An innovative SNP genotyping method adapting to multiple platforms and throughputs.

KEY MESSAGE: An innovative genotyping method designated as semi-thermal asymmetric reverse PCR (STARP) was developed for genotyping individual SNPs with improved accuracy, flexible throughputs, low operational costs, and high platform compatibility. Multiplex chip-based technology for genome-scale genotyping of single nucleotide polymorphisms (SNPs) has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs as well as the compatibility with multiple platforms. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP). In this method, genotyping assay was performed under unique PCR conditions using two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers each were substituted one base in different positions at their 3' regions to significantly increase the amplification specificity of the two alleles and tailed at 5' ends to provide priming sites for PEA-primers. The two PEA-primers were developed for common use in all genotyping assays to stringently target the PCR fragments generated by the two AMAS-primers with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. The state-of-the-art primer design and unique PCR conditions endowed STARP with all the major advantages of high accuracy, flexible throughputs, simple assay design, low operational costs, and platform compatibility. In addition to SNPs, STARP can also be employed in genotyping of indels (insertion-deletion polymorphisms). As vast variations in DNA sequences are being unearthed by many genome sequencing projects and genotyping by sequencing, STARP will have wide applications across all biological organisms in agriculture, medicine, and forensics.

Leucine aminopeptidases: the ubiquity of LAP-N and the specificity of LAP-A.

The wound-induced leucine aminopeptidase (EC 3.4.11.1) genes, LapA1 and LapA2, from tomato (Lycopersicon esculentum Mill.) were isolated and characterized. The genes were organized in a tandem array with approximately 6 kb separating their coding regions. Quantitation of LapA mRNA levels in conjunction with nuclear run-on experiments indicated that LapA genes were primarily under transcriptional control after wounding and infection with Pseudomonas syringae pv. tomato. In contrast, actin genes were down-regulated after pathogen infection. The sequences of the LapA1 and LapA2 5'-flanking regions were determined and several potential regulatory motifs were identified. Ribonuclease protection studies revealed that LapA1 and LapA2 had short 18-bp 5'-untranslated regions (UTR), both genes were expressed after wounding, and LapA1 mRNAs were 3.3-fold more abundant than LapA2 transcripts. While the region surrounding LapA1 was conserved, the 3'-UTRs and 3'-flanking regions of LapA2 had diverged in two inbred tomato lines. The accumulation of LapA mRNAs and of LAP-A (acidic pI), LAP-N (neutral pI) and LAP-related proteins were examined in two monocot and five dicot species. The LAP-N and 66- and 77-kDa LAP-related proteins were detected in healthy and wounded leaves of all plants examined. The LAP-A proteins were only detected in nightshade and their accumulation was distinct from that observed in tomato.

A plasma membrane sucrose-binding protein that mediates sucrose uptake shares structural and sequence similarity with seed storage proteins but remains functionally distinct.

Photoaffinity labeling of a soybean cotyledon membrane fraction identified a sucrose-binding protein (SBP). Subsequent studies have shown that the SBP is a unique plasma membrane protein that mediates the linear uptake of sucrose in the presence of up to 30 mM external sucrose when ectopically expressed in yeast. Analysis of the SBP-deduced amino acid sequence indicates it lacks sequence similarity with other known transport proteins. Data presented here, however, indicate that the SBP shares significant sequence and structural homology with the vicilin-like seed storage proteins that organize into homotrimers. These similarities include a repeated sequence that forms the basis of the reiterated domain structure characteristic of the vicilin-like protein family. In addition, analytical ultracentrifugation and nonreducing SDS-polyacrylamide gel electrophoresis demonstrate that the SBP appears to be organized into oligomeric complexes with a Mr indicative of the existence of SBP homotrimers and homodimers. The structural similarity shared by the SBP and vicilin-like proteins provides a novel framework to explore the mechanistic basis of SBP-mediated sucrose uptake. Expression of the maize Glb protein (a vicilin-like protein closely related to the SBP) in yeast demonstrates that a closely related vicilin-like protein is unable to mediate sucrose uptake. Thus, despite sequence and structural similarities shared by the SBP and the vicilin-like protein family, the SBP is functionally divergent from other members of this group.

Localization and post-translational processing of the wound-induced leucine aminopeptidase proteins of tomato.

Leucine aminopeptidase (LAP) is induced by wounding and bacterial pathogen infection in tomato. DNA blot analysis of XbaI-digested lambdalap genomic clones demonstrated that LapA1 and LapA2 cDNAs were encoded by two different LapA genes in the tomato genome. The coding and untranslated regions of LapA1 and LapA2 mRNAs shared more than 93% identity. The deduced amino acid sequences of LapA cDNA clones and in vitro translation of LapA1 mRNA indicated that LAP-A was synthesized as a 60-kDa precursor protein. The processing of a 60-kDa preLAP-A into the mature 55-kDa LAP-A was demonstrated in vivo by expression of the full-length LapA1 cDNA in insect cells. Sequencing of a single LAP-A form isolated from a two-dimensional polyacrylamide gel indicated that LAP-A proteins had two different N termini that were separated by two residues. The LAP-A presequence had features similar to chloroplast transit peptides. Comparison of LAP-A levels in chloroplast and total protein extracts from methyl jasmonate-treated leaves indicated that a small proportion of the LAP-A proteins was detected in the plastids. Inspection of the LAP-A presequence indicated the presence of a dibasic protease (Kex2/furin) processing site motif 6-8 residues upstream from the LAP-A N termini. Its potential role in LAP-A precursor biogenesis is discussed.

The Impact of Chlorophyll-Retention Mutations, d1d2 and cyt-G1, during Embryogeny in Soybean.

The ultrastructural, physiological, and molecular changes in developing and mature seeds were monitored in a control line (Glycine max [L.] Merr., cv Clark) that exhibited seed degreening and two mutant lines (d1d2 and cyt-G1) that retained chlorophyll upon seed maturation. Ultrastructural studies showed that the control line had no internal membranes, whereas stacked thylakoid membranes were detected in the green seed from the mutant lines. Pigment analyses indicated that total chlorophyll was lowest in the mature seeds of the control line. Mature d1d2 and cyt-G1 seed had elevated Chl a and Chl b levels, respectively. In both control and mutant lines, Lhcb1, Lhcb2, and RbcS mRNAs were abundant in embryos prior to cotyledon filling, declined after the onset of storage protein accumulation, and were barely detectable or undetectable in all later stages of seed development. Therefore, the chlorophyll-retention phenotype must be a result of the alteration of a process that occurs after translation of photosynthesis-related mRNAs to stabilize apoprotein and pigment levels. Furthermore, different elements controlling either the synthesis or turnover of Chl a and Chl b must be impaired in the d1d2 and cyt-G1 lines. No reproducible differences in total leaf, embryonic, and chloroplast protein profiles and plastid DNAs could be correlated with the mutations that induced chlorophyll retention.