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CBF Genomic Clone Update 04-04-03

Solanaceae:

Primary genotypes of interest:

Lycopersicon esculentum cv. TA491:

1. Three linked genes 2. DNA sequence of 19kb λ insert completed

Solanum commersonii Oka 4583 clone:

1. Four genes (one may be unlinked? 2. Relevant λ clones subcloned into plasmid 3. Shotgun subclone library completed 4. DNA sequencing in progress

S. tuberosum cv Umatilla:

1. Numerous genes 2. Complete coverage possible with all λ clones in hand 3. Shotgun subclone libraries and DNA sequence determination of one clone in progress 4. Subcloning remainder of relevant λ clones into plasmid vector in progressseduction girls pickup dating tips Attract Women Body Language

Additional species of relevance to manuscript:

Lycopersicon pimpinellifolium:

1. λ clones in hand 2. Subcloning λ clones into plasmid in progress

Lycopersicon hirsutum:

1. λ clones in hand 2. Subcloning λ clones into plasmid in progress

Manuscript in preparation:

Mapping of the CBF locus in tomato to determine if CBF locus maps to previously identified low temperature tolerance QTL.

Observation that the only cold regulated gene, LeCBF1 appears to be absent in L. pimpinellifolium.

Cloning and sequencing of the CBF locus in L. pimpinellifolium to determine exact deletion end points.

Genetic analysis of segregating populations to determine whether LeCBF absence in L. pimpinellifolium linked to extreme chilling sensitivity in L. pimpinellifolium vs. L. esculentum.

Examination of additional Lycopersicon species, including cold tolerant genotypes of L. hirsutum, S. commersonii and S. tuberosum for CBF gene copy number and individual CBF gene expression patterns in response to cold temperatures.

DNA sequence determination of relevant Lycopersicon genotypes and comparative analyses with S. commersonii and S. tuberosum.

Legumes:

Medicago truncatula (barrel medic):

1. Using M. truncatula EST database identified 3 unique CBFs 2. DNA blots indicate each likely a small multigene family 3. Complete coverage appears possible with all λ clones in hand 4. Complete λ clone series subcloned into plasmid vector 5. Relevant subclones selected and sent to MSU GTSF for shotgun subclone library preparation and DNA sequence determination. GTSF is slow

Medicago falcata (alfalfa):

1. Using M. truncatula clones as probe; DNA blots indicate numerous genes in genome, perhaps significantly more than truncatula 2. Complete coverage appears possible with all λ clones in hand 3. Complete λ clone series subcloned into plasmid vector 4. Relevant subclones selected and sent to MSU GTSF for shotgun subclone library preparation and DNA sequence determination. GTSF is slow.

Soybean cv Stout:

1. EST database revealed 3 unique soybean CBFs 2. DNA blots indicate each likely a small multigene family 3. Complete coverage appears possible with all λ clones in hand 4. Subcloning complete series into plasmid vector in progress 5. Shotgun subclone libraries will be initiated soon 6. None appear to be expressed in response to cold or drought

Manuscript in preparation:

Previous work (Dhindsa) indicated alfalfa CAS genes may be functional homologs of Arabidopsis COR genes.

Abundance of CAS gene transcripts appears to be positively correlated with LT50 of different genotypes.

To determine whether alfalfa CAS genes might be part of a Medicago CBF regulon, we used the M. truncatula database and identified 3 unique Medicago CBFs. DNA blot analysis indicates that the alfalfa genome may have a significantly expanded CBF locus over that of M. truncatula. To determine complexity of Medicago CBF locus (loci) we have cloned the entire genomic region from both M. truncatula and M. falcata and are in the process of determining the DNA sequence of said region.

Expression analyses of both M. truncatula and M falcata indicate that two of these Medicago CBF transcripts accumulate at 1h (earliest time point examined) and continue to accumulate at least to 6h. Expression at 24 h significantly reduced relative to 6h time point, similar to Arabidopsis CBF expression profile.

Expression analyses of both M. truncatula and M falcata indicate that Medicago CAS15 gene transcripts begin to accumulate at about 6 h after being exposed to 6oC in M. falcata but not at all in M truncatula, whereas CAS17 transcripts are expressed in both M. truncatula and M falcata.

Absence of expression of Medicago CAS15 gene transcripts in M. truncatula and presence of CAS17 transcripts invokes the possibility that M. truncatula CAS15 genes may lack CRT/DRE sequences. To determine as much CAS15 and CAS17 genomic regions are being sequenced from both M. truncatula and M. falcata.

Barley:

Dicktoo:

1. Bacteriophage λ library constructed and screened with HvCBF3 and HvCBF4 (akaCBF1) 2. 19 clones identified in primary 3. 11 brought through to 3o screen 4. 4 clones selected for shotgun subclone library construction and sequence determination 5. Awaiting sequence from GTSF 6. Will rescreen for missing CBFs; need DNA sequence data and/or Jeff’s PCR results

Morex: Bacteriophage lambda library constructed; will screen concomitantly with Nure and Tremoir

Nure & Tremoir:

1. Library construction and screening to begin in 1-2 weeks.

Attention..!!

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