Updated guidelines for gene nomenclature in wheat.

KEY MESSAGE: Here, we provide an updated set of guidelines for naming genes in wheat that has been endorsed by the wheat research community. The last decade has seen a proliferation in genomic resources for wheat, including reference- and pan-genome assemblies with gene annotations, which provide new opportunities to detect, characterise, and describe genes that influence traits of interest. The expansion of genetic information has supported growth of the wheat research community and catalysed strong interest in the genes that control agronomically important traits, such as yield, pathogen resistance, grain quality, and abiotic stress tolerance. To accommodate these developments, we present an updated set of guidelines for gene nomenclature in wheat. These guidelines can be used to describe loci identified based on morphological or phenotypic features or to name genes based on sequence information, such as similarity to genes characterised in other species or the biochemical properties of the encoded protein. The updated guidelines provide a flexible system that is not overly prescriptive but provides structure and a common framework for naming genes in wheat, which may be extended to related cereal species. We propose these guidelines be used henceforth by the wheat research community to facilitate integration of data from independent studies and allow broader and more efficient use of text and data mining approaches, which will ultimately help further accelerate wheat research and breeding.

Transcriptomic and biochemical investigations support the role of rootstock-scion interaction in grapevine berry quality.

BACKGROUND: In viticulture, rootstock genotype plays a critical role to improve scion physiology, berry quality and to adapt grapevine (Vitis vinifera L.) to different environmental conditions. This study aimed at investigating the effect of two different rootstocks (1103 Paulsen - P - and Mgt 101-14 - M) in comparison with not grafted plants - NGC - on transcriptome (RNA-seq and small RNA-seq) and chemical composition of berry skin in Pinot noir, and exploring the influence of rootstock-scion interaction on grape quality. Berry samples, collected at veraison and maturity, were investigated at transcriptional and biochemical levels to depict the impact of rootstock on berry maturation. RESULTS: RNA- and miRNA-seq analyses highlighted that, at veraison, the transcriptomes of the berry skin are extremely similar, while variations associated with the different rootstocks become evident at maturity, suggesting a greater diversification at transcriptional level towards the end of the ripening process. In the experimental design, resembling standard agronomic growth conditions, the vines grafted on the two different rootstocks do not show a high degree of diversity. In general, the few genes differentially expressed at veraison were linked to photosynthesis, putatively because of a ripening delay in not grafted vines, while at maturity the differentially expressed genes were mainly involved in the synthesis and transport of phenylpropanoids (e.g. flavonoids), cell wall loosening, and stress response. These results were supported by some differences in berry phenolic composition detected between grafted and not grafted plants, in particular in resveratrol derivatives accumulation. CONCLUSIONS: Transcriptomic and biochemical data demonstrate a stronger impact of 1103 Paulsen rootstock than Mgt 101-14 or not grafted plants on ripening processes related to the secondary metabolite accumulations in berry skin tissue. Interestingly, the MYB14 gene, involved in the feedback regulation of resveratrol biosynthesis was up-regulated in 1103 Paulsen thus supporting a putative greater accumulation of stilbenes in mature berries.

Development of a deletion and genetic linkage map for the 5A and 5B chromosomes of wheat (Triticum aestivum).

The aims of the present study were to provide deletion maps for wheat ( Triticum aestivum L.) chromosomes 5A and 5B and a detailed genetic map of chromosome 5A enriched with popular microsatellite markers, which could be compared with other existing maps and useful for mapping major genes and quantitative traits loci (QTL). Physical mapping of 165 gSSR and EST-SSR markers was conducted by amplifying each primer pair on Chinese Spring, aneuploid lines, and deletion lines for the homoeologous group 5 chromosomes. A recombinant inbred line (RIL) mapping population that is recombinant for only chromosome 5A was obtained by crossing the wheat cultivar Chinese Spring and the disomic substitution line Chinese Spring-5A dicoccoides and was used to develop a genetic linkage map of chromosome 5A. A total of 67 markers were found polymorphic between the parental lines and were mapped in the RIL population. Sixty-three loci and the Q gene were clustered in three linkage groups ordered at a minimum LOD score of 5, while four loci remained unlinked. The whole genetic 5A chromosome map covered 420.2 cM, distributed among three linkage groups of 189.3, 35.4, and 195.5 cM. The EST sequences located on chromosomes 5A and 5B were used for comparative analysis against Brachypodium distachyon (L.) P. Beauv. and rice ( Oryza sativa L.) genomes to resolve orthologous relationships among the genomes of wheat and the two model species.

The e3 ubiquitin ligase gene family in plants: regulation by degradation.

The regulation of protein expression and activity has been for long time considered only in terms of transcription/translation efficiency. In the last years, the discovery of post-transcriptional and post-translational regulation mechanisms pointed out that the key factor in determining transcript/protein amount is the synthesis/degradation ratio, together with post-translational modifications of proteins. Polyubiquitinaytion marks target proteins directed to degradation mediated by 26S-proteasome. Recent functional genomics studies pointed out that about 5% of Arabidopsis genome codes for proteins of ubiquitination pathway. The most of them (more than one thousand genes) correspond to E3 ubiquitin ligases that specifically recognise target proteins. The huge size of this gene family, whose members are involved in regulation of a number of biological processes including hormonal control of vegetative growth, plant reproduction, light response, biotic and abiotic stress tolerance and DNA repair, indicates a major role for protein degradation in control of plant life.

Two loci on chromosome 5H determine low-temperature tolerance in a 'Nure' (winter) x 'Tremois' (spring) barley map.

Barley ( Hordeum vulgare subsp. vulgare) is an economically important diploid model for the Triticeae; and a better understanding of low-temperature tolerance mechanisms could significantly improve the yield of fall-sown cereals. We developed a new resource for genetic analysis of winter hardiness-related traits, the 'Nure' x 'Tremois' linkage map, based on a doubled-haploid population that is segregating for low-temperature tolerance and vernalization requirement. Three measures of low-temperature tolerance and one measure of vernalization requirement were used and, for all traits, QTLs were mapped on chromosome 5H. The vernalization response QTL coincides with previous reports at the Vrn-1/Fr1 region of the Triticeae. We also found coincident QTLs at this position for all measures of low-temperature tolerance. Using Composite Interval Mapping, a second proximal set, of coincident QTLs for low-temperature tolerance, and the accumulation of two different COR proteins (COR14b and TMC-Ap3) was identified. The HvCBF4 locus, or another member of the CBF loci clustered in this region, is the candidate gene underlying this QTL. There is a CRT/DRE recognition site in the promoter of cor14b with which a CBF protein could interact. These results support the hypothesis that highly conserved regulatory factors, such as members of the CBF gene family, may regulate the stress responses of a wide range of plant species.

The cold-regulated transcriptional activator Cbf3 is linked to the frost-tolerance locus Fr-A2 on wheat chromosome 5A.

Wheat chromosome 5A plays a key role in cold acclimation and frost tolerance. The major frost tolerance gene Fr-A1(formerly Fr1) and two loci that regulate the transcription of cold- regulated genes (Cor) have previously been mapped on the long arm of this chromosome. In this study we report the discovery of a new locus for frost tolerance designated Fr-A2. This new locus was mapped on the long arm of chromosome 5A of diploid wheat (T. monococcum), 40 cM from the centromere and 30 cM proximal to the major frost tolerance locus Fr-A1. We found also that frost-tolerant and frost-susceptible T. monococcum parental lines differed in the transcription level of the cold induced gene Cor14b when plants were grown at 15 degrees C. Transcription levels of this gene were measured in each of the recombinant inbred lines and mapped as a QTL that perfectly overlapped the QTL for frost survival at the Fr-A2 locus. This result suggested that frost tolerance in this cross was mediated by differential regulation of the expression of the Corgenes. In a previous study in hexaploid wheat (T. aestivum) we had shown that Cor14b was regulated by two loci located on chromosome 5A, one in the same chromosome region as the T. monococcum Fr-A2 locus and the other one closely linked to Fr-A1. Since CBF transcriptional activators in Arabidopsis regulate Corgenes and are involved in frost tolerance, we decided to localize the cold-regulated CBF-like barley gene Cbf3 on the T. monococcum map. This gene was mapped on the peak of the Fr-A2 QTL for frost tolerance. This result suggests that the observed differential regulation of Cor14b at the Fr-A2 locus is due to allelic variation at the XCbf3 locus, and that this transcriptional activator gene might be a candidate gene for the Fr-A2 frost tolerance locus on wheat chromosome 5A.

The cold dependent accumulation of COR TMC-AP3 in cereals with contrasting, frost tolerance is regulated by different mRNA expression and protein turnover.

The accumulation of specific cold-regulated (COR) proteins is a component of the hardening process and different amount of COR proteins has been related to different degrees of cold tolerance. A number of different mechanisms controls the accumulation of the COR proteins in the plant cells. In this work we describe the mechanisms controlling the accumulation of the COR protein TMC-AP3, a putative chloroplastic amino acid selective channel protein [1] in barley, durum, wheat, emmer and bread wheat. Winter barley and, to less extent, winter bread wheat showed a higher cor tmc-ap3 expression at low temperature than the spring one while no significant differences were detected between the emmer and the durum. wheat genotypes. After 2 days of de-hardening the transcript level dropped down in the same way in all tested genotypes, nevertheless the decrease in protein content was genotype dependent. In all frost resistant genotypes the amount of COR TMC-AP3 after 9 days of de-hardening was higher compared with that of susceptible ones. These findings suggest that resistant and susceptible genotypes have different protein degradation rate and/or mRNA translational efficiency. Differences in the protein degradation rate were not dependent from the amino acidic sequence of the protein, being extremely similar in all tested genotypes. A genetic study based on Chinese spring/Cheyenne chromosome substitution lines showed that the turnover of TMC-AP3 is a polygenic trait controlled by a number of loci being the most important located on chromosomes 1B, 2B, 2D and 4D.

Two loci on wheat chromosome 5A regulate the differential cold-dependent expression of the cor14b gene in frost-tolerant and frost-sensitive genotypes.

Although cold acclimation in cereals involves the expression of many cold-regulated genes, genetic studies have shown that only very few chromosomal regions carry loci that play an important role in frost tolerance. To investigate the genetic relationship between frost tolerance and the expression of cold-regulated genes, the expression and regulation of the wheat homolog of the barley cold-regulated gene cor14b was studied at various temperatures in frost-sensitive and frost-tolerant wheat genotypes. At 18/15 degrees C (day/night temperatures) frost-tolerant plants accumulated cor14b mRNAs and expressed COR14b proteins, whereas the sensitive plants did not. This result indicates that the threshold temperature for induction of the wheat cor14b homolog is higher in frost-resistant plants, and allowed us to use this polymorphism in a mapping approach. Studies made with chromosome substitution lines showed that the polymorphism for the threshold induction temperature of the wheat cor14b homolog is controlled by a locus(i) located on chromosome 5A of wheat, while the cor14b gene was mapped in Triticum monococcum on the long arm of chromosome 2Am. The analysis of single chromosome recombinant lines derived from a cross between Chinese Spring/Triticum spelta 5A and Chinese Spring/Cheyenne 5A identified two loci with additive effects that are involved in the genetic control of cor14b mRNA accumulation. The first locus was tightly linked to the marker psr911, while the second one was located between the marker Xpsr2021 and Frost resistance 1 (Fr1).

High expression level of a gene coding for a chloroplastic amino acid selective channel protein is correlated to cold acclimation in cereals.

A cold-regulated gene (cor tmc-ap3) coding for a putative chloroplastic amino acid selective channel protein was isolated from cold-treated barley leaves combining the differential display and the 5'-RACE techniques. Cor tmc-ap3 is expressed at low level under normal growing temperature, and its expression is strongly enhanced after cold treatment. A positive correlation between the expression of cor tmc-ap3 and frost tolerance was found both among barley cultivars and among cereal species. The COR TMC-AP3 protein was expressed in vitro, purified and used to raise a polyclonal antibody. Western analysis showed that the cor tmc-ap3 gene product is localized to the chloroplastic outer envelope fraction, supporting its putative function. The frost-resistant winter cultivar Onice accumulated COR TMC-AP3 more rapidly and at a higher level than the frost-susceptible spring cultivar Gitane. After 28 days of cold acclimation the winter cultivar had about 2-fold more protein than the spring genotype. All these results suggest that an increased amount of a chloroplastic amino acid selective channel protein could be required for cold acclimation in cereals. Hypotheses about the role of COR TMC-AP3 during the hardening process are discussed.

A leucine-rich repeat receptor-like protein kinase (LRPKm1) gene is induced in Malus x domestica by Venturia inaequalis infection and salicylic acid treatment.

A cDNA clone encoding a leucine-rich repeat (LRR) receptor-like protein kinase (LRPKm1) of Malus x domestica cv. Florina has been isolated using as a heterologous probe a cloned gene encoding a polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L. A genomic clone containing the 5'-regulatory region and a 5' portion of the open reading frame of the LRPKm1 gene has also been isolated. An open reading frame of 2997 nt (999 amino acids) was present in the cDNA clone, encoding a receptor-like protein comprising a 21 amino acid signal peptide for secretion, a leucine zipper, 23 LRRs, a putative membrane-spanning region and a serine/threonine protein kinase domain. LRPKm1 shows homology to the A. thaliana receptor-like protein kinase RLK5 and, to a minor extent, to PGIP. The LRPKm1 region from +5 to +600 exhibits an alternative reading frame that encodes a product corresponding to a proline-rich protein fragment homologous to several hydroxyproline-rich proteins. Southern blot analysis showed that LRPKm1 belongs to a multigene family and that there is length polymorphism of the hybridizing restriction fragments among different M. x domestica cultivars. Northern blot analysis was carried out on mRNA extracted from infected leaves of either cv. Florina (resistant to Venturia inaequalis) or cv. Golden Delicious (susceptible to V. inaequalis), and from tissues treated with salicylic acid. A 3500 bp transcript hybridizing at high stringency with the LRPKm1 cDNA accumulated in response to infection or salicylic acid treatment. Transcript accumulation was more intense in the incompatible interaction than in the compatible one. The possible involvement of this receptor-like protein kinase in resistance of apple to phytopathogenic fungi is discussed.

The interaction between cold and light controls the expression of the cold-regulated barley gene cor14b and the accumulation of the corresponding protein.

We report the expression of the barley (Hordeum vulgare L.) COR (cold-regulated) gene cor14b (formerly pt59) and the accumulation of its chloroplast-localized protein product. A polyclonal antibody raised against the cor14b-encoded protein detected two chloroplast COR proteins: COR14a and COR14b. N-terminal sequencing of COR14a and expression of cor14b in Arabidopsis plants showed that COR14a is not encoded by the cor14b sequence, but it shared homology with the wheat (Triticum aestivum L.) WCS19 COR protein. The expression of cor14b was strongly impaired in the barley albino mutant an, suggesting the involvement of a plastidial factor in the control of gene expression. Low-level accumulation of COR14b was induced by cold treatment in etiolated plants, although cor14b expression and protein accumulation were enhanced after a short light pulse. Light quality was a determining factor in regulating gene expression: red or blue but not far-red or green light pulses were able to promote COR14b accumulation in etiolated plants, suggesting that phytochrome and blue light photoreceptors may be involved in the control of cor14b gene expression. Maximum accumulation of COR14b was reached only when plants were grown and/or hardened under the standard photoperiod. The effect of light on the COR14b stability was demonstrated by using transgenic Arabidopsis. These plants constitutively expressed cor14b mRNAs regardless of temperature and light conditions; nevertheless, green plants accumulated about twice as much COR14b protein as etiolated plants.

Wild and cultivated barleys show differences in the expression pattern of a cold-regulated gene family under different light and temperature conditions.

Cold acclimation in plants involves the expression of many genes and gene families. The present study reports the expression analysis of three members of the blt14 gene family in barley. Gene-specific antisense oligonucleotides were used as probes in northern experiments so as to follow independently the expression of individual members of the gene family. Each clone revealed different accumulation kinetics when a spring and a winter cultivar were compared, suggesting that the different regulatory mechanisms leading to mRNA accumulation of an individual member of the blt14 gene family are genotype-dependent. In a collection of Hordeum spontaneum genotypes both qualitative and quantitative polymorphisms were found for the accumulation of blt14-related mRNAs, although no clear relationships were found between blt14 expression and frost resistance. The accumulation of the blt14-related mRNAs was also modulated by light and by the albino mutation a(n). The effects of light on the accumulation of the transcripts corresponding to the blt14 gene family were evaluated by comparing etiolated and green plants. Etiolated plants accumulate the blt14-related mRNAs at a detectable level already at 22 degrees C. When the same plants are exposed to cold in absence of light an increased mRNA accumulation above the level present in green cold-treated plants can be detected. On the contrary, etiolated plants showed a reduced blt14 accumulation when exposed to cold in the presence of light. Cold-induced expression of the blt14 gene family was strongly reduced in plants carrying the albino mutation a(n). This mutant showed a defective molecular response to cold even when probed with a cDNA coding for LEA type protein (paf93). The albino mutant a(n) was not able to harden when exposed to low temperature providing a direct evidence of the relationship between expression of cold-regulated (COR) genes and the development of cold hardening. Failure of cold acclimation in the mutant cannot be merely ascribed to the absence of photosynthetic activity, since etiolated wild-type plants accumulated COR mRNAs and improved frost resistance when exposed to cold.

Genetic analysis of the accumulation of COR14 proteins in wild (Hordeum spontaneum) and cultivated (Hordeum vulgare) barley.

The cold-regulated (COR14) protein of 14 kDa is a polypeptide accumulated under low-temperature conditions in the chloroplasts of barley leaves. In H. vulgare the COR14 antibody cross-reacts with two proteins, with a slightly different relative molecular weight around the marker of 14.4 kDa, referred to as COR14a and COR14b (high and low relative molecular weight, respectively). In a collection of H. spontaneum genotypes a clear polymorphism was found for the corresponding COR proteins. While some accessions showed the same COR pattern as cultivated barley, in 38 out of 61 accessions examined the COR14 antibody cross-reacted with an additional coldregulated protein with a relative molecular weight of about 24 kDa (COR24). The accumulation of COR24 was often associated with the absence of COR14b; the relationship between the COR14b/COR24 polymorphism and the adaptation of H. spontaneum to different environments is discussed. By studying COR14 accumulation in cultivated barley we have found that the threshold induction-temperature of COR14a is associated with the loci controlling winter hardiness. This association was demonstrated by using either a set of 30 cultivars of different origin, or two sets of frost-tolerant and frost-sensitive F1 doubled-haploid lines derived from the cross Dicktoo (winter type) x Morex (spring type). These results suggest that the threshold induction-temperature of COR14a can be a potential biochemical marker for the identification of superior frostresistant barley genotypes.

The accumulation of a cold-regulated chloroplastic protein is light-dependent.

The protein encoded by cDNA clone pt59 and induced in barley (Hordeum vulgare L.) by cold was over-expressed in coli to produce the matching antibody, which in vivo recognized a cold-induced protein of 14 kDa (COR14) that was found in the chloroplast stroma. The accumulation of COR14 occurred only at low temperatures after even a brief exposure of the plants to light. Plants grown and fully hardened in the dark accumulated a reduced amount of pt59-corresponding mRNA and only traces of COR14. Light exposure for as short as 5 min was enough to normalize the expression of pt59-corresponding mRNA and increase the accumulation of COR14. These findings indicate that one or more light-dependent factors are involved in transcription of the gene and accumulation of the protein. The COR14 protein was stored in amounts only slightly greater in the resistant barley cultivar. Onice than in the susceptible cultivar Gitane, although the former had a higher induction-temperature threshold for COR14 than the latter. This fact is an evolutionary advantage, enabling the resistant varieties in the field to prepare the cold well ahead of the susceptible ones.

RFLP analysis of highly polymorphic loci in barley.

Barley middle-repeat sequences were screened for their ability to discriminate 51 barley commercial varieties. Two hordein clones, a clone encoding a leaf-specific thionin, a desiccation induced cDNA clone, a clone coding for 5S-rRNA and one corresponding to ubiquitin genes were tested. A very sensitive RFLP technique including four cutter restriction enzymes and denaturing 4% polyacrylamide gels were used to evidence the highest level of polymorphism.The RFLP data were analyzed by computer. Some probe/enzyme combinations were able to differentiate a large number of the cultivars tested, whereas three probe/enzyme combinations succeeded in identifying all the varieties. The use of this RFLP method can thus be suggested for cultivar identification in barley.

Molecular cloning and characterization of cold-regulated genes in barley.

Five different cDNA clones have been isolated which are homologous to cold-regulated mRNAs in barley (Hordeum vulgare L.). The analyses of their hybridizations indicate that the transcripts accumulate to different levels during cold-treatment. Hybridization experiments using RNAs isolated from different plant tissues indicate that several cold-regulated genes are expressed in a tissue-specific manner. The expression studies suggest that in barley several different genes are involved in the cold hardening process depending on developmental stages and tissues involved. Homology has been found between the isolated cDNAs and cold-induced transcripts of related cereals. DNA sequence analysis of the clones pT59 and pA086 reveals that the proteins deduced from the longest open reading frame contain arginine rich basic domains.

Cold-induced mRNAs accumulate with different kinetics in barley coleoptiles.

The effect of cold treatment on gene expression in two different barley (Hordeum vulgare L.) cultivars has been studied. Cold stress induced a set of new mRNAs as determined by in-vitro translation of coleoptile RNA obtained from control and stressed seedlings. These mRNAs accumulated with different kinetics, and the cold-induced proteins could be grouped into five categories. The first category (a) is represented by a single protein with Mr of 75 kDa that reaches its highest level of expression after 6 h at 5°C. This polypeptide readily accumulates in the plant tissues and it can be detected when proteins separated by two-dimensional electrophoresis are stained with silver nitrate. The other polypeptides appear later during the 1- to 4-d stress period (protein groups b and c), increase (group d), or decrease during the period of treatment (group e). Only minor differences between the two cultivars with different cold-resistance capacities were found when the in-vitro translation products were compared. The results obtained demonstrate that several mRNAs are specifically expressed as a response to cold treatment in barley coleoptiles.