Background: Although Single Nucleotide Polymorphism (SNP) marker is an invaluable tool for positional cloning, association study and evolutionary analysis, low SNP detection efficiency by Allele-Specific PCR (AS-PCR) still restricts its application as molecular marker like other markers such as Simple Sequence Repeat (SSR). To overcome this problem, primers with a single nucleotide artificial mismatch introduced within the three bases closest to the 3’end (SNP site) have been used in AS-PCR. However, for one SNP site, nine possible mismatches can be generated among the three bases and how to select the right one to increase primer specificity is still a challenge. Results: In this study, different from the previous reports which used a limited quantity of primers randomly (several or dozen pairs), we systematically investigated the effects of mismatch base pairs, mismatch sites and SNP types on primer specificity with 2071 primer pairs, which were designed based on SNPs from Brassica oleracea 01-88 and 02-12. According to the statistical results, we (1) found that the primers designed with SNP (A/T), in which the mismatch (CA) in the 3^rd nucleotide from the 3’ end, had the highest allele-specificity (81.9%). This information could be used when designing primers from a large quantity of SNP sites; (2) performed the primer design principle which forms the one and only best primer for every SNP type. This is never reported in previous studies. Additionally, we further identified its availability in rapeseed (Brassica napus L.) and sesame (Sesamum indicum). High polymorphism percent (75%) of the designed primers indicated it is a general method and can be applied in other species. Conclusion: The method provided in this study can generate primers more effectively for every SNP site compared to other AS-PCR primer design methods. The high allele-specific efficiency of the SNP primer allows the feasibility for low- to moderate- throughput SNP analyses and is much suitable for gene mapping, map-based cloning, and marker-assisted selection in crops.

Background: Brassica oleracea is a morphologically diverse species in the family Brassicaceae and contains a group of nutrition-rich vegetable crops, including common heading cabbage, cauliflower, broccoli, kohlrabi, kale, Brussels sprouts. This diversity along with its phylogenetic membership in a group of three diploid and three tetraploid species, and the recent availability of genome sequences within Brassica provide an unprecedented opportunity to study intra- and inter-species divergence and evolution in this species and its close relatives. Description: We have developed a comprehensive database, Bolbase, which provides access to the B. oleracea genome data and comparative genomics information. The whole genome of B. oleracea is available, including nine fully assembled chromosomes and 1,848 scaffolds, with 45,758 predicted genes, 13,382 transposable elements, and 3,581 non-coding RNAs. Comparative genomics information is available, including syntenic regions among B. oleracea, Brassica rapa and Arabidopsis thaliana, synonymous (Ks) and non-synonymous (Ka) substitution rates between orthologous gene pairs, gene families or clusters, and differences in quantity, category, and distribution of transposable elements on chromosomes. Bolbase provides useful search and data mining tools, including a keyword search, a local BLAST server, and a customized GBrowse tool, which can be used to extract annotations of genome components, identify similar sequences and visualize syntenic regions among species. Users can download all genomic data and explore comparative genomics in a highly visual setting. Conclusions: Bolbase is the first resource platform for the B. oleracea genome and for genomic comparisons with its relatives, and thus it will help the research community to better study the function and evolution of Brassica genomes as well as enhance molecular breeding research. This database will be updated regularly with new features, improvements to genome annotation, and new genomic sequences as they become available. Bolbase is freely available at http://ocri-genomics.org/bolbase.

Background: Brassica oleraceaencompass a family of vegetables and cabbage that are among the most widely cultivated crops. In 2009, theB. oleraceaGenome Sequencing Project was launched using next generation sequencing technology. None of the available maps were detailed enough to anchor the sequence scaffolds for the Genome Sequencing Project. This report describes the development of a large number of SSR and SNP markers from the whole genome shotgun sequence data ofB. oleracea, and the construction of a high-density genetic linkage map using a double haploid mapping population.Results: TheB. oleraceahigh-density genetic linkage map that was constructed includes 1,227 markers in nine linkage groups spanning a total of 1197.9 cM with an average of 0.98 cM between adjacent loci. There were 602 SSR markers and 625 SNP markers on the map. The chromosome with the highest number of markers (186) was C03, and the chromosome with smallest number of markers (99) was C09.Conclusions: This first high-density map allowed the assembled scaffolds to be anchored to pseudochromosomes. The map also provides useful information for positional cloning, molecular breeding, and integration of information of genes and traits inB. oleracea. All the markers on the map will be transferable and could be used for the construction of other genetic maps.

Sulforaphane is abundant in broccoli (Brassica oleracea var. italica) and has been widely studied since it may reduce the risk of several types of cancer. Here, the variation of sulforaphane levels during different stages of broccoli flower bud development was studied by high-performance liquid chromatography (HPLC). At the same time, the expressions of gene AOP2 in all the materials were studied by real-time Polymerase chain reaction (RT-PCR), and the purpose was to reveal the correlation of AOP2 and sulforaphane contents in different organs of broccoli flower bud development. Sulforaphane levels were evaluated in broccoli florets, buds in bolting, flowers, and ripe seed. The results showed that there was a good linear equation, Y = 3754.22X − 2072.47, R² = 0.9991, and sulforaphane levels increased during development from florets to young buds in bolting plants, thereafter decreased gradually in older buds to flowers. The ripe seed was the highest organ (13.35g/kg DW (dry weight)) with sulforaphane content in all the materials. RT-PCR result showed an opposite relationship of sulforaphane contents and the expression levels of gene AOP2 from young buds to flowers in bolting period. The study demonstrated that the over-expression of the gene AOP2 leads to a peak level of sulforaphane content in organs of broccoli.