Telomere sequence variability in genotypes from natural plant populations: unusual block-organized double-monomer terminal telomeric arrays.

BACKGROUND: Telomeres are the nucleoprotein complexes that physically cap the ends of eukaryotic chromosomes. Most plants possess Arabidopsis-type telomere sequences (TSs). In addition to terminal TSs, more diverse interstitial TSs exists in plants. Although telomeres have been sufficiently studied, the actual diversity of TSs in land plants is underestimated. RESULTS: We investigate genotypes from seven natural populations with contrasting environments of four Chenopodium species to reveal the variability in TSs by analyzing Oxford Nanopore reads. Fluorescent in situ hybridization was used to localize telomeric repeats on chromosomes. We identified a number of derivative monomers that arise in part of both terminal and interstitial telomeric arrays of a single genotype. The former presents a case of block-organized double-monomer telomers, where blocks of Arabidopsis-type TTTAGGG motifs were interspersed with blocks of derivative TTTAAAA motifs. The latter is an integral part of the satellitome with transformations specific to the inactive genome fraction. CONCLUSIONS: We suggested two alternative models for the possible formation of derivative monomers from telomeric heptamer motifs of Arabidopsis-type. It was assumed that derivatization of TSs is a ubiquitous process in the plant genome but occurrence and frequencies of derivatives may be genotype-specific. We also propose that the formation of non-canonical arrays of TSs, especially at chromosomal termini, may be a source for genomic variability in nature.

Regulation of plant epigenetic memory in response to cold and heat stress: towards climate resilient agriculture.

Plants have evolved to adapt and grow in hot and cold climatic conditions. Some also adapt to daily and seasonal temperature changes. Epigenetic modifications play an important role in regulating plant tolerance under such conditions. DNA methylation and post-translational modifications of histone proteins influence gene expression during plant developmental stages and under stress conditions, including cold and heat stress. While short-term modifications are common, some modifications may persist and result in stress memory that can be inherited by subsequent generations. Understanding the mechanisms of epigenomes responding to stress and the factors that trigger stress memory is crucial for developing climate-resilient agriculture, but such an integrated view is currently limited. This review focuses on the plant epigenetic stress memory during cold and heat stress. It also discusses the potential of machine learning to modify stress memory through epigenetics to develop climate-resilient crops.

Isolation of High-Molecular-Weight DNA for Long-Read Sequencing Using a High-Salt Gel Electroelution Trap.

Long-read sequencing technologies require high-molecular-weight (HMW) DNA of sufficient purity and integrity, which can be difficult to obtain from complex biological samples. We propose a method for purifying HMW DNA that takes advantage of the fact that DNA's electrophoretic mobility decreases in a high-ionic-strength environment. The method begins with the separation of HMW DNA from various impurities by electrophoresis in an agarose gel-filled channel. After sufficient separation, a high-salt gel block is placed ahead of the DNA band of interest, leaving a gap between the separating gel and the high-salt gel that serves as a reservoir for sample collection. The DNA is then electroeluted from the separating gel into the reservoir, where its migration slows due to electrostatic shielding of the DNA's negative charge by excess counterions from the high-salt gel. As a result, the reservoir accumulates HMW DNA of high purity and integrity, which can be easily collected and used for long-read sequencing and other demanding applications without additional desalting. The method is simple and inexpensive, yields sequencing-grade HMW DNA even from difficult plant and soil samples, and has the potential for automation and scalability.

The role of LTR retrotransposons in plant genetic engineering: how to control their transposition in the genome.

KEY MESSAGE: We briefly discuss that the similarity of LTR retrotransposons to retroviruses is a great opportunity for the development of a genetic engineering tool that exploits intragenic elements in the plant genome for plant genetic improvement. Long terminal repeat (LTR) retrotransposons are very similar to retroviruses but do not have the property of being infectious. While spreading between its host cells, a retrovirus inserts a DNA copy of its genome into the cells. The ability of retroviruses to cause infection with genome integration allows genes to be delivered to cells and tissues. Retrovirus vectors are, however, only specific to animals and insects, and, thus, are not relevant to plant genetic engineering. However, the similarity of LTR retrotransposons to retroviruses is an opportunity to explore the former as a tool for genetic engineering. Although recent long-read sequencing technologies have advanced the knowledge about transposable elements (TEs), the integration of TEs is still unable either to control them or to direct them to specific genomic locations. The use of existing intragenic elements to achieve the desired genome composition is better than using artificial constructs like vectors, but it is not yet clear how to control the process. Moreover, most LTR retrotransposons are inactive and unable to produce complete proteins. They are also highly mutable. In addition, it is impossible to find a full active copy of a LTR retrotransposon out of thousands of its own copies. Theoretically, if these elements were directly controlled and turned on or off using certain epigenetic mechanisms (inducing by stress or infection), LTR retrotransposons could be a great opportunity to develop a genetic engineering tool using intragenic elements in the plant genome. In this review, the recent developments in uncovering the nature of LTR retrotransposons and the possibility of using these intragenic elements as a tool for plant genetic engineering are briefly discussed.

Correction: Ramakrishnan et al. The Dynamism of Transposon Methylation for Plant Development and Stress Adaptation. Int. J. Mol. Sci. 2021, 22, 11387.

The authors would like to make the following corrections to the original publication [...].

Long terminal repeats (LTR) and transcription factors regulate PHRE1 and PHRE2 activity in Moso bamboo under heat stress.

BACKGROUND: LTR retrotransposons play a significant role in plant growth, genome evolution, and environmental stress response, but their regulatory response to heat stress remains unclear. We have investigated the activities of two LTR retrotransposons, PHRE1 and PHRE2, of moso bamboo (Phyllostachys edulis) in response to heat stress. RESULTS: The differential overexpression of PHRE1 and PHRE2 with or without CaMV35s promoter showed enhanced expression under heat stress in transgenic plants. The transcriptional activity studies showed an increase in transposition activity and copy number among moso bamboo wild type and Arabidopsis transgenic plants under heat stress. Comparison of promoter activity in transgenic plants indicated that 5'LTR promoter activity was higher than CaMV35s promoter. Additionally, yeast one-hybrid (Y1H) system and in planta biomolecular fluorescence complementation (BiFC) assay revealed interactions of heat-dependent transcription factors (TFs) with 5'LTR sequence and direct interactions of TFs with pol and gag. CONCLUSIONS: Our results conclude that the 5'LTR acts as a promoter and could regulate the LTR retrotransposons in moso bamboo under heat stress.

The Dynamism of Transposon Methylation for Plant Development and Stress Adaptation.

Plant development processes are regulated by epigenetic alterations that shape nuclear structure, gene expression, and phenotypic plasticity; these alterations can provide the plant with protection from environmental stresses. During plant growth and development, these processes play a significant role in regulating gene expression to remodel chromatin structure. These epigenetic alterations are mainly regulated by transposable elements (TEs) whose abundance in plant genomes results in their interaction with genomes. Thus, TEs are the main source of epigenetic changes and form a substantial part of the plant genome. Furthermore, TEs can be activated under stress conditions, and activated elements cause mutagenic effects and substantial genetic variability. This introduces novel gene functions and structural variation in the insertion sites and primarily contributes to epigenetic modifications. Altogether, these modifications indirectly or directly provide the ability to withstand environmental stresses. In recent years, many studies have shown that TE methylation plays a major role in the evolution of the plant genome through epigenetic process that regulate gene imprinting, thereby upholding genome stability. The induced genetic rearrangements and insertions of mobile genetic elements in regions of active euchromatin contribute to genome alteration, leading to genomic stress. These TE-mediated epigenetic modifications lead to phenotypic diversity, genetic variation, and environmental stress tolerance. Thus, TE methylation is essential for plant evolution and stress adaptation, and TEs hold a relevant military position in the plant genome. High-throughput techniques have greatly advanced the understanding of TE-mediated gene expression and its associations with genome methylation and suggest that controlled mobilization of TEs could be used for crop breeding. However, development application in this area has been limited, and an integrated view of TE function and subsequent processes is lacking. In this review, we explore the enormous diversity and likely functions of the TE repertoire in adaptive evolution and discuss some recent examples of how TEs impact gene expression in plant development and stress adaptation.

High-throughput retrotransposon-based genetic diversity of maize germplasm assessment and analysis.

Maize is one of the world's most important crops and a model for grass genome research. Long terminal repeat (LTR) retrotransposons comprise most of the maize genome; their ability to produce new copies makes them efficient high-throughput genetic markers. Inter-retrotransposon-amplified polymorphisms (IRAPs) were used to study the genetic diversity of maize germplasm. Five LTR retrotransposons (Huck, Tekay, Opie, Ji, and Grande) were chosen, based on their large number of copies in the maize genome, whereas polymerase chain reaction primers were designed based on consensus LTR sequences. The LTR primers showed high quality and reproducible DNA fingerprints, with a total of 677 bands including 392 polymorphic bands showing 58% polymorphism between maize hybrid lines. These markers were used to identify genetic similarities among all lines of maize. Analysis of genetic similarity was carried out based on polymorphic amplicon profiles and genetic similarity phylogeny analysis. This diversity was expected to display ecogeographical patterns of variation and local adaptation. The clustering method showed that the varieties were grouped into three clusters differing in ecogeographical origin. Each of these clusters comprised divergent hybrids with convergent characters. The clusters reflected the differences among maize hybrids and were in accordance with their pedigree. The IRAP technique is an efficient high-throughput genetic marker-generating method.

Long Tandem Arrays of Cassandra Retroelements and Their Role in Genome Dynamics in Plants.

Retrotransposable elements are widely distributed and diverse in eukaryotes. Their copy number increases through reverse-transcription-mediated propagation, while they can be lost through recombinational processes, generating genomic rearrangements. We previously identified extensive structurally uniform retrotransposon groups in which no member contains the gag, pol, or env internal domains. Because of the lack of protein-coding capacity, these groups are non-autonomous in replication, even if transcriptionally active. The Cassandra element belongs to the non-autonomous group called terminal-repeat retrotransposons in miniature (TRIM). It carries 5S RNA sequences with conserved RNA polymerase (pol) III promoters and terminators in its long terminal repeats (LTRs). Here, we identified multiple extended tandem arrays of Cassandra retrotransposons within different plant species, including ferns. At least 12 copies of repeated LTRs (as the tandem unit) and internal domain (as a spacer), giving a pattern that resembles the cellular 5S rRNA genes, were identified. A cytogenetic analysis revealed the specific chromosomal pattern of the Cassandra retrotransposon with prominent clustering at and around 5S rDNA loci. The secondary structure of the Cassandra retroelement RNA is predicted to form super-loops, in which the two LTRs are complementary to each other and can initiate local recombination, leading to the tandem arrays of Cassandra elements. The array structures are conserved for Cassandra retroelements of different species. We speculate that recombination events similar to those of 5S rRNA genes may explain the wide variation in Cassandra copy number. Likewise, the organization of 5S rRNA gene sequences is very variable in flowering plants; part of what is taken for 5S gene copy variation may be variation in Cassandra number. The role of the Cassandra 5S sequences remains to be established.

Probing the Proton-Loading Site of Cytochrome C Oxidase Using Time-Resolved Fourier Transform Infrared Spectroscopy.

Crystal structure analyses at atomic resolution and FTIR spectroscopic studies of cytochrome c oxidase have yet not revealed protonation or deprotonation of key sites of proton transfer in a time-resolved mode. Here, a sensitive technique to detect protolytic transitions is employed. In this work, probing a proton-loading site of cytochrome c oxidase from Paracoccus denitrificans with time-resolved Fourier transform infrared spectroscopy is presented for the first time. For this purpose, variants with single-site mutations of N131V, D124N, and E278Q, the key residues in the D-channel, were studied. The reaction of mutated CcO enzymes with oxygen was monitored and analyzed. Seven infrared bands in the "fast" kinetic spectra were found based on the following three requirements: (1) they are present in the "fast" phases of N131V and D124N mutants, (2) they have reciprocal counterparts in the "slow" kinetic spectra in these mutants, and (3) they are absent in "fast" kinetic spectra of the E278Q mutant. Moreover, the double-difference spectra between the first two mutants and E278Q revealed more IR bands that may belong to the proton-loading site protolytic transitions. From these results, it is assumed that several polar residues and/or water molecule cluster(s) share a proton as a proton-loading site. This site can be propionate itself (holding only a fraction of H+), His403, and/or water cluster(s).

Genetic analysis of the grapevine genotypes of the Russian Vitis ampelographic collection using iPBS markers.

Cultivated grapevine (Vitis vinifera L. ssp. sativa D.C.) is one of the oldest agricultural crops, each variety comprising an array of clones obtained by vegetative propagation from a selected vine grown from a single seedling. Most clones within a variety are identical, but some show a different form of accession, giving rise to new divergent phenotypes. Understanding the associations among the genotypes within a variety is crucial to efficient management and effective grapevine improvement. Inter-primer binding-site (iPBS) markers may aid in determining the new clones inside closely related genotypes. Following this idea, iPBS markers were used to assess the genetic variation of 33 grapevine genotypes collected from Russia. We used molecular markers to identify the differences among and within five grapevine clonal populations and analysed the variation, using clustering and statistical approaches. Four of a total of 30 PBS primers were selected, based on amplification efficiency. Polymerase chain reaction (PCR) with PBS primers resulted in a total of 1412 bands ranging from 300 to 6000 bp, with a polymorphism ratio of 44%, ranging from 58 to 75 bands per group. In total, were identified seven private bands in 33 genotypes. Results of molecular variance analysis showed that 40% of the total variation was observed within groups and only 60% between groups. Cluster analysis clearly showed that grapevine genotypes are highly divergent and possess abundant genetic diversities. The iPBS PCR-based genome fingerprinting technology used in this study effectively differentiated genotypes into five grapevine groups and indicated that iPBS markers are useful tools for clonal selection. The number of differences between clones was sufficient to identify them as separate clones of studied varieties containing unique mutations. Our previous phenotypic and phenological studies have confirmed that these genotypes differ from those of maternal plants. This work emphasized the need for a better understanding of the genotypic differences among closely related varieties of grapevine and has implications for the management of its selection processes.

Natural History of a Satellite DNA Family: From the Ancestral Genome Component to Species-Specific Sequences, Concerted and Non-Concerted Evolution.

Satellite DNA (satDNA) is the most variable fraction of the eukaryotic genome. Related species share a common ancestral satDNA library and changing of any library component in a particular lineage results in interspecific differences. Although the general developmental trend is clear, our knowledge of the origin and dynamics of satDNAs is still fragmentary. Here, we explore whole genome shotgun Illumina reads using the RepeatExplorer (RE) pipeline to infer satDNA family life stories in the genomes of Chenopodium species. The seven diploids studied represent separate lineages and provide an example of a species complex typical for angiosperms. Application of the RE pipeline allowed by similarity searches a determination of the satDNA family with a basic monomer of ~40 bp and to trace its transformation from the reconstructed ancestral to the species-specific sequences. As a result, three types of satDNA family evolutionary development were distinguished: (i) concerted evolution with mutation and recombination events; (ii) concerted evolution with a trend toward increased complexity and length of the satellite monomer; and (iii) non-concerted evolution, with low levels of homogenization and multidirectional trends. The third type is an example of entire repeatome transformation, thus producing a novel set of satDNA families, and genomes showing non-concerted evolution are proposed as a significant source for genomic diversity.

Allelic Diversity of Acetyl Coenzyme A Carboxylase accD/bccp Genes Implicated in Nuclear-Cytoplasmic Conflict in the Wild and Domesticated Pea (Pisum sp.).

Reproductive isolation is an important component of species differentiation. The plastid accD gene coding for the acetyl-CoA carboxylase subunit and the nuclear bccp gene coding for the biotin carboxyl carrier protein were identified as candidate genes governing nuclear-cytoplasmic incompatibility in peas. We examined the allelic diversity in a set of 195 geographically diverse samples of both cultivated (Pisum sativum, P. abyssinicum) and wild (P. fulvum and P. elatius) peas. Based on deduced protein sequences, we identified 34 accD and 31 bccp alleles that are partially geographically and genetically structured. The accD is highly variable due to insertions of tandem repeats. P. fulvum and P. abyssinicum have unique alleles and combinations of both genes. On the other hand, partial overlap was observed between P. sativum and P. elatius. Mapping of protein sequence polymorphisms to 3D structures revealed that most of the repeat and indel polymorphisms map to sequence regions that could not be modeled, consistent with this part of the protein being less constrained by requirements for precise folding than the enzymatically active domains. The results of this study are important not only from an evolutionary point of view but are also relevant for pea breeding when using more distant wild relatives.

FastPCR: An in silico tool for fast primer and probe design and advanced sequence analysis.

Polymerase chain reaction (PCR) is one of the most important laboratory techniques used in molecular biology, genetics and molecular diagnostics. The success of a PCR-based method largely depends on the correct nucleic acid sequence analysis in silico prior to a wet-bench experiment. Here, we report the development of an online Java-based software for virtual PCR on linear or circular DNA templates and multiple primer or probe search from large or small databases. Primer or probe sensitivity and specificity are predicted by searching a database to find sequences with an optimal number of mismatches, similarity and stability. The software determines primer location, orientation, efficiency of binding and calculates primer melting temperatures for standard and degenerate oligonucleotides. The software is suitable for batch file processing, which is essential for automation when working with large amounts of data. The online Java software is available for download at http://primerdigital.com/tools/pcr.html. Accession numbers for the sequences resulting from this study: EU140956 EU177767 EU867815 EU882730 FJ975775-FJ975780 HM481419 HM481420 KC686837-KC686839 KM262797.

Copy-number variation of housekeeping gene rpl13a in rat strains selected for nervous system excitability.

We evaluated copy number variation (CNV) for four genes in rat strains differing in nervous system excitability. rpl13a copy number is significantly reduced in hippocampus and bone marrow in rats with a high excitability threshold and stress. The observed phenomenon may be associated with a role for rpl13a in lipid metabolism.

Novel alleles of the VERNALIZATION1 genes in wheat are associated with modulation of DNA curvature and flexibility in the promoter region.

BACKGROUND: In wheat, the vernalization requirement is mainly controlled by the VRN genes. Different species of hexaploid and tetraploid wheat are widely used as genetic source for new mutant variants and alleles for fundamental investigations and practical breeding programs. In this study, VRN-A1 and VRN-B1 were analysed for 178 accessions representing six tetraploid wheat species (Triticum dicoccoides, T. dicoccum, T. turgidum, T. polonicum, T. carthlicum, T. durum) and five hexaploid species (T. compactum, T. sphaerococcum, T. spelta, T. macha, T. vavilovii). RESULTS: Novel allelic variants in the promoter region of VRN-A1 and VRN-B1 were identified based on the change in curvature and flexibility of the DNA molecules. The new variants of VRN-A1 (designated as Vrn-A1a.2, Vrn-A1b.2 - Vrn-A1b.6 and Vrn-A1i) were found to be widely distributed in hexaploid and tetraploid wheat, and in fact were predominant over the known VRN-A1 alleles. The greatest diversity of the new variants of VRN-B1 (designated as VRN-B1.f, VRN-B1.s and VRN-B1.m) was found in the tetraploid and some hexaploid wheat species. For the first time, minor differences within the sequence motif known as the VRN-box of VRN1 were correlated with wheat growth habit. Thus, vrn-A1b.3 and vrn-A1b.4 were revealed in winter wheat in contrast to Vrn-A1b.2, Vrn-A1b.5, Vrn-A1b.6 and Vrn-A1i. It was found that single nucleotide mutation in the VRN-box can influence the vernalization requirement and growth habit of wheat. Our data suggest that both the A-tract and C-rich segment within the VRN-box contribute to its functionality, and provide a new view of the hypothesised role of the VRN-box in regulating transcription of the VRN1 genes. Specifically, it is proposed that combination of mutations in this region can modulate vernalization sensitivity and flowering time of wheat. CONCLUSIONS: New allelic variants of the VRN-A1 and VRN-B1 genes were identified in hexaploid and tetraploid wheat. Mutations in A-tract and C-rich segments within the VRN-box of VRN-A1 are associated with modulation of the vernalization requirement and flowering time. New allelic variants will be useful in fundamental investigations into the regulation of VRN1 expression, and provide a valuable genetic resource for practical breeding of wheat.

Allelic variation at the VERNALIZATION-A1, VRN-B1, VRN-B3, and PHOTOPERIOD-A1 genes in cultivars of Triticum durum Desf.

MAIN CONCLUSION: The durum wheat varieties from Ukraine, Russia, and Kazakhstan are characterized by the specific allelic composition of the VRN genes that sharply distinguish them from the Triticum durum varieties from other countries. For numerous varieties, the VRN alleles which previously were not found in tetraploid wheat were identified. The ability of wheat to adapt to a wide range of environmental conditions is mostly determined by the allelic diversity within genes regulating the vernalization requirement (VRN) and photoperiod response (PPD). In the present study, allelic variation in the VRN1, VRN3, and PPD-A1 genes was investigated for 134 varieties of Triticum durum from different eco-geographic areas. It was shown that varieties from Russia and Ukraine have a specific allelic composition at the VRN genes, which in quantity and quality differed from European and American cultivars. A large number of varieties of T. durum from Russia carry the dominant Vrn-A1a.1 allele, previously identified mainly in hexaploid wheat. For some varieties from Eastern Europe and Asia, Vrn-A1i and vrn-A1b.3 recently revealed in wheat were also identified. Polymorphism of the VRN-B1 promoter region, distinguishing all three variants of this sequence (VRN-B1.f, VRN-B1.s, and VRN-B1.m), was detected. It was found that the dominant Vrn-B1c allele is commonly found in varieties of T. durum from Russia and Ukraine, but not Europe or USA. Furthermore, many Ukrainian and Russian varieties carry the dominant alleles of the both VRN-A1 and VRN-B1 genes simultaneously, while varieties from Europe and America carry the dominant allele of VRN-A1 alone. Finally, a high frequency of the Vrn-B3a allele, which previously was found only in some accessions of hexaploid wheat, was observed for varieties from Ukraine and Russia. It was revealed that the Ukrainian pool of T. durum varieties is currently the largest genetic source of the dominant Vrn-B3a allele in wheat in the worldwide.

Design and validation of an STR hexaplex assay for DNA profiling of grapevine cultivars.

Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser-induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high-throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling.

Assessment of genetic diversity in Nordic timothy (Phleum pratense L.).

BACKGROUND: Timothy (Phleum pratense L.), a cool-season hexaploid perennial, is the most important forage grass species in Nordic countries. Earlier analyses of genetic diversity in a collection of 96 genebank accessions of timothy with SSR markers demonstrated high levels of diversity but could not resolve population structure. Therefore, we examined a subset of 51 accessions with REMAP markers, which are based on retrotransposons, and compared the diversity results with those obtained with SSR markers. RESULTS: Using four primer combinations, 533 REMAP markers were analyzed, compared with 464 polymorphic alleles in the 13 SSR loci previously. The average marker index, which describes information obtained per experiment (per primer combination or locus) was over six times higher with REMAPs. Most of the variation found was within accessions, with somewhat less, 89 %, for REMAPs, than for SSR, with 93 %. CONCLUSIONS: SSRs revealed differences in the level of diversity slightly better than REMAPs but neither marker type could reveal any clear clustering of accessions based on countries, vegetation zones, or different cultivar types. In our study, reliable evaluation of SSR allele dosages was not possible, so each allele had to be handled as a dominant marker. SSR and REMAP, which report from different mechanisms of generating genetic diversity and from different genomic regions, together indicate a lack of population structure. Taken together, this likely reflects the outcrossing and hexaploid nature of timothy rather than failures of either marker system.

Discovery, evaluation and distribution of haplotypes and new alleles of the Photoperiod-A1 gene in wheat.

Photoperiod response in wheat is determined to a large extent by the homoeologous series of Photoperiod 1 (Ppd1) genes. In this study, Ppd-A1 genomic sequences from the 5' UTR and promoter region were analysed in 104 accessions of six tetraploid wheat species (Triticum dicoccoides, T. dicoccum, T. turgidum, T. polonicum, T. carthlicum, T. durum) and 102 accessions of six hexaploid wheat species (T. aestivum, T. compactum, T. sphaerococcum, T. spelta, T. macha, T. vavilovii). This data was supplemented with in silico analysis of publicly available sequences from 46 to 193 accessions of diploid and tetraploid wheat, respectively. Analysis of a region of the Ppd-A1 promoter identified thirteen haplotypes, which were divided in two haplogroups. Distribution of the Ppd-A1 haplogroups and haplotypes in wheat species, and their geographical distributions were analysed. Polymerase chain reaction combined with a heteroduplex mobility assay was subsequently used to efficiently discriminate between Ppd-A1 alleles, allowing identification of the Ppd-A1b haplotypes and haplogroups. The causes of anomalous migration of Ppd-A1 heteroduplexes in gels were found to be the localization of mismatches relative to the center of fragment, the cumulative effect of neighbouring polymorphic sites, and the location of mismatches within A/T-tracts. Analysis of the Ppd-A1 5' UTR in hexaploid wheat revealed a novel mutation within the "photoperiod critical" region in a subset of T. compactum accessions. This putative photoperiod insensitive allele (designated Ppd-A1a.4) includes a 684 bp deletion which spans region in common with deletions previously identified in other photoperiod insensitive Ppd1 alleles.