Paul A. Wiersma

Identification of new self-incompatibility alleles in sweet cherry (Prunus avium L.) and clarification of incompatibility groups by PCR and sequencing analysis

Abstract Correct assignment of sweet cherry cultivars to cross-compatibility groups is important for the efficient production of cherry fruit. Despite considerable confusion in the literature, these groups continue to be an effective tool for predicting pollination effectiveness for breeders and growers. PCR fragments generated from cherry S-RNase sequences coincided with specific S-allele phenotypes. Twenty five genomic DNA fragments, representing the six most common alleles, were cloned and sequenced. In addition, fragments were characterized from four new S-alleles. These genomic and cDNA sequences were invariant among cultivars with the same S-allele. Using the sequence data, PCR and restriction enzyme-based methodology was developed for rapid analysis of S-genotypes. Analysis and description of fragmentation patterns for S-allele determination are discussed. The method was utilized to characterize the S-allele composition of 70 sweet cherry cultivars obtained from collections in North America, including many of the named releases from the Canadian breeding programs at Agriculture and Agri-Food Canada in Summerland, B.C., and Vineland, Ontario. A number of differences between published S-allele assignments and PCR data were discovered and a new listing of cultivar S-allele assignments is presented.

Reduced polyphenol oxidase gene expression and enzymatic browning in potato ( Solanum tuberosum L.) with artificial microRNAs

BackgroundPolyphenol oxidase (PPO), often encoded by a multi-gene family, causes oxidative browning, a significant problem in many food products. Low-browning potatoes were produced previously through suppression of PPO gene expression, but the contribution of individual PPO gene isoform to the oxidative browning process was unknown. Here we investigated the contributions of different PPO genes to total PPO protein activity, and the correlations between PPO protein level, PPO activity and tuber tissue browning potential by suppression of all previously characterized potato PPO genes, both individually and in combination using artificial microRNAs (amiRNAs) technology.ResultsSurvey of the potato genome database revealed 9 PPO-like gene models, named StuPPO1 to StuPPO9 in this report. StuPPO1, StuPPO2, StuPPO3 and StuPPO4 are allelic to the characterized POTP1/P2, POT32, POT33 and POT72, respectively. Fewer ESTs were found to support the transcriptions of StuPPO5 to StuPPO8. StuPPO9 related ESTs were expressed at significant higher levels in pathogen-infected potato tissues. A series of browning phenotypes were obtained by suppressing StuPPO1 to StuPPO4 genes alone and in combination. Down-regulation of one or several of the PPO genes did not usually cause up-regulation of the other PPO genes in the transgenic potato tubers, but resulted in reduced PPO protein levels. The different PPO genes did not contribute equally to the total PPO protein content in the tuber tissues, with StuPPO2 accounting for ~ 55% as the major contributor, followed by StuPPO1, ~ 25-30% and StuPPO3 and StuPPO4 together with less than 15%. Strongly positive correlations between PPO protein level, PPO activity and browning potential were demonstrated in our analysis. Low PPO activity and low-browning potatoes were produced by simultaneous down-regulation of StuPPO2 to StuPPO4, but the greatest reduction occurred when StuPPO1 to StuPPO4 were all suppressed.ConclusionStuPPO1 to StuPPO4 genes contributed to browning reactions in tuber tissues but their effect was not equal. Different PPO genes may be regulated independently reflecting their diversified functions. Our results show that amiRNAs can be used to suppress closely related members of highly conserved multi-gene family. This approach also suggests a new strategy for breeding low-browning crops using small DNA inserts.

The complete genome sequence of a new polerovirus in strawberry plants from eastern Canada showing strawberry decline symptoms

Strawberry decline disease, probably caused by synergistic reactions of mixed virus infections, threatens the North American strawberry industry. Deep sequencing of strawberry plant samples from eastern Canada resulted in the identification of a new virus genome resembling poleroviruses in sequence and genome structure. Phylogenetic analysis suggests that it is a new member of the genus Polerovirus, family Luteoviridae. The virus is tentatively named “strawberry polerovirus 1” (SPV1).

The complete nucleotide sequence and genome organization of pea streak virus (genus Carlavirus)

Pea streak virus (PeSV) is a member of the genus Carlavirus in the family Betaflexiviridae. Here, the first complete genome sequence of PeSV was determined by deep sequencing of a cDNA library constructed from dsRNA extracted from a PeSV-infected sample and Rapid Amplification of cDNA Ends (RACE) PCR. The PeSV genome consists of 8041 nucleotides excluding the poly(A) tail and contains six open reading frames (ORFs). The putative peptide encoded by the PeSV ORF6 has an estimated molecular mass of 6.6 kDa and shows no similarity to any known proteins. This differs from typical carlaviruses, whose ORF6 encodes a 12- to 18-kDa cysteine-rich nucleic-acid-binding protein.