Cold Tolerance in Vaccinium corymbosum Shamita Punjabi Lab Methods in Genomics BIO 343 Davidson College

Starting point: CBF genes Polashock et al (2010)Polashock et al (2010) The CBF (C-repeat binding factor) genes have a host of downstream targets that help plants acclimate to the cold CBF genes are found in many species and are therefore a good target for understanding how cold acclimation can occur in a variety of plants

CBFs and COR genes in other species Cold Acclimation/Freezing Tolerance in Blueberries Polashock et al (2010) -COR6.6 -COR78 -COR15A etc.. Polashock et al (2010) Frost Tolerance in Temperate Cereals Galiba et al (2009) -FR2 -TaCBF14 -TaCBF15 Galiba et al (2009) Cold Tolerance in Eucalyptus Species Navarro et al (2009) -EguCBF1c -EguCBF1d Navarro et al (2009) Cold Tolerance signaling in Arabidopsis Lissarre et al, ICE1 -ICE2 Zhou et al, 2010 Lissarre et al, 2010 Zhou et al, SIZ1

Research Scheme Choose at least 1 CBF gene and 1 other frost tolerance gene to find in blueberry Obtained mRNA sequences for the EguCBF1c in Eucalyptus, TaCBF14 in common wheat and ICE1 and SIZ1 in Arabidopsis from NCBI Use tBLASTx in Vaccinium Database to find matches of mRNA sequences to amino acid sequences of blueberry scaffolds Submit best scaffolds to SSR database and choose primers based on vicinity to gene matches on scaffold Ultimately, devise a schematic pathway for activation of cold tolerance genes in blueberry Links to Sequences for all genes used are provided on the Wiki

Primers from SSR Search EguCBF1c & TaCBF14 3 Primer Matches on Scaffold (~488,000 bp) E score = 2e-17 Forward Primer: AGTTCTAAACCGATTGTGCGTT Reverse Primer: AATTCCAACCTAACTGCCAGAA TG 479,956 bp, Product: 291 bp Forward Primer: TCTCTCTCAGATCTCTGATCCGT Reverse Primer: AAAGCAAGAAGAGAAATGGTGG TCT 479,466 bp, Product: 110 bp Forward Primer: AATCTGCAAATCTCCATCACCT Reverse Primer: TCCTAAAAACCAAAGCATGTCC CT 463,925 bp, Product: 226 bp

3 Primer Matches on Scaffold (~55, ,000 bp) E score = 4e-80 Forward Primer: CGCATCTTTACTCCACTAACCC Reverse Primer: AATCCCTGCTGTGTATCTTGGT TC 55,088 bp, Product: 127 bp Forward Primer: GTGGGGAGCAAACTCACTAATC Reverse Primer: AATAACAAAAACTCGCTCTCGC CA 67,058 bp, Product: 186 bp Forward Primer: GAGAAGTGAAGGAATGGAGGTG Reverse Primer: CGAAATGGGTTCACTCTCTACC TGT 60,104 bp, Product: 259 bp Primers from SSR Search ICE1

Primers from SSR Search SIZ1 3 Primer Matches on Scaffold (~85, ,000 bp) E score = 0.0 Forward Primer: AAGCCGCATATTAGAGCGTATC Reverse Primer: CCTCCCTCCTCTCTCTCTCTCT AG 86,562 bp, Product: 300 bp Forward Primer: ATTGCAATCTTGCACAGAGAGA Reverse Primer: CTACATAGGATACGCATTGGCA AG 86,761 bp, Product: 279 bp Forward Primer: CATTTGTACCCCCTCAAGTAGC Reverse Primer: TTTCCCTAGTGGTGAAGTGTGA GA 107,162 bp, Product: 157 bp

Phospholipid Signaling Pathway Byeong-ha Lee et al used microarrays to find genes induced and repressed in cold environments Byeong-ha Lee et al Phospholipid signaling is one of many pathways is affected by CBF transcription factors Early induced genes: IP5PII and ADTGK1 Late induced genes: IPK2a KEGG Map for “phosphatidylinositol signaling” includes all of these genes

Primers from SSR Search IP5PII ( ) 3 Primer Matches on Scaffold (~93, ,000) E score = e-154 Forward Primer: GATTCGAACGGCAGTATAAACC Reverse Primer: GCCCTTATCAATCTCCAAATGA AT 106,789, Product: 222 bp Forward Primer: ATGGAGTACCAAGGAAAAACGA Reverse Primer: CCATTTTTATCGGGGTGAGTAA TC 81,787, Product: 246 bp Forward Primer: TCTCTTCTACTGTCAGAGGCCC Reverse Primer: CACTCTGTTTGGAAAATGTGGA ATA 86,548, Product: 231 bp

Primers from SSR Search ATDGK1 ( ) 3 Primer Matches on Scaffold (~355, ,000) E score = 0.0 Forward Primer: CTAGCCTACCAACTACCTCCGA Reverse Primer: GGATTGCTTCTCTGTTTCTGCT AG 352,411, Product: 214 bp Forward Primer: AGCAGAAACAGAGAAGCAATCC Reverse Primer: CAAGGCAAACCCTAGAGAGAGA CT 352,582, Product: 143 bp Forward Primer: TTGAACATGCTCTTGAATCCTG Reverse Primer: TACGTGAGTATCATCCACAGCC AATA 355,495, Product: 131 bp

Primers from SSR Search IPK2a ( ) 4 Primer Matches on Scaffold (~2,000-3,000) E score = 4e-81 Forward Primer: AATCAATCAGTTGACATGCGTC Reverse Primer: GCTTAAAGCTTAACAAGCCCAA CT 7,764, Product: 197 bp Forward Primer: ATCTAAATGTTTAATCGGGGGC Reverse Primer: ATCTAGGGAGACTGTTGGGGAT TG 17,950, Product: 143 bp Forward Primer: CCAATGCTGCTTCACTGTACTC Reverse Primer: TACTTGTCGGTTGCAGATTCAC AAAC 7,124, Product: 229 bp Forward Primer: ACCCATCCGAGGTATGTTACAG Reverse Primer: AAAGATTAAAGGCGGATAAGGC TTCGG 791, Product: 108 bp

KEGG Map for Phosphatidylinositol signaling pathway

Rough Scheme of Blueberry Cold Response Pathway CBF COLD! ICE1 (Sumoylation) IP5PII ADTGK1 IPK2a SIZ1 Primers via SSR search have been provided for all subjects above