Homoeologous Sequences Research Articles

Reshaped DNA methylation cooperating with homoeolog-divergent expression promotes improved root traits in synthesized tetraploid wheat.

Polyploidization is a major event driving plant evolution and domestication. However, how reshaped epigenetic modifications coordinate gene transcription to generate phenotypic variations during wheat polyploidization is currently elusive. Here, we profiled transcriptomes and DNA methylomes of two diploid wheat accessions (SlSl and AA) and their synthetic allotetraploid wheat line (SlSlAA), which displayed elongated root hair and improved root capability for nitrate uptake and assimilation after tetraploidization. Globally decreased DNA methylation levels with a reduced difference between subgenomes were observed in the roots of SlSlAA. DNA methylation changes in first exon showed strong connections with altered transcription during tetraploidization. Homoeolog-specific transcription was associated with biased DNA methylation as shaped by homoeologous sequence variation. The hypomethylated promoters showed significantly enriched binding sites for MYB, which may affect gene transcription in response to root hair growth. Two master regulators in root hair elongation pathway, AlCPC and TuRSL4, exhibited upregulated transcription levels accompanied by hypomethylation in promoter, which may contribute to the elongated root hair. The upregulated nitrate transporter genes, including NPFs and NRTs, also are significantly associated with hypomethylation, indicating an epigenetic-incorporated regulation manner in improving nitrogen use efficiency. Collectively, these results provided new insights into epigenetic changes in response to crop polyploidization and underscored the importance of epigenetic regulation in improving crop traits.

The structure of the tetraploid sour cherry 'Schattenmorelle' (Prunus cerasus L.) genome reveals insights into its segmental allopolyploid nature.

Sour cherry (Prunus cerasus L.) is an important allotetraploid cherry species that evolved in the Caspian Sea and Black Sea regions from a hybridization of the tetraploid ground cherry (Prunus fruticosa Pall.) and an unreduced pollen of the diploid sweet cherry (P. avium L.) ancestor. Details of when and where the evolution of this species occurred are unclear, as well as the effect of hybridization on the genome structure. To gain insight, the genome of the sour cherry cultivar 'Schattenmorelle' was sequenced using Illumina NovaSeqTM and Oxford Nanopore long-read technologies, resulting in a ~629-Mbp pseudomolecule reference genome. The genome could be separated into two subgenomes, with subgenome PceS_a originating from P. avium and subgenome PceS_f originating from P. fruticosa. The genome also showed size reduction compared to ancestral species and traces of homoeologous sequence exchanges throughout. Comparative analysis confirmed that the genome of sour cherry is segmental allotetraploid and evolved very recently in the past.

Recent advances in the genetics underlying wheat grain protein content and grain protein deviation in hexaploid wheat.

Wheat is one of the most important global crops and selection for better performance has been ongoing since ancient times. As a quantitative trait controlled by the interplay of several genomic loci and under the strong influence of the environment, grain protein content (GPC) is of major interest in breeding programs. Here, we review the most recent contributions to the genetics underlying wheat GPC and grain protein deviation (GPD, representing the relationship between grain protein content and yield), together with the performance of genomic prediction models characterizing these traits. A total of 364 significant loci related to GPC and GPD are positioned on the hexaploid wheat genome, highlighting genomic regions where significant independent QTL overlap, with special focus on two regions located on chromosomes 3A and 5A. Some of the corresponding homoeologous sequences co-locate with significant independent QTL reported on the B and D subgenomes. Overlapping independent QTL from different studies are indicative of genomic regions exhibiting stability across environments and genotypes, with promising candidates for improving grain quality.

Ectopic expression of TaOEP16-2-5B, a wheat plastid outer envelope protein gene, enhances heat and drought stress tolerance in transgenic Arabidopsis plants

Abiotic stresses, such as heat and drought, are major environmental factors restricting crop productivity and quality worldwide. A plastid outer envelope protein gene, TaOEP16-2, was identified from our previous transcriptome analysis [1,2]. In this study, the isolation and functional characterization of the TaOEP16-2 gene was reported. Three homoeologous sequences of TaOEP16-2 were isolated from hexaploid wheat, which were localized on the chromosomes 5A, 5B and 5D, respectively. These three homoeologues exhibited different expression patterns under heat stress conditions, TaOEP16-2-5B was the dominant one, and TaOEP16-2-5B was selected for further analysis. Compared with wild type (WT) plants, transgenic Arabidopsis plants overexpressing the TaOEP16-2-5B gene exhibited enhanced tolerance to heat stress, which was supported by improved survival rate, strengthened cell membrane stability and increased sucrose content. It was also found that TaOEP16-2 was induced by drought stress and involved in drought stress tolerance. TaOEP16-2-5B has the same function in ABA-controlled seed germination as AtOEP16-2. Our results suggest that TaOEP16-2-5B plays an important role in heat and drought stress tolerance, and could be utilized in transgenic breeding of wheat and other crop plants.

High Transferability of Homoeolog-Specific Markers between Bread Wheat and Newly Synthesized Hexaploid Wheat Lines.

Bread wheat (Triticum aestivum, 2n = 6x = 42, AABBDD) has a complex allohexaploid genome, which makes it difficult to differentiate between the homoeologous sequences and assign them to the chromosome A, B, or D subgenomes. The chromosome-based draft genome sequence of the ‘Chinese Spring’ common wheat cultivar enables the large-scale development of polymerase chain reaction (PCR)-based markers specific for homoeologs. Based on high-confidence ‘Chinese Spring’ genes with known functions, we developed 183 putative homoeolog-specific markers for chromosomes 4B and 7B. These markers were used in PCR assays for the 4B and 7B nullisomes and their euploid synthetic hexaploid wheat (SHW) line that was newly generated from a hybridization between Triticum turgidum (AABB) and the wild diploid species Aegilops tauschii (DD). Up to 64% of the markers for chromosomes 4B or 7B in the SHW background were confirmed to be homoeolog-specific. Thus, these markers were highly transferable between the ‘Chinese Spring’ bread wheat and SHW lines. Homoeolog-specific markers designed using genes with known functions may be useful for genetic investigations involving homoeologous chromosome tracking and homoeolog expression and interaction analyses.

Phylogeny and molecular evolution of the DMC1 gene in the polyploid genus Leymus (Triticeae: Poaceae) and its diploid relatives

AbstractThe level and pattern of nucleotide variation in duplicate genes provide important information on the evolutionary history of polyploids and divergent processes between homoeologous loci within lineages. Leymus, a group of allopolyploid species with the NsXm genomes, is a perennial genus with a diverse array of morphology, ecology, and distribution in Triticeae. To estimate the phylogeny and molecular evolution of a single‐copy DMC1 gene in Leymus and its diploid relatives, DMC1 homoeologous sequences were isolated from the sampled Leymus species and were analyzed with those from 30 diploid taxa representing 18 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that: (i) different Leymus species might derive their Ns genome from different Psathyrostachys species; (ii) Pseudoroegneria has contributed to the nuclear genome of some Leymus species, which might result from recurrent hybridization or incomplete lineage sorting; (iii) the Xm genome origin of Leymus could differ among species; (iv) rapid radiation and multiple origin might account for the rich diversity, numbers of species, and wide ecological adaptation of Leymus species; and (v) the DMC1 sequence diversity of the Ns genome in Leymus species was lower than that in the Psathyrostachys diploids, while the level of DMC1 sequence diversity in Leymus was higher than that in diploid Pseudoroegneria. Our results provide new insight on the evolutionary dynamics of duplicate DMC1 genes, polyploid speciation, and the phylogeny of Leymus species.

Sequencing-based high throughput mutation detection in bread wheat

BackgroundForward genetic approaches have limited use for agronomic traits that can’t be reliably scored on a single plant basis. Thus, mutants in wheat and other crops are more useful for gene function studies by reverse genetic approach. With a long-term goal to develop a sequence-based mutation detection resource in hexaploid wheat, we conducted a feasibility study to accurately differentiate induced mutations from the homoeologs’ sequence variations present among the three wheat genomes.ResultsA reduced representation ApeKI library consisting of 21 Ethylmethane Sulfonate (EMS) induced mutants and two wild type cv. Indian plants was developed using individual barcode adapters and sequenced. A novel bioinformatics pipeline was developed to identify sequence variants using 178,464 wheat unigenes as a reference wheat transcriptome. In total, 14,130 mutational changes [Single Nucleotide Polymorphisms (SNPs) and Insertions/Deletions (INDELs)] and 150,511 homoeologous sequence changes were detected. On an average, 662 SNPs (ranging from 46 to 1,330) and 10 small INDELs (ranging from 0 to 23) were identified for each of the mutants. A mutation frequency of one per 5 Kb was observed with 70 % being transitions and 30 % transversions. The pipeline was tested using the known sequence changes in the three wheat genes. Genes present in the distal regions of the chromosomes were found to be more prone to EMS compared to genes present in the proximal regions. Redefined parameters identified a total of 28,348 mutational changes (1,349/plant).ConclusionsWe conclude that sequencing based mutation detection is a valuable method to identify induced mutations at large.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2112-1) contains supplementary material, which is available to authorized users.

A high-throughput pipeline for detecting locus-specific polymorphism in hexaploid wheat (Triticum aestivum L.).

BackgroundBread wheat (Triticum aestivum L., 2n = 6x = 42) is an allohexaploid with a huge genome. Due to the presence of extensive homoeologs and paralogs, generating locus-specific sequences can be challenging, especially when a large number of sequences are required. Traditional methods of generating locus-specific sequences are rather strenuous and time-consuming if large numbers of sequences are to be handled.ResultsTo improve the efficiency of isolating sequences for targeted loci, a time-saving and high-throughput pipeline integrating orthologous sequence alignment, genomic sequence retrieving, and multiple sequence alignment was developed. This pipeline was successfully employed in retrieving and aligning homoeologous sequences and 83% of the primers designed based on the pipeline successfully amplified fragments from the targeted subgenomes.ConclusionsThe high-throughput pipeline developed in this study makes it feasible to efficiently identify locus-specific sequences for large numbers of sequences. It could find applications in all research projects where locus-specific sequences are required. In addition to generating locus-specific markers, the pipeline was also used in our laboratory to identify differentially expressed genes among the three subgenomes of bread wheat. Importantly, the pipeline is not only valuable for research in wheat but should also be applicable to other allopolyploid species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-015-0082-6) contains supplementary material, which is available to authorized users.

HaploSNP affinities and linkage map positions illuminate subgenome composition in the octoploid, cultivated strawberry (Fragaria × ananassa)

The cultivated strawberry, Fragaria×ananassa possesses a genetically complex allo-octoploid genome. Advances in genomics research in Fragaria, including the release of a genome sequence for F. vesca, have permitted the development of a high throughput whole genome genotyping array for strawberry, which promises to facilitate genetics and genomics research. In this investigation, we used the Axiom® IStraw90® array for linkage map development, and produced a linkage map containing 8,407 SNP markers spanning 1,820cM. Whilst the linkage map provides good coverage of the genome of both parental genotypes, the map of ‘Monterey’ contained significantly fewer mapped markers than did that of ‘Darselect’. The array contains a novel marker class known as haploSNPs, which exploit homoeologous sequence variants as probe destabilization sites to effectively reduce marker ploidy. We examined these sites as potential indicators of subgenomic identities by using comparisons to allele states in two ancestral diploids. On this basis, haploSNP loci could be inferred to be derived from F. vesca, F. iinumae, or from an unknown source. When the identity classifications of haploSNPs were considered in conjunction with their respective linkage map positions, it was possible to define two discrete subgenomes, while the remaining homoeologues of each chromosome could not be partitioned into two discrete subgenomic groupings. These findings suggested a novel hypothesis regarding octoploid strawberry subgenome structure and evolutionary origins.

A bi-filtering method for processing single nucleotide polymorphism array data improves the quality of genetic map and accuracy of quantitative trait locus mapping in doubled haploid populations of polyploid Brassica napus

BackgroundSingle nucleotide polymorphism (SNP) markers have a wide range of applications in crop genetics and genomics. Due to their polyploidy nature, many important crops, such as wheat, cotton and rapeseed contain a large amount of repeat and homoeologous sequences in their genomes, which imposes a huge challenge in high-throughput genotyping with sequencing and/or array technologies. Allotetraploid Brassica napus (AACC, 2n = 4x = 38) comprises of two highly homoeologous sub-genomes derived from its progenitor species B. rapa (AA, 2n = 2x = 20) and B. oleracea (CC, 2n = 2x = 18), and is an ideal species to exploit methods for reducing the interference of extensive inter-homoeologue polymorphisms (mHemi-SNPs and Pseudo-simple SNPs) between closely related sub-genomes.ResultsBased on a recent B. napus 6K SNP array, we developed a bi-filtering procedure to identify unauthentic lines in a DH population, and mHemi-SNPs and Pseudo-simple SNPs in an array data matrix. The procedure utilized both monomorphic and polymorphic SNPs in the DH population and could effectively distinguish the mHemi-SNPs and Pseudo-simple SNPs that resulted from superposition of the signals from multiple SNPs. Compared with conventional procedure for array data processing, the bi-filtering method could minimize the pseudo linkage relationship caused by the mHemi-SNPs and Pseudo-simple SNPs, thus improving the quality of SNP genetic map. Furthermore, the improved genetic map could increase the accuracies of mapping of QTLs as demonstrated by the ability to eliminate non-real QTLs in the mapping population.ConclusionsThe bi-filtering analysis of the SNP array data represents a novel approach to effectively assigning the multi-loci SNP genotypes in polyploid B. napus and may find wide applications to SNP analyses in polyploid crops.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1559-4) contains supplementary material, which is available to authorized users.

Intergenomic single nucleotide polymorphisms as a tool for bacterial artificial chromosome contig building of homoeologous Brassica napus regions.

BackgroundHomoeologous sequences pose a particular challenge if bacterial artificial chromosome (BAC) contigs shall be established for specific regions of an allopolyploid genome. Single nucleotide polymorphisms (SNPs) differentiating between homoeologous genomes (intergenomic SNPs) may represent a suitable screening tool for such purposes, since they do not only identify homoeologous sequences but also differentiate between them.ResultsSequence alignments between Brassica rapa (AA) and Brassica oleracea (CC) sequences mapping to corresponding regions on chromosomes A1 and C1, respectively were used to identify single nucleotide polymorphisms between the A and C genomes. A large fraction of these polymorphisms was also present in Brassica napus (AACC), an allopolyploid species that originated from hybridisation of A and C genome species. Intergenomic SNPs mapping throughout homoeologous chromosome segments spanning approximately one Mbp each were included in Illumina’s GoldenGate® Genotyping Assay and used to screen multidimensional pools of a Brassica napus bacterial artificial chromosome library with tenfold genome coverage. Based on the results of 50 SNP assays, a BAC contig for the Brassica napus A subgenome was established that spanned the entire region of interest. The C subgenome region was represented in three BAC contigs.ConclusionsThis proof-of-concept study shows that sequence resources of diploid progenitor genomes can be used to deduce intergenomic SNPs suitable for multiplex polymerase chain reaction (PCR)-based screening of multidimensional BAC pools of a polyploid organism. Owing to their high abundance and ease of identification, intergenomic SNPs represent a versatile tool to establish BAC contigs for homoeologous regions of a polyploid genome.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-560) contains supplementary material, which is available to authorized users.

Phylogeny and molecular evolution of the Acc1 gene within the StH genome species in Triticeae (Poaceae)

To estimate the phylogeny and molecular evolution of a single-copy gene encoding plastid acetyl-CoA carboxylase (Acc1) within the StH genome species, two Acc1 homoeologous sequences were isolated from nearly all the sampled StH genome species and were analyzed with those from 35 diploid taxa representing 19 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1) the StH genome species from the same areas or neighboring geographic regions are closely related to each other; (2) the Acc1 gene sequences of the StH genome species from North America and Eurasia are evolutionarily distinct; (3) Dasypyrum has contributed to the nuclear genome of Elymus repens and Elymus mutabilis; (4) the StH genome polyploids have higher levels of sequence diversity in the H genome homoeolog than the St genome homoeolog; and (5) the Acc1 sequence may evolve faster in the polyploid species than in the diploids. Our result provides some insight on evolutionary dynamics of duplicate Acc1 gene, the polyploidy speciation and phylogeny of the StH genome species.

Molecular characterisation of two homoeologous elicitor-responsive lipin genes in cotton

The identification and molecular characterisation of two lipin-like gene copies (GhLIPN) in cotton, Gossypium hirsutum, an allotetraploid derived from two progenitor diploid Gossypium species, is described. Sequence analyses of the GhLIPN copies, designated GhLIPN-1 and -2, revealed that they contain 11 exons, separated by ten introns. They each have a 2,643bp open reading frame that encodes 880 aa proteins, and share a 97.7 and 95.5% sequence similarity at the translated nucleotide and amino acid level, respectively. The GhLIPN genes have a distinct domain architecture consisting of an archetypical N-terminal lipin domain, followed by a haloacid dehalogenase (HAD) domain towards the C-terminus. A Southern blot did not distinguish between the two gene copies, which suggests that they may be homoeologs rather than paralogs. GhLIPN-2 is more similar to a homoeologous sequence from G. raimondii, representing the ancestral D-genome, compared to GhLIPN-1 that matches G. herbaceum and that represents the A-genome. Our data indicate that GhLIPN-1 and GhLIPN-2 are homoeologs that derive from the A- and the D-diploid genomes, respectively. The promoter sequences of GhLIPN-1 and -2 differ by 56%, as a result of multiple indels. In silico analysis of the promoter regions revealed that both genes contain numerous putative defence-related and elicitor-responsive cis-elements that support a role for GhLIPN in defence responses. Relative quantification real-time PCR confirmed the up-regulation in response to a cell-wall-derived V. dahliae elicitor, which supported the association of GhLIPN with defence signalling. The results add a new dimension to the proposed roles of lipins in plants by suggesting that lipins may have a role in defence signalling.

Detection and genotyping of restriction fragment associated polymorphisms in polyploid crops with a pseudo-reference sequence: a case study in allotetraploid Brassica napus

BackgroundThe presence of homoeologous sequences and absence of a reference genome sequence make discovery and genotyping of single nucleotide polymorphisms (SNPs) more challenging in polyploid crops.ResultsTo address this challenge, we constructed reduced representation libraries (RRLs) for two Brassica napus inbred lines and their 91 doubled haploid (DH) progenies using a modified ddRADseq technique. A bioinformatics pipeline termed RFAPtools was developed to discover and genotype SNPs and presence/absence variations (PAVs). Using this pipeline, a pseudo-reference sequence (PRF) containing 180,991 sequence tags was constructed. By aligning sequence reads to the pseudo-reference sequence, allelic SNPs as well as PAVs were identified and genotyped with RFAPtools. Two parallel linkage maps, one SNP bin map containing 8,780 SNP loci and one PAV linkage map containing 12,423 dominant loci, were constructed. By aligning marker sequences to B. rapa sequence scaffolds, whose genome is available, we assigned 44 unassembled sequence scaffolds comprising 8.15 Mb onto the B. rapa chromosomes, and also identified 14 instances of misassembly and eight instances of mis-ordering sequence scaffolds.ConclusionsThese results indicate that the modified ddRADseq approach is a cost-effective and simple method to genotype tens of thousands SNPs and PAV markers in a polyploidy plant species. The results also demonstrated that RFAPtools developed in this study are powerful to mine allelic SNPs from homoeologous sequences in polyploids, therefore they are generally applicable in either diploid or polyploid species with or without a reference genome sequence.

Transcriptome de novo assembly from next-generation sequencing and comparative analyses in the hexaploid salt marsh species Spartina maritima and Spartina alterniflora (Poaceae)

Spartina species have a critical ecological role in salt marshes and represent an excellent system to investigate recurrent polyploid speciation. Using the 454 GS-FLX pyrosequencer, we assembled and annotated the first reference transcriptome (from roots and leaves) for two related hexaploid Spartina species that hybridize in Western Europe, the East American invasive Spartina alterniflora and the Euro-African S. maritima. The de novo read assembly generated 38 478 consensus sequences and 99% found an annotation using Poaceae databases, representing a total of 16 753 non-redundant genes. Spartina expressed sequence tags were mapped onto the Sorghum bicolor genome, where they were distributed among the subtelomeric arms of the 10 S. bicolor chromosomes, with high gene density correlation. Normalization of the complementary DNA library improved the number of annotated genes. Ecologically relevant genes were identified among GO biological function categories in salt and heavy metal stress response, C4 photosynthesis and in lignin and cellulose metabolism. Expression of some of these genes had been found to be altered by hybridization and genome duplication in a previous microarray-based study in Spartina. As these species are hexaploid, up to three duplicated homoeologs may be expected per locus. When analyzing sequence polymorphism at four different loci in S. maritima and S. alterniflora, we found up to four haplotypes per locus, suggesting the presence of two expressed homoeologous sequences with one or two allelic variants each. This reference transcriptome will allow analysis of specific Spartina genes of ecological or evolutionary interest, estimation of homoeologous gene expression variation using RNA-seq and further gene expression evolution analyses in natural populations.

Phylogeny and molecular evolution of the DMC1 gene within the StH genome species in Triticeae (Poaceae)

To estimate the phylogeny and molecular evolution of a single-copy nuclear disrupted meiotic cDNA (DMC1) gene within the StH genome species, two DMC1 homoeologous sequences were isolated from nearly all the sampled StH genome species and were analyzed with those from seven diploid taxa representing the St and H genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1) there is a close relationship among North American StH genome species; (2) the DMC1 gene sequences of the StH genome species from North America and Eurasia are evolutionarily distinct; (3) the StH genome polyploids have higher levels of sequence diversity in the St genome homoeolog than the H genome homoeolog; (4) the DMC1 sequence may evolve faster in the polyploid species than in the diploids; (5) high dN and dN/dS values in the St genome within polyploid species could be caused by low selective constraints or AT-biased mutation pressure. Our result provides some insight on evolutionary dynamics of duplicate DMC1 gene, the polyploidization events and phylogeny of the StH genome species.

Evolutionary Relationship of Wheat Protein Disulphide Isomerase (PDI) Gene Promoter Sequence Based on Phylogenetic Analysis

Protein disulphide isomerase (PDI) is an oxidoreductase enzyme abundant in the endoplasmic reticulum (ER). In plants, PDIs have been shown to assist the folding and deposition of seed storage proteins during the biogenesis of protein bodies in the endosperm. 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 were reported in our previous publications. Promoter sequences of three homoeologous genes encoding typical PDI, located on chromosome group 4 of bread wheat, and PDI promoter sequence analysis of Triticum urartu, Aegilops speltoides and Aegilops tauschii had also been reported previously. In this study, we report the isolation and sequencing of a ~700 bp region, comprising ~600 bp of the putative promoter region and 88 bp of the first exon of the typical PDI gene, in five accessions each from Triticum urartu (AA), Aegilops speltoides (BB) and Aegilops tauschii (DD). Sequence analysis indicated large variation among sequences belonging to the different genomes, while close similarity was found within each species and with the corresponding homoeologous PDI sequences of Triticum aestivum cv. CS (AABBDD) resulting in an overall high conservation of the sequence conferring endosperm-specific expression.

Discrimination of alleles and copy numbers at the Q locus in hexaploid wheat using quantitative pyrosequencing

Speltoid spikes are characterized by pyramidal spike morphology featuring an elongated rachis and tenacious glumes. Speltoids are considered undesirable spike aberrants in wheat breeding leading to increased heterogeneity within a cultivar candidate. As a consequence, the presence of speltoids may result in rejection of a cultivar candidate during official field trials or denial of cultivar certification during seed multiplication. A reliable method is, thus, required to assess the occurrence of speltoids, early on in a wheat breeding program. The domestication gene Q located on the long arm of wheat chromosome 5A is known to suppress the speltoid phenotype in wheat. Here, a quantitative pyrosequencing assay was developed to distinguish between normal wheat plants, which possess two copies of the Q allele, and aberrants, which are either aneuploids lacking the correct number of chromosome 5A copies or plants which carry the primitive q allele. An accurate and reproducible determination of the Q gene copy number was achieved for different wheat genotypes based on homoeologous sequence quantification with two primer combinations at the Q locus. Single plants with one to four copies of the Q allele could be detected by quantitative pyrosequencing which corresponded to the occurrence of speltoid (1 Q allele), normal (2 Q alleles), and compact (more than 2 Q alleles) spikes. Q and q specific alleles could be differentiated at SNP position 2299 of the Q gene. This SNP is assumed to be related to the emergence of free-threshing wheat forms. To our knowledge this is the first report for detection of aneuploids and differentiation of Q alleles in bread wheat using pyrosequencing technology. In future, quantitative pyrosequencing assay can be applied in wheat breeding programs to carry out marker-assisted selection against the presence of speltoid spike aberrants.

Identification, characterization and interpretation of single‐nucleotide sequence variation in allopolyploid crop species

An understanding of nature and extent of nucleotide sequence variation is required for programmes of discovery and characterization of single nucleotide polymorphisms (SNPs), which provide the most versatile class of molecular genetic marker. A majority of higher plant species are polyploids, and allopolyploidy, because of hybrid formation between closely related taxa, is very common. Mutational variation may arise both between allelic (homologous) sequences within individual subgenomes and between homoeologous sequences among subgenomes, in addition to paralogous variation between duplicated gene copies. Successful SNP validation in allopolyploids depends on differentiation of the sequence variation classes. A number of biological factors influence the feasibility of discrimination, including degree of gene family complexity, inbreeding or outbreeding reproductive habit, and the level of knowledge concerning progenitor diploid species. In addition, developments in high-throughput DNA sequencing and associated computational analysis provide general solutions for the genetic analysis of allopolyploids. These issues are explored in the context of experience from a range of allopolyploid species, representing grain (wheat and canola), forage (pasture legumes and grasses), and horticultural (strawberry) crop. Following SNP discovery, detection in routine genotyping applications also presents challenges for allopolyploids. Strategies based on either design of subgenome-specific SNP assays through homoeolocus-targeted polymerase chain reaction (PCR) amplification, or detection of incremental changes in nucleotide variant dosage, are described.

Protein disulphide isomerase family in bread wheat (Triticum aestivum L.): genomic structure, synteny conservation and phylogenetic analysis

Eight genes encoding protein disulphide isomerase (PDI)-like proteins in bread wheat were cloned and characterized and their genomic structure was compared with that of homoeologous genes isolated from other plant species. Fourteen wheat cDNA sequences of PDI-like genes were amplified and cloned; eight of them were relative to distinct PDI-like genes, whereas six corresponded to homoeologous sequences. Also, the genomic sequences of the eight non-homoeologous genes were amplified and cloned. Phylogenetic analysis, which included eight genes encoding PDI-like proteins and the gene encoding the typical PDI, assigned at least one of them to each of the eight major clades identified in the phylogenetic tree of the PDI gene family of plants. The close chromosome synteny between wheat and rice was confirmed by the location of the homoeologous genes of the PDI family in syntenic regions of the two species. Within the same phylogenetic group, a high level of conservation, in terms of sequence homology, genomic structure and domain organization, was detected between wheat and the other plant species. The high level of conservation of sequence and genomic organization within the PDI gene family, even between distant plant species, might be ascribed to the key metabolic roles of their protein products.