POSITIONAL CLONING OF WHEAT AND BARLEY VERNALIZATION GENES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0185870
• Grant No.: 00-35300-9565
• Project No.: CA-D*-ARS-6762-CG
• Proposal No.: 2000-01678
• Project Start Date: Sep 1, 2000
• Project End Date: Aug 31, 2003
• Grant Year: 2000

Project Director
Dubcovsky, J.

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
PLANT SCIENCES

Non Technical Summary
The adaptability of wheat and barley to a large range of environments is partially due to genetic variation in sensitivity to vernalization (acceleration of the ability to flower by a chilling treatment). The difference in vernalization requirement divides wheat and barley cultivars into winter and spring classes, and is mainly determined by vernalization genes Vrn-1 and Vrn-2. The objective of this proposal is to clone these two genes using a positional cloning approach. We will first delimit the minimum regions encompassing Vrn-1 and Vrn-2 using large insert clones (BAC clones) from wheat, barley, and rice and then we will sequence them. We will identify the genes present in these regions using gene-finding computer programs; comparative sequencing of wheat, barley and rice; and hybridization of the DNA from this region with available cDNA libraries (expressed genes). To identify Vrn-1 and Vrn-2 among the candidate genes we will analyze the expression patterns of the candidate genes, their genetic variation in induced and natural mutants, and their complementation ability in transformation experiments. Cloned vernalization genes will allow a fast transformation of winter to spring genotypes and vice versa, accelerate the development of spring genotypes resistant to frost damage, and make possible the development of molecular markers for the different vernalization alleles to study their agronomic value. Besides their practical applications, the Vrn clones will facilitate the elucidation of the basic mechanisms of vernalization in temperate cereals.

Animal Health Component

(N/A)

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
203	1549	1080	50%
203	1550	1080	50%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1549 - Wheat, general/other; 1550 - Barley;

Field Of Science
1080 - Genetics;

Keywords

plant biology

wheat

barley

vernalization

plant genetics

gene cloning

genomes

genetic mapping

position effects

flowering

triticum

artificial chromosomes

genetic markers

dna sequences

gene analysis

hexaploids

genetic variance

gene loci

chromosome walking

rice

computer programs

gene expression

Goals / Objectives
The long-term goal of this project is to provide a better understanding of the vernalization process in the Triticeae species. The specific objectives are: 1) Complete the physical contigs encompassing the vernalization genes using large insert clones. 2) Delimit the minimum region within the contigs encompassing Vrn-1 and Vrn-2 and sequence the complete contig. 3) Identify all the genes present in these regions 4) Identify Vrn-1 and Vrn-2 among the candidate genes. 5) Create triple-recessive vrn-2 hexaploid wheats to analyze natural variation at this locus.

Project Methods
We will complete the physical contigs for the vernalization genes using simultaneous chromosome walking in wheat, barley and rice. After completing the contigs we will delimit the minimum region between the crossovers flanking Vrn-1 and Vrn-2 genes in our high-density mapping populations. We will sequence the complete contig and assemble the sequence using computer programs. We will identify the genes present in the contigs using genefinding computer programs; comparative sequencing of wheat, barley and rice large insert clones; and hybridization of these clones with available cDNA libraries. To identify Vrn-1 and Vrn-2 among the candidate genes we will analyze the expression patterns of the candidate genes, their genetic variation in induced and natural mutants, and their complementation ability in transformation experiments. To create triple-recessive vrn-2 hexaploid wheats we will screen and combine natural deletions using the Vrn-2 gene as a probe, or alternatively we will transfer the vrn-Am2 gene from T. monococcum to hexaploid wheat lines with deletions for the Vrn-B2 and Vrn-D2 genes.

Progress 09/01/00 to 08/31/03

Outputs
The objective of this project was to clone vernalization genes Vrn1 and Vrn2 in diploid wheat and barley using a positional cloning approach. High-density genetic maps and physical maps were completed for both genes. The physical maps were sequenced and candidate genes were identified for both genes. Quantitative PCR was used to characterize the expression profile of both genes and to demonstrate that the RNA levels of Vrn1 were upregulated by vernalization and the RNA levels of Vrn2 were downregualted by vernalization. This was the expected result because Vrn2 is a dominant repressor of flowering and Vrn1 is a dominant promoter of flowering. Analysis of the allelic variation at the Vrn1 locus showed that the spring growth habit was generally associated with deletions in the promoter region of the meristem identity gene Apetala1. The information from the genetic studies, the transcription profiles, and the allelic variation was combined to demonstrate that the Apetala1 gene was the Vrn1 gene from wheat. Results from this study were published in the Proceedings of the National Academy of Science. The vernalization gene Vrn1 is also involved in the regulation of frost tolerance in wheat. We used the information generated from the positional cloning of the Vrn1 gene to participate in an international project aimed to elucidate the mechanisms of cold tolerance in wheat (Crosatti et al. in press). During the positional cloning studies we generated a large amount of sequence information from orthologous regions in different wheat genomes. These results were used to establish the rapid divergence of the intergenic regions among Triticeae genomes (Wicker at al. 2003).

Impacts
Vernalization genes Vrn1 and Vrn2 play a central role in determining growth habit in many temperate cereals. The recent cloning of these two genes in our laboratory will provide the means to modify wheat growth habit by transformation and to modulate the strength of the vernalization response to regulate flowering date. The promoters identified in these two genes will be useful tools to manipulate other traits at specific stages of the growing season.

Publications

• Peer reviewed journals Yan L, Loukoianov A, Tranquilli G, Helguera M, Fahima T, and Dubcovsky J. 2003. Positional cloning of wheat vernalization gene VRN1. Proc. Natl. Acad. Sci. US 100:6263-6268.
• Crosatti C, Mare C, Mazzucotelli E, Belloni S, Barilli S, Bassi R, Dubcovsky J, Galiba G, Stanca AM, and Cattivelli L. 2003. Genetic analysis of the expression of the cold-regulated gene cor14b: a way toward the identification of components of the cold response signal transduction in Triticeae. Canadian Journal of Botany. In press.
• Wicker T, Yahiaoui N, Guyot R, Schlagenhauf E, Liu E, Dubcovsky J, and Keller B. 2003. Rapid genome divergence at orthologous LMW Glutenin loci of the A and Am genomes of wheat. The Plant Cell 15: 1186-1197.
• Presentations in meetings and symposiums Galiba G, Vagujfalvi A, Kerepesi I, Crosatti C, Cattivelli L, Dubcovsky J, Kocsy G, Sutka J. 2003. Mapping of traits affecting the cold acclimation of wheat. EUCARPIA Cereal Section Meeting 21-25 November 2002 Salsomaggiore (Italy) From biodiversity to genomics: breeding strategies for small grain cereals in the third Millenium

Progress 01/01/02 to 12/31/02

Outputs
The objective of this project was to clone vernalization genes Vrn1 and Vrn2 in diploid wheat and barley using a positional cloning approach. During this period we identified two transcription factors as candidate genes for the main vernalization genes (Vrn1 and Vrn2) in wheat and barley by a map based approach. This identification was based on detailed molecular maps based on 6000 gametes each, construction of physical BAC contigs in Triticum monococcum, and sequencing of approximately 10 BACs resulting in 1 Mb of wheat sequence. Sequence analysis in the colinear regions in barley, rice and sorghum revealed new aspects of retrotransposon activity in wheat and genome evolution in grasses. One of the genes identified in the Vrn2 region was a member of the SNF2 family of global transcription regulators. The expression patterns of the Snf2-like gene and the comparison of winter and spring accessions confirmed that this gene was not Vrn2. Phylogenetic analyses of the Snf2-like genes find in wheat, barley, rice and Arabidopsis define a new subfamily that includes human and mouse genes designated Snf2P. Quantitative PCR studies provided additional evidence supporting the identification of the two new transcription factors as candidates for the two vernalization genes. The transcripts of Vrn2 decrease with vernalization whereas the transcripts of the Vrn1 candidate gene increase after vernalization in both apexes and leaves. This was the expected pattern of expression for these tow genes according to previous studies on the epistatic interactions between these two genes.

Impacts
Vernalization genes Vrn1 and Vrn2 play a central role in determining growth habit in many temperate cereals. The recent cloning of these two genes in our laboratory will provide the means to modify growth habit by transformation and to modulate the strength of the vernalization response to regulate flowering date. Understanding of this regulatory pathway may also be useful to manipulate other traits at specific stages of the growing season.

Publications

• Reports: W. Ramakrishna, J. Ma, P. SanMiguel, J. Emberton, J. Dubcovsky, B. A. Shiloff, Z. Jiang, N. Rostoks, C. S. Busso, M. Ogden, E. Linton, A. Kleinhofs, K. M. Devos, J. Messing, J. L. Bennetzen. 2002. Frequent genic rearrangements in two regions of grass genomes identified by comparative sequence analysis. Comparative and Functional Genomics 3: 165-166.
• Presentation in meetings and symposiums: J. Ma, P. SanMiguel, J. Dubcovsky, B. A. Shiloff, N. Rostoks, Z. Jiang, C. S. Busso, A. Kleinhofs, K. M. Devos, W. Ramakrishna, J. L. Bennetzen. 2002. Comparative sequence analysis of homologous wx1 regions in barley, maize, pearl millet, rice, sorghum and wheat. Plant and Animal Genome X Conference, January, 2002 San Diego, CA.
• Ramakrishna, W., J. Dubcovsky, P. SanMiguel, Y.J. Park, C. S. Busso, L. Yan, J. Emberton and J. L. Bennetzen. 2002. Genic and genomic evolution in a colinear region of four cereal species. Plant and Animal Genome X Conference, January, 2002 San Diego, CA.
• W. Ramakrishna, J. Ma, P. SanMiguel, J. Emberton, J. Dubcovsky, B. A. Shiloff, Z. Jiang, N. Rostoks, C. S. Busso, M. Ogden, E. Linton, A. Kleinhofs, K. M Devos, J. Messing, J. L. Bennetzen. 2002. Frequent genic rearrangements in two regions of grass genomes identified by comparative sequence analysis. Plant and Animal Genome X Conference, January, 2002 San Diego, CA.
• Yan, L., V. Echenique, C. S. Busso, W. R.amakrishna, P. SanMiguel, J. L. Bennetzen, S. Harrington, and J. Dubcovsky. 2002. Organization, structure and expression of the genes encoding SNF2-like proteins in cereal plants. Plant and Animal Genome X Conference, January, 2002 San Diego, CA.
• SanMiguel, P., W. Ramakrishna, J. L. Bennetzen, C. Busso, and J. Dubcovsky. 2002. Transposable elements, genes and recombination in a 215-kb contig from wheat chromosome 5A. Functional and Integrative Genomics. 2: 70-80.
• Ramakrishna W., J. Dubcovsky, Park Y.-J., Busso C. S., Emberton J., SanMiguel P., and Bennetzen J. L. 2002 - 2003. Different types and rates of genome evolution detected by comparative sequence analysis of orthologous segments from four cereal genomes. Genetics in press.
• Yan L., V. Echenique, C. S. Busso, P. SanMiguel, W. Ramakrishna, J.L. Bennetzen, S. Harrington, and J. Dubcovsky. 2002 - 2003. Cereal genes similar to Snf2 define a new subfamily that includes human and mouse genes. Molecular and General Genetics. In press.
• A. Vagujfalvi, G. Galiba, L. Cattivelli, J. Dubcovsky. 2002 - 2003. Two frost-tolerance genes on wheat chromosome 5AL are linked to CBF-like transcriptional activators. Molecular and General Genetics. In press

Progress 01/01/01 to 12/31/01

Outputs
The objective of this project was to clone vernalization genes Vrn1 and Vrn2 in diploid wheat and barley using a positional cloning approach. The number of chromosomes analyzed with the markers flanking these genes was increased to 3840 for Vrn2 and to 2200 for Vrn1 to increase the resolution of the high-density maps. New recombinant plants were characterized and crossovers were found at less than 0.1 cM from the targeted genes. In the Vrn-1 regions in wheat and barley 500-kb contigs were constructed starting from a marker distal to the gene. A second contig of 150-kb was started from markers proximal to the gene. The gap between the proximal and distal contigs was not closed in wheat but a Sorghum BAC was identified that includes genes at both sides of the gap. This Sorghum BAC is being sequenced to find additional genes in the gap region. Two barley BACs, 4 wheat BACs and one rice BAC from the proximal region were completely sequenced to a 10-X coverage and 5 of them were completely finished (two were published and 2 are in press). In the distal region, one wheat BAC was sequenced and linked to a sequenced contig in the rice genome project. The gap present in the physical contigs of wheat and barley has not been closed yet in the rice genome project. Sequence analysis revealed interesting aspects of retrotransposon activity in wheat. The same analysis identified 8 genes in the region, two of which were identified as putative candidate genes for Vrn1. In the Vrn-2 region, complete contigs including the Vrn-2 flanking markers were constructed in barley and rice. A gap present in the contigs of T. monococcum could not be completed because of the presence of numerous retrotransposons. To solve this problem a 200-kb contig was constructed using BACs from the A genome of tetraploid wheat Langdon that included both flanking markers. The complete rice, barley, T. monococcum, and Langdon contigs were sequenced. Six genes were identified in the regions between the flanking crossovers. One of them was a member of the SNF2 family of global transcription regulators. The expression patterns of the Snf2-like gene and the comparison of winter and spring accessions suggested that this gene was not Vrn2. Phylogenetic analyses of the Snf2-like genes find in wheat, barley, rice and Arabidopsis define a new subfamily that includes human and mouse genes designated Snf2P. Two additional genes have been also identified as putative candidates for Vrn2 and expression studies were started to test this hypothesis.

Impacts
This study provides evidence of the possibility to perform positional cloning of genes important for agriculture in the large genome of wheat. Vernalization genes Vrn1 and Vrn2 play a central role in determining growth habit in many temperate cereals. Cloning of these genes will provide the means to modify growth habit by transformation and to modulate the strength of the vernalization response to regulate flowering date. Understanding of this regulatory pathway may also be useful to manipulate other traits at specific stages of the growing season.

Publications

• Dubcovsky J., W. Ramakrishna, P. SanMiguel, C. S. Busso, L. Yan, B. A. Shiloff, & J. L. Bennetzen. 2001. Comparative sequence analysis of colinear barley and rice BACs. Plant Physiology. 125:1342-1353.
• Lagudah E., J. Dubcovsky, & W. Powell. 2001. Wheat Genomics. Plant Physiology and Biochemistry. 39:335-344.
• Echenique V.C., B. Stamova, P. Wolters, G. R. Lazo, & J. Dubcovsky. 2001. Frequencies of Ty1-copia and Ty3-gypsy retroelements within the Triticeae EST databases. Theor. Appl. Genet. In press.
• Dubcovsky J. 2001. Plant gene cloning may lead to better timing of flowering. NRI Research Highlights. NRI-CGP USDA CSREES. No. 2. Stamova, B., V. C. Echenique, P. Wolters, G. R. Lazo, & J. Dubcovsky. 2001. Frequency of retroelements within the Triticeae EST databases. Plant & Animal Genome IX Conference, January 13-17, 2001 San Diego, CA
• Ramakrishna W., J. Dubcovsky, P. SanMiguel, J. Ma, B.Shillof, J. Emberton, C. S. Busso, L. Yan, N. Rostoks, K. Devos, A. Kleinhofs, C. Bell and J. L. Bennetzen. 2001. Comparative Sequence Analysis of Grass Genomes using Orthologous Regions from Barley, Maize, Pearl Millet, Sorghum, Rice and Wheat. . Plant & Animal Genome IX Conference, January 13-17, 2001 San Diego, CA.
• L.Yan, V. Echenique, C. S. Busso, W. R.amakrishna, P. SanMiguel, J. L. Bennetzen, S. Harrington, & J. Dubcovsky. 2002. Organization, structure and expression of the genes encoding SNF2-like proteins in cereal plants. Plant & Animal Genome X Conference, January, 2002 San Diego, CA.