic work and to accelerate improvement of the Triticeae crops. Comparative genomics and integration of genomic resources from Triticeae plants and the model grass Brachypodium distachyon are aiding the discovery of new genes and functional analyses of genes in Triticeae crops. Innovative approaches and tools such as analysis of next-generation populations, evolutionary genomics and systems approaches with mathematical modeling are new strategies that will help us discover alleles for adaptive traits to future agronomic environments. In this review, we provide an update on genomic tools for use with Triticeae plants and Brachypodium and describe emerging approaches toward crop improvements in Triticeae.
doi:10.1093/pcp/pct163
PMCID: PMC3856857  PMID: 24204022
Barley; Brachypodium; Crop improvement; Next-generation sequencing; Triticeae; Wheat
25.  Backup without redundancy: genetic interactions reveal the cost of duplicate gene loss 
We show that genetic interaction profiles offer a powerful approach to elicit phenotypes that are far richer than is attainable using single gene deletions. This has allowed us to address the long-standing question of the role played by duplicate genes (paralogs) in robustness against deletion.We provide for the first time direct evidence that the capacity of some duplicates to cover for the loss of their paralogs can account for the observed difference in fitness between duplicate and singleton deletions mutants, but that the overall contribution of this effect to dispensability is small.More broadly, we demonstrate that paralogs possessing apparent backup capacity in some environments have in fact distinct and non-overlapping functions, and are unable to provide backup across a range of compromising conditions. This resolves the previous paradox of how backup genes conferring dispensability can nevertheless be independently maintained in the population.From a practical point of view, our findings suggest efficient strategies to elicit rich deletion phenotypes that should be highly relevant for the design of future phenotypic screens.
Much of our understanding of biological processes has been derived from the characterization of the functional consequence to an organism of altering one or more of its genes. Efforts to systematically evaluate the phenotypic effects of gene loss, however, have been hampered by the fact that the disruption of most genes has surprisingly modest effects on cell growth and viability. The high proportion of genes with no apparent deletion effect has wide-ranging practical and theoretical implications and has been the subject of considerable interest (Wagner, 2000, 2005; Giaever et al, 2002; Gu et al, 2003; Papp et al, 2004; Kafri et al, 2005). One factor that has been implicated as contributing to the high degree of dispensability is the abundance of closely related paralogs present in most genomes (Winzeler et al, 1999; Wagner, 2000; Giaever et al, 2002). Indeed, recent work in S. cerevisiae has shown that the existence of a paralog elsewhere in the genome significantly increases the chance that deletion of a given gene has little effect on growth (Gu et al, 2003). However, current analyses have been mostly correlative, and direct mechanistic evidence supporting or refuting the role of backup compensation in mutational robustness is still largely missing. Furthermore, backup between duplicates is not easily justified in evolutionary terms, in that a genuine ability to comprehensively cover for the loss of another gene is evolutionarily unstable (Brookfield, 1992).
Here, we exploit the recent availability of high-density quantitative genetic interaction profiles (EMAPs) to address these issues directly. To test whether SSL paralogs can account for the excess fitness of duplicates, we classified genes into fitness categories according to their deletion growth defect (Materials and methods). The subset of genes covered by our combined data set exhibits an over-representation of duplicate genes in the weak/no deletion phenotype (WNP) class similar to that reported previously (Gu et al, 2003) (Figure 1B). Strikingly, this difference corresponds to the number of WNP duplicates that have an SSL interaction with their corresponding paralog (Figure 1C). Our data thus provide direct evidence that it is indeed duplicate compensation that accounts for the observed difference in deletion growth defect between duplicates and singletons, at least for the genes covered by our data set.
Apart from the mechanism itself, the characteristic features of buffering duplicates have received considerable attention (Gu et al, 2003; Kafri et al, 2005; Wagner, 2005). Our data allowed us to unambiguously distinguish the subset of duplicates whose dispensability can be attributed to the existence of a backup paralog. The ability to identify backup duplicates directly put us in a position to study their features, and how they differ from other duplicates without buffering properties. In particular, we asked to what extent the observed buffering in rich media reflects functional similarity and a genuine ability to cover for the loss of a paralog in a broader range of conditions.
To assess the extent to which SSL duplicates can provide genuine backup under compromising conditions, we fist used genetic interaction profiles as a more stringent test for redundancy that assesses the effect of gene loss in the background of additional gene deletions. In contrast to the expectation that truly buffered duplicates should have few if any synthetic interactions, we find that the number is in fact substantial and often exceeds that of random genes and non-SSL duplicates (Figure 2B). Similarly, using a recent data set of sensitivity profiles of deletion strains to a range of agents and environments (Brown et al, 2006), we find that the deletion of SSL duplicates across a range of environments has on average no weaker (and in fact a slightly stronger) effect on cellular growth rate than that of non-SSL duplicates or random genes. Taken together, these findings suggest that the backup capacity of SSL duplicates is limited and not indicative of a comprehensive ability to cover for the loss of the paralogous partner.
We next tested the degree of functional similarity of buffering duplicates using similarity in genetic interaction as well as environmental sensitivity profiles as indicators of shared functionality (Tong et al, 2004; Schuldiner et al, 2005; Brown et al, 2006; Pan et al, 2006). In spite of their rich media buffering properties, we find that the interaction and sensitivity patterns of most SSL duplicates are divergent and are usually more similar to those of other, non-paralogous genes (Figure 2C and D; Supplementary Figure 10).
Lastly, in addition to our analysis of duplicate phenotypes, we used genetic interaction spectra as deletion phenotypes for generic genes whose single deletion in standard conditions has little measurable effect. As expected, genetic interactions provide a deletion phenotype for many more genes (80–90%) than single gene deletions in standard growth environments (Steinmetz et al, 2002), which yield a detectable growth defect only for 30–40% (Figure 4B). To assess whether these interactions reflect the cost of gene loss (gene importance), we asked if there is a relationship between the probability of a gene being retained between related species and its number of genetic interactions. Indeed, genetic interactivity exhibits a strong correlation with gene retention across related phyla (Figure 4C and Supplementary Figure 7), and predicts the likelihood of gene loss better than lethality/viability, quantitative growth deficiency or environmental specificity (Supplementary Figure 8). Thus, genetic interactions provide a cost of gene loss that effectively recapitulates evolutionary constraints. This is further supported by the observation that genetic interactions are significantly correlated with environmental sensitivity across a range of conditions. Thus, our findings suggest that for most genes there is a substantial cost of gene loss, even though this is often not reflected in single gene deletion tests carried out in standard conditions.
Many genes can be deleted with little phenotypic consequences. By what mechanism and to what extent the presence of duplicate genes in the genome contributes to this robustness against deletions has been the subject of considerable interest. Here, we exploit the availability of high-density genetic interaction maps to provide direct support for the role of backup compensation, where functionally overlapping duplicates cover for the loss of their paralog. However, we find that the overall contribution of duplicates to robustness against null mutations is low (∼25%). The ability to directly identify buffering paralogs allowed us to further study their properties, and how they differ from non-buffering duplicates. Using environmental sensitivity profiles as well as quantitative genetic interaction spectra as high-resolution phenotypes, we establish that even duplicate pairs with compensation capacity exhibit rich and typically non-overlapping deletion phenotypes, and are thus unable to comprehensively cover against loss of their paralog. Our findings reconcile the fact that duplicates can compensate for each other's loss under a limited number of conditions with the evolutionary instability of genes whose loss is not associated with a phenotypic penalty.
doi:10.1038/msb4100127
PMCID: PMC1847942  PMID: 17389874
duplication; evolution; genetic interactions; redundancy

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