E. A. Salina

Relationship between homoeologous regulatory and structural genes in allopolyploid genome – A case study in bread wheat

BackgroundThe 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).ResultsPCR-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.ConclusionThe 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.

Microsatellite mapping of the induced sphaerococcoid mutation genes in Triticum aestivum

Abstract The S1, S2 and S3 genes of the induced sphaerococcoid mutation in common wheat (Triticum aestivum) were mapped using three different F2 populations consisting of 71–96 individual plants. Twenty-four microsatellite markers from homeologous group 3 of T. aestivum were used to map the S1, S2 and S3 genes on chromosomes 3D, 3B and 3A, respectively. The S1 locus was found to be closely linked to the centromeric marker Xgwm456 of the long arm (2.9 cM) and mapped not far (8.0 cM) from the Xgdm72 marker of the short arm of chromosome 3D. The S2 gene was tightly linked to 2 centromeric markers (Xgwm566, Xgwm845) of chromosome 3B. S3 was located between Xgwm2 (5.1 cM), the marker of the short arm, and Xgwm720 (6.6 cM), the marker of the long arm, both of chromosome 3A. Mapping the S1, S2 and S3 loci of the induced sphaerococcoid mutation near the centromeric regions supports the hypothesis that the sphaerococcum type may be due to gene duplication resulting from DNA recombination in the centromeric region.

Wheat genome structure: translocations during the course of polyploidization.

The genomic organization of Triticum timopheevii (2n=28, AtAtGG) was compared with hexaploid wheat T. aestivum (2n=42, AABBDD) by comparative mapping using microsatellites derived from bread wheat. Genetic maps for the two crosses T. timopheevii var. timopheevii × T. timopheevii var. typica and T. timopheevii K-38555×T. militinae were constructed. On the first population, 121 loci were mapped, and on the second population 103 loci. The transferability of the wheat markers to T. timopheevii was generally better for the A genome-specific markers (76–78% produced amplification products; 26 and 29% were polymorphic) than for B genome-specific markers (54% produced amplification products; 14 and 16% were polymorphic). Of the D genome-specific markers, one third produced amplification products in T. timopheevii, but only 5 and 2% were polymorphic in the corresponding mapping populations. The maps constructed confirmed the previously described translocation between chromosome arms 6AtS and 1GS and revealed at least two yet unknown rearrangements on chromosomes 4At and 6At. The presence of other translocations and rearrangements between T. timopheevii and T. aestivum was demonstrated by a variety of markers mapping to nonhomoeologous positions.

SNP Markers: Methods of Analysis, Ways of Development, and Comparison on an Example of Common Wheat

SNPs (single nucleotide polymorphisms), which belong to the last-generation molecular markers, occur at high frequencies in both animal and plant genomes. The development of SNP markers allows to automatize and enhance tenfolds the effectiveness of genotype analysis. This review summarizes literature data on methods of SNP polymorphism analysis. Various methods of developing SNP markers are considered, taking common wheat Triticum aestivum L. as an example. These markers are compared to other DNA markers, in order to ensure adequate choice of marker type for solving various molecular genetic problems.

Elimination of a tandem repeat of telomeric heterochromatin during the evolution of wheat

Abstract An analysis of accessions of Triticum and Aegilops species (86 diploid, 91 tetraploid and 109 hexaploid) was performed using squash-dot hybridization with the tandem repeat Spelt1 sequence as a probe. The Spelt1 sequence is a highly species-specific repeat associated with the telomeric heterochromatin of Aegilops speltoides Boiss. in which its copy numbers vary from 1.5×105 to 5.3×105. The amounts of Spelt1 are sharply decreased in tetraploid and hexaploid species and vary widely from less than 102 to 1.2×104. Two tetraploid wheats, Triticum timopheevii Zhuk. and T. carthlicum Nevski, are exceptional endemic species and within their restricted geographical distributions maintain the amounts of Spelt1 unaltered. The Spelt1 repetitive sequence was localized on the 6BL chromosome of tetraploid wheat Triticum durum Desf. cv ‘Langdon’ by dot-hybridization using D-genome disomic substitution lines. The possible causes of the loss of the telomere-associated tandem repeat Spelt1 in the process of wheat evolution and polyploidization are discussed.

FRIZZY PANICLE Drives Supernumerary Spikelets in Bread Wheat

Wheat transcription factors located on chromosome group 2 drive the yield-related production of supernumerary spikelets. Bread wheat (Triticum aestivum) inflorescences, or spikes, are characteristically unbranched and normally bear one spikelet per rachis node. Wheat mutants on which supernumerary spikelets (SSs) develop are particularly useful resources for work towards understanding the genetic mechanisms underlying wheat inflorescence architecture and, ultimately, yield components. Here, we report the characterization of genetically unrelated mutants leading to the identification of the wheat FRIZZY PANICLE (FZP) gene, encoding a member of the APETALA2/Ethylene Response Factor transcription factor family, which drives the SS trait in bread wheat. Structural and functional characterization of the three wheat FZP homoeologous genes (WFZP) revealed that coding mutations of WFZP-D cause the SS phenotype, with the most severe effect when WFZP-D lesions are combined with a frameshift mutation in WFZP-A. We provide WFZP-based resources that may be useful for genetic manipulations with the aim of improving bread wheat yield by increasing grain number.

Microsatellites confirm the authenticity of inter-varietal chromosome substitution lines of wheat (Triticum aestivum L.)

Abstract Ninety-five wheat microsatellite markers (WMS) were used to verify the authenticity of the set of Saratovskaya 29/Yanetzkis Probat inter-varietal wheat chromosome substitution lines developed using Saratovskaya 29 as the recipient variety. Polymorphic markers were available for all chromosome arms except 4DS, 6DS and 7DS. Each chromosome substitution line was tested by 2–8 microsatellite markers. The results demonstrate that most of the lines are correct. Out of 21 lines tested 17 showed the expected microsatellite pattern of the donor variety. Two entire chromosomes, 1B and 7A, and two chromosome arms, 3AL and 6DS, were not substituted with Yanetzkis Probat in their respective lines. Three microsatellite markers located in the distal regions of chromosome arms 4AL, 3BS and 5BL in the corresponding substitution lines did not reveal the expected microsatellite pattern of the recipient variety. The possible causes of the incorrect substitution line development and the appearance of incorrect distal microsatellite markers are discussed. The data confirm the idea that microsatellite markers provide ideal tools for testing the authenticity of genetic stocks of wheat.

[Production of wheat-rye substitution lines and identification of chromosome composition of karyotypes using C-banding, GISH, and SSR markers].

Based on the cross (Triticum aestivum L. × Secale cereale L.) × T. aestivum L., wheat-rye substitution lines (2n = 42) were produced with karyotypes containing, instead of a pair of homologous wheat chromosomes, a homeologous pair of rye chromosomes. The chromosome composition of these lines was described by GISH and C-banding methods, and SSR analysis. The results of genomic in situ hybridization demonstrated that karyotype of these lines included one pair of rye chromosomes each and lacked wheat-rye translocations. C-banding and SSR markers were used to identify rye chromosomes and determine the wheat chromosomes at which the substitution occurred. The lines were designated 1R(1D), 2R(2D)2, 2R(2D)3, 3R(3B), 6R(6A)2. The chromosome composition of lines 1R(1A), 2R(W)1, 5R(W), 5R(5A), and 6R(W)1, which were earlier obtained according to the same scheme for crossing, was characterized using methods of telocentric analysis, GISH, C-banding, and SSR analysis. These lines were identified as 1R(1A), 2R(2D)1, 5R(5D), 5R(5A), and 6R(6A)1, C-banding of chromosomes belonging to line 1R(1A) revealed the presence of two translocated chromosomes (3DS.3DL-del. and 4AL.W) during simultaneous amplification of SSR markers located on 3DL and 4AS arms. The “combined” long arm of the newly derived chromosome 4A is assumed to be formed from the long arm of chromosome 4AS itself and a deleted segment 3DL. All examined lines are cytologically stable, except for 3R(3B), which does not affect the stability of rye 3R chromosome transfer. Chromosome identification and classification of the lines will permit them to be models for genetic studies that can be used thereafter as promising “secondary gene pools” for the purpose of plant breeding.

Anthocyanin biosynthesis genes location and expression in wheat-rye hybrids.

Studies into gene expression in a foreign background contribute toward understanding of how genes derived from different species or genera manages to co-exist in a common nucleus, on the one hand, and help to estimate possible effectiveness of wide hybridization for cultivated plant improvement, on the other hand. The aim of this study was to investigate conservation of wheat and rye expression networks, using the anthocyanin biosynthesis pathway (ABP) genes as a model system. We isolated and analyzed ABP genes encoding enzymes acting at different steps of the pathway: chalcone-flavanone isomerase (CHI), flavanone 3-hydroxylase (F3H), anthocyanidin synthase (ANS), and anthocyanidin-3-glucoside rhamnosyltransferase (3RT). The rye ABP genes locations we determined (Chi on chromosome 5RL, F3h on 2RL, Ans on 6RL, 3Rt on 5RL, the regulatory Rc—red coleoptile—gene on 4RL) were in agreement with the rearrangements established between rye and wheat chromosomes. Expression of the ABP structural genes was studied in wheat–rye chromosome addition and substitution lines. F3h activation by the Rc gene was found to be critical for the red coleoptile trait formation. It was shown that the rye regulatory Rc gene can activate the wheat target gene F3h and vice versa wheat Rc induces expression of rye F3h. However, lower level of expression of rye F3h in comparison with that of the two wheat orthologues in the wheat–rye chromosome substitution line 2R(2D) was observed. Thus, although work of the wheat and rye ABP gene systems following the formation of wheat–rye hybrids is finely coordinated, some divergence exists between rye and wheat ABP genes, affecting level of gene expression.

The homoeologous genes encoding chalcone–flavanone isomerase in Triticum aestivum L.: Structural characterization and expression in different parts of wheat plant

Chalcone-flavanone isomerase (CHI; EC 5.5.1.6.) participates in the early step of flavonoid biosynthesis, related to plant adaptive and protective responses to environmental stress. The bread wheat genomic sequences encoding CHI were isolated, sequenced and mapped to the terminal segment of the long arms of chromosomes 5A, 5B and 5D. The loss of the final Chi intron and junction of the two last exons was found in the wheat A, B and D genomes compared to the Chi sequences of most other plant species. Each of the three diploid genomes of hexaploid wheat encodes functional CHI; however, transcription of the three homoeologous genes is not always co-regulated. In particular, the three genes demonstrated different response to salinity in roots: Chi-D1 was up-regulated, Chi-A1 responds medially, whereas Chi-B1 was not activated at all. The observed variation in transcriptional activity between the Chi homoeologs is in a good agreement with structural diversification of their promoter sequences. In addition, the correlation between Chi transcription and anthocyanin pigmentation in different parts of wheat plant has been studied. The regulatory genes controlling anthocyanin pigmentation of culm and pericarp modulated transcription of the Chi genes. However, in other organs, there was no strong relation between tissue pigmentation and the transcription of the Chi genes, suggesting complex regulation of the Chi expression in most parts of wheat plant.