Development of a diagnostic DNA marker for the geographic origin of Shorea leprosula

The development of sequence characterized amplified region (SCAR) markers derived from amplified fragment length polymorphisms (AFLPs) is described for Shorea leprosula. An AFLP fragment that showed nearly complete differentiation between Borneo and Sumatra was gel-extracted, sequenced, and converted into a SCAR marker using the inverse polymerase chain reaction (PCR) technique. The single nucleotide polymorphism (SNP) that originally caused the AFLP was found in the MseI restriction site. Differentiation between islands was detected either as size variation of the codominant SCAR marker or after digestion of the PCR products with the restriction enzyme MseI (PCR-RFLP). Size variation was due to insertions/deletions found within the sequenced region that flanked the original AFLP fragment. After genotyping 151 samples of S. leprosula from 14 populations in Sumatra and Borneo, all but one sample from Sumatra were homozygous for one size variant (427 bp), while S. leprosula populations from Borneo showed different genotypes than Sumatra populations and variation not only among populations but also within populations. Complete differentiation and fixation on alternative variants was found for the geographic regions of Sumatra and Borneo by the PCR-RFLP method. The SCAR marker did not amplify in Shorea parvifolia and thus can also be used to distinguish between S. leprosula and S. parvifolia. The marker was successfully amplified from wood DNA extracts suggesting its applicability to track the geographic origin of timber.

Identification of genes associated with low furanocoumarin content in grapefruit

Some furanocoumarins in grapefruit (Citrus paradisi) are associated with the so-called grapefruit juice effect. Previous phytochemical quantification and genetic analysis suggested that the synthesis of these furanocoumarins may be controlled by a single gene in the pathway. In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis of fruit tissues was performed to identify the candidate gene(s) likely associated with low furanocoumarin content in grapefruit. Fifteen tentative differentially expressed fragments were cloned through the cDNA-AFLP analysis of the grapefruit variety Foster and its spontaneous low-furanocoumarin mutant Low Acid Foster. Sequence analysis revealed a cDNA-AFLP fragment, Contig 6, was homologous to a substrate-proved psoralen synthase gene, CYP71A22, and was part of citrus unigenes Cit.3003 and Csi.1332, and predicted genes Ciclev10004717m in mandarin and orange1.1g041507m in sweet orange. The two predicted genes contained the highly conserved motifs at one of the substrate recognition sites of CYP71A22. Digital gene expression profile showed the unigenes were expressed only in fruit and seed. Quantitative real-time PCR also proved Contig 6 was down-regulated in Low Acid Foster. These results showed the differentially expressed Contig 6 was related to the reduced furanocoumarin levels in the mutant. The identified fragment, homologs, unigenes, and genes may facilitate further furanocoumarin genetic study and grapefruit variety improvement.

In silico analysis of a putative SPIRAL gene related to strawberry ripening

The aim of this study was to characterize a gene associated with ripening in strawberry, a non-climacteric fruit. Differently expressed transcripts of candidate genes functioning in fruit development and ripening were identified from strawberry ( Fragaria × ananassa Duch.) in four ripening stages using the cDNA-AFLP method. The cDNA fragment designated C11M32M003 was selected from the putative ripening-related genes for further analysis. This transcript accumulated in the green receptacle, and the achene, but gene expression decreased in both tissues in parallel with the progress of ripening (Balogh, 2006). In silico analysis revealed that both the cDNA-AFLP fragment (C11M32M003) and the full-length cDNA AY695666 showed over 60% homology at the nucleotide level with two gene groups found in various plant species, including Arabidopsis thaliana . One of the candidate groups consisted of NITRILASE sequences thought to be related to auxin biosynthesis. As an alternative, a lesser known gene group named SPIRAL was suggested. The results of the detailed bioinformatic comparisons presented in this paper prove that the strawberry sequence analysed belongs to the SPIRAL gene family.

Comparative AFLP reveals paternal sex ratio chromosome specific DNA sequences in the parasitoid wasp Trichogramma kaykai

The parasitoid wasp Trichogramma kaykai with a haplo-diploid sex determination has a B chromosome called the paternal sex ratio (PSR) chromosome that confers paternal genome loss during early embryogenesis, resulting in male offspring. So far, it is not well known whether the PSR chromosome has unique DNA sequence characteristics. By comparative AFLP fingerprinting of genomic DNA from wasps with and without the PSR chromosome, we isolated DNA from PSR-specific bands. Fourteen of such DNA fragments were analysed to confirm their PSR specificity. Seven were sequenced and two (PT-AFLP 1 and PT-AFLP1 3) were identified as parts of retrotransposon genes based on BLAST searches. Internal primers designed from a third AFLP fragment allowed PCR amplification of a PSR chromosome specific marker, which can be used to screen for the PSR trait in male wasps. Southern analysis revealed a dispersed repetitive nature of this third sequence in the T. kaykai genome, suggesting that it is part of a transposon. A fourth AFLP fragment (PT-AFLP 5) appears to be a large repetitive sequence on the PSR chromosome. This sequence is also found in the genome of both T. kaykai and the closely related species Trichogramma deion , but its distribution on the PSR chromosome strongly resembles that of T. deion rather than that of T. kaykai. Our results provide further insight into the repetitive nature of sequences comprising B chromosomes and their similarities with their host and closely related species.

Identification of an AFLP Fragment Linked to Rust Resistance in Asparagus Bean and Its Conversion to a SCAR Marker

Rust disease, incited by the fungus Uromyces vignae, adversely affects production and quality of asparagus bean and other types of cowpea in many parts of the world. Genetic resistance to the rust pathogen has been identified in a few accessions, but it is difficult to efficiently transfer the resistance to a broad range of asparagus bean cultivars using traditional breeding approaches. We determined that rust resistance was controlled by a single dominant gene designated Rr1 in the cross of a highly resistant cultivar ZN016 and highly susceptible cultivar Zhijiang 282. Bulked segregant analysis was applied to an F2 population derived from these parents, and an AFLP marker (E-AAG/M-CTG), 150 bp in size, was detected in the resistant bulk. The AFLP fragment was then converted to a SCAR marker, named ABRSAAG/CTG98, and the genetic distance between the marker and the Rr1 gene was estimated to be 5.4 cM. This SCAR marker could be used effectively for MAS of Rr1 in breeding programs to develop rust-resistant asparagus bean cultivars and potentially more widely to breed rust-resistant cultivars of other types of cowpea.