Genomic Fingerprinting of <i>Camelina</i>Species Using cTBP as Molecular Marker

Interest on the genus Camelina has recently increased due to the biofuel, or jet fuel, potential of the oil extracted from seeds of the cultivated species Camelina sativa (L.) Crantz. While our knowledge on C. sativa is constantly augmenting, only few studies have been performed on the other species of the genus, which could be a potentially useful material for the genetic improvement of C. sativa. The genus Camelina consists of 11 species, but only six (C. sativa, C. microcarpa, C. alyssum, C. rumelica, C. hispida and C. laxa) could be retrieved from germplasm banks to carry out genomic fingerprinting studies based on the use of the cTBP molecular marker. Each species, with the exception of C. alyssum that is proposed to be a subspecies of C. sativa, shows a distinct cTBP profile resulting from multiple DNA length polymorphisms present in the second intron of the members of the β-tubulin gene family. In contrast to the high level of genetic diversity detected among the six Camelina species, low variability is observed among and within the accessions of the same species, except for C. hispida that is characterized by an intra-accession high number of cTBP polymorphic bands. In addition, cTBP is also able to identify incorrectly classified accessions and provide information on the ploidy level of each species.

<i>In Silico</i>Exploration of <i>Cannabis sativa</i>L. Genome for Simple Sequence Repeats (SSRs)

Simple sequence repeat (SSR) or microsatellite markers, are a valuable tool for several purposes such as evaluation of genetic diversity, fingerprinting, marker assisted selection, and breeding. Recent developments in sequencing technologies and bioinformatics analyses provide new opportunity to produce a high number of less costly SSRs. Here, we used for the first time a wholegenome shotgun sequencing of the nuclear genome and transcriptome of hemp to develop microsatellite markers for C. sativa L. (hemp). Hemp is an ancient crop that is widely cultivated as a source of fiber, seeds and medicine. The analysis using the MISA program revealed a total of 407,491 SSRs (from mono-nucleotide to deca-nucleotide) in the hemp genome and 15,655 SSRs in the transcriptome. Analysis of the frequency and distribution of SSRs showed that the mono-nucleotide repeats were the most abundant (55.4%) in the genome whereas the tri-nucleotide motifs (30.4%) resulted highly predominant in the transcriptome. Poly A/T was predominant over poly G/C in both genome and transcriptome sequences. Among the tri-nucleotide repeats AAG/CTT (34.5%) resulted the most abundant in the transcriptome. Repeats larger than tri-nucleotide were also observed in the hemp genome and transcriptome. Dinucleotide and tri-nucleotide repeat expansion of 8605 and 1401 times iteration were observed however, other SSR expansion more than 387 times repetition was not found. Primers were designed for amplification of few long microsatellite sequences which could be used to identify polymorphism and to study genetic diversity among hemp cultivars.

Bovine <i>β</i>-casein: Detection of two single nucleotide polymorphisms by bidirectional allele specific polymerase chain reaction (BAS-PCR) and monitoring of their variation

Due to the functional importance of bovine milk protein polymorphisms, their correct discrimination is of great interest both from a scientific and practical point of view. Nowadays a large number of commercial platforms are available for semiautomated or fully automated SNP geno-typing. However, in some cases the use of simple and rather cheap methods is an effective tool to be implemented within one’s own laboratory for the routine analysis of a specific SNP. The present paper describes two simple tests based on the bidirectional allele-specific polymerase chain reaction (BAS-PCR) developed for the identification of β-casein (CSN2) B and I genetic variants. The practical application of the two methods on a panel of 84 Italian Brown bulls and 100 Italian Friesian cows is also discussed, including the biological significance of the two genetic variants and the importance of taking their occurrence into account when linkage analyses are performed on milk functional properties. A combined system for analysing milk protein variants by isoelectrofocusing (IEF) and the BAS-PCR assay developed for CSN2*I is described.