Carlos F. Quiros

Comparative analysis of methylthioalkylmalate synthase (MAM) gene family and flanking DNA sequences in Brassica oleracea and Arabidopsis thaliana

Gene BoGSL-PRO is associated with presence of 3-carbon side-chain glucosinolates (GSL). This gene is a member of the methylthioalkylmalate synthase (MAM) gene family. A BAC clone of Brassica oleracea, B21F5, containing this gene, was sequenced, annotated and compared to its corresponding region in Arabidopsis thaliana. Twelve protein-coding genes and 10 transposable elements were found in this clone. The corresponding region in A. thaliana chromosome I has 14 genes and no transposable elements. Analysis of MAM gene family in both species, which also include genes controlling 4-carbon side-chain GSL, separated the genes in two groups based on exon numbers and function. Phylogenetic analysis of the amino acid sequences encoded by these genes suggest that these two groups were produced by a duplication that must have occurred before the divergence of the Rosid and Asterid lineages of angiosperms. Comparison with putative orthologs from several prokaryotes further suggest that the members of the gene family with 10 exons, which encode proteins involved in 4-carbon side-chain GSL biosynthesis, were derived via truncation of the 3′ end from ancestral genes more similar in length to those with 12 exons, which encode proteins involved in 3-carbon side-chain GSL biosynthesis. Lower gene density in B. oleracea compared to A. thaliana is due in part to presence of transposable elements (TE) mostly in inter-genic regions.

Analysis of a set of RAPD markers by hybridization and sequencing in Brassica: a note of caution

A series of RAPD markers generated by a single 10-mer primer were analyzed by hybridization to amplified and genomic DNA and by sequencing in two Brassica species. Primer B18 produced different profiles of nine major bands each in both Brassica nigra (B genome) and B. napus (AC genomes). Cloning and sequencing of five B18 B. nigra amplification products revealed that they were all unrelated to each other. Only limited stretches of high similarity of up to 69 nucleotides were shared by some of these clones. Hybridization to genomic DNA indicated that only two corresponded to a highly repeated sequence, whereas the rest were low copy sequences. In spite of their lack of homology, when these clones were used as probes to amplified B. nigra DNA, they hybridized to multiple bands in the profile. Hybridization of B. nigra clones for bands of similar sizes in both species, failed to hybridize in B. napus, revealing lack of homology between the DNAs of the two species. Because of these inconsistencies, it is concluded that RAPD markers, although useful for genetical studies, should be used with caution specially when basing homology on cross-hybridization and fragment sizes.

SRAP Molecular Marker Technology in Plant Science

Molecular markers are commonly used in genetic diversity analysis, genetic map construc‐ tion, gene mapping and cloning, and marker assisted selection in plant breeding. Based on detection procedure, most molecular marker technologies can be classified into hybridiza‐ tion-based or PCR-based systems. Restriction fragment length polymorphism (RFLP) is the first hybridization-based molecular marker system that was intensively used at the begin‐ ning of the molecular biology era in life science while hybridization-based marker methods such as microarrays and diversity array technology (DArT) are used currently to detect sin‐ gle nucleotide polymorphisms (SNP). In contrast, many PCR-based molecular marker detec‐ tion methods have been developed. For example, amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSR) and sequence related amplified polymorphism (SRAP), inter-simple sequence repeat (ISSR), se‐ quence tagged site (STS), and sequence characterized amplification region (SCAR), are com‐ monly used in genomic analysis (Jones et al., 2009).

The Genetics of Brassica oleracea

Brassica oleracea is one of the most important species of the Brassicaceae family because the species includes some of the most economically important vegetables in the world. Common heading cabbage and cauliflower are the most widely grown crops of this species, but broccoli is also now emerging rapidly as a world vegetable. The wide center of origin for this species is the Mediterranean Basin, and primitive forebears of our modern B. oleracea crop forms have been cultivated and selected for several millennia. Undoubtedly, the diverse array of wild forms found in this species and other very closely related species played very important roles in stimulating the occurrence of morphological variation within and among the B. oleracea crops as they underwent development. In the years following the rediscovery of Mendel’s work, many scientists studied the underlying genetic factors controlling the divergent morphologies within the species. This was of interest not only from a basic scientific standpoint, but also due to the practical necessity of understanding the complex sets of genes that combine and give rise to a specific crop form like heading cabbage or cauliflower. This knowledge is crucial in moving genes between crops in the process of breeding improved varieties. Secondary plant metabolites have emerged as key components of crops within this species because they appear to contribute added-value to the various crops by conferring intrinsic healthful effects on populations that consume these vegetables. Among the various components believed to confer a chemoprotective effect in B. oleracea, glucosinolates, and isothiocyanates have received the most attention in recent years and are considered in detail herein. The study of B. oleracea genetics has been greatly advanced during the modern era of gene study at the molecular level. Although the species has presented challenges, scientists focused on these crops are now mapping genes to specific chromosomes and the genome is well on its way to being sequenced. As knowledge advances at the molecular level, a fuller understanding of gene sequences and there relations to morphology, disease resistance, phytochemical make-up, and other important traits are being realized in B. oleracea.

Sequence comparison of two codominant RAPD markers in Brassica nigra: deletions, substitutions and microsatellites.

SummaryTwo RAPD fragments segregating codominantly were investigated in a F2 population of Brassica nigra. Southern hybridization of these DNA fragments to genomic B. nigra DNA digested with several endonucleases revealed similar restriction profiles. Sequencing of the two fragments disclosed 93% homology. The differences were due mainly to an internal 41 nucleotide deletion in one of the fragments. Minor deletions of one to three bases, including a microsatellite of CTT motif were also observed. In addition, base substitutions, mostly transitions were detected. These relatively small differences suggested that the two RAPD products were indeed different versions of the same sequence. The larger fragment of 1154 bp was denominated A051 whereas the shorter one, denominated A052, had 1116 bp.