Statistical Approaches to Analyse Gene Bank Data Using a Lentil Germplasm Collection as a Case Study

Normally in a plant gene bank a large number of accessions per each crop and/ or taxon is stored. During their characterization and preliminary evaluation, several quantitative and qualitative data are recorded and, usually, a wide intra accession variation is observed. Th e management of all this information becomes very difficult without effective statistical methods combining these different types of data. At the Institute of Plant Genetics, CNR, in Bari (Italy) this problem has been tackled by testing many statistical approaches. The present contribution describes one of these approaches, which to date has proven to be highly adequate; a case study describing a lentil germplasm collection has been used for demonstration. A valuable application of this method is the determination of core subsets important to increase the utilization and accessibility of plant genetic resources. In the presented case study a subset of the lentil germplasm collection was chosen to perform molecular analysis based on ISSR markers. The samples were selected on the basis of both morpho-agronomic evaluation and geographical origin. These markers proved to be useful for distinguishing among closely related genotypes and for possibly substantiating the genetic peculiarity of some interesting material

Statistical Approaches to Analyse Gene Bank Data Using a Lentil Germplasm Collection as a Case Study

Normally in a plant gene bank a large number of accessions per each crop and/ or taxon is stored. During their characterization and preliminary evaluation, several quantitative and qualitative data are recorded and, usually, a wide intra accession variation is observed. Th e management of all this information becomes very difficult without effective statistical methods combining these different types of data. At the Institute of Plant Genetics, CNR, in Bari (Italy) this problem has been tackled by testing many statistical approaches. The present contribution describes one of these approaches, which to date has proven to be highly adequate; a case study describing a lentil germplasm collection has been used for demonstration. A valuable application of this method is the determination of core subsets important to increase the utilization and accessibility of plant genetic resources. In the presented case study a subset of the lentil germplasm collection was chosen to perform molecular analysis based on ISSR markers. The samples were selected on the basis of both morpho-agronomic evaluation and geographical origin. These markers proved to be useful for distinguishing among closely related genotypes and for possibly substantiating the genetic peculiarity of some interesting material

The miRNAome of globe artichoke: conserved and novel micro RNAs and target analysis

<p>Abstract</p> <p>Background</p> <p>Plant microRNAs (miRNAs) are involved in post-transcriptional regulatory mechanisms of several processes, including the response to biotic and abiotic stress, often contributing to the adaptive response of the plant to adverse conditions. In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found. Due to low abundance, non conserved miRNAs are difficult to identify and isolate using conventional approaches. Conversely, deep-sequencing of small RNA (sRNA) libraries can detect even poorly expressed miRNAs.</p> <p>No miRNAs from globe artichoke have been described to date. We analyzed the miRNAome from artichoke by deep sequencing four sRNA libraries obtained from NaCl stressed and control leaves and roots.</p> <p>Results</p> <p>Conserved and novel miRNAs were discovered using accepted criteria. The expression level of selected miRNAs was monitored by quantitative real-time PCR. Targets were predicted and validated for their cleavage site. A total of 122 artichoke miRNAs were identified, 98 (25 families) of which were conserved with other plant species, and 24 were novel. Some miRNAs were differentially expressed according to tissue or condition, magnitude of variation after salt stress being more pronounced in roots. Target function was predicted by comparison to <it>Arabidopsis </it>proteins; the 43 targets (23 for novel miRNAs) identified included transcription factors and other genes, most of which involved in the response to various stresses. An unusual cleaved transcript was detected for miR393 target, transport inhibitor response 1.</p> <p>Conclusions</p> <p>The miRNAome from artichoke, including novel miRNAs, was unveiled, providing useful information on the expression in different organs and conditions. New target genes were identified. We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant kingdom.</p

Whole-exome sequencing of selected bread wheat recombinant inbred lines as a useful resource for allele mining and bulked segregant analysis

Although wheat (Triticum aestivum L.) is the main staple crop in the world and a major source of carbohydrates and proteins, functional genomics and allele mining are still big challenges. Given the advances in next-generation sequencing (NGS) technologies, the identification of causal variants associated with a target phenotype has become feasible. For these reasons, here, by combining sequence capture and target-enrichment methods with high-throughput NGS re-sequencing, we were able to scan at exome-wide level 46 randomly selected bread wheat individuals from a recombinant inbred line population and to identify and classify a large number of single nucleotide polymorphisms (SNPs). For technical validation of results, eight randomly selected SNPs were converted into Kompetitive Allele-Specific PCR (KASP) markers. This resource was established as an accessible and reusable molecular toolkit for allele data mining. The dataset we are making available could be exploited for novel studies on bread wheat genetics and as a foundation for starting breeding programs aimed at improving different key agronomic traits

Overexpression of the MYB29 transcription factor affects aliphatic glucosinolate synthesis in Brassica oleracea

Isothiocyanates, the bio-active hydrolysis products of glucosinolates, naturally produced by several Brassicaceae species, play an important role in human health and agriculture. This study aims at correlating the content of aliphatic glucosinolates to the expression of genes involved in their synthesis in Brassica oleracea, and perform functional analysis of BoMYB29 gene. To this purpose, three genotypes were used: a sprouting broccoli, a cabbage, and a wild genotype (Winspit), a high glucosinolate containing accession. Winspit showed the highest transcript level of BoMYB28, BoMYB29 and BoAOP2 genes, and BoAOP2 expression was positively correlated with that of the two MYB genes. Further analyses of the aliphatic glucosinolates also showed a positive correlation between the expression of BoAOP2 and the production of sinigrin and gluconapin in Winspit. The Winspit BoMYB29 CDS was cloned and overexpressed in Winspit and in the DH AG1012 line. Overexpressing Winspit plants produced higher quantities of alkenyl glucosinolates, such as sinigrin. Conversely, the DH AG1012 transformants showed a higher production of methylsulphinylalkyl glucosinolates, including glucoraphanin, and, despite an up-regulation of the aliphatic glucosinolate genes, no increase in alkenyl glucosinolates. The latter may be explained by the absence of a functional AOP2 gene in DH AG1012. Nevertheless, an extract of DH AG1012 lines overexpressing BoMYB29 provided a chemoprotective effect on human colon cells. This work exemplifies how the genetic diversity of B. oleracea may be used by breeders to select for higher expression of transcription factors for glucosinolate biosynthesis to improve its natural, health-promoting properties

Capturing variation in Lens (Fabaceae): Development and utility of an exome capture array for lentil

Premise of the Study: Lentil is an important legume crop with reduced genetic diversity caused by domestication bottlenecks. Due to its large and complex genome, tools for reduced representation sequencing are needed. We developed an exome capture array for use in various genetic diversity studies. Methods: Based on the CDC Redberry draft genome, we developed an exome capture array using multiple sources of transcript resources. The probes were designed to target not only the cultivated lentil, but also wild species. We assessed the utility of the developed method by applying the generated data set to population structure and phylogenetic analyses. Results: The data set includes 16 wild lentils and 22 cultivar accessions of lentil. Alignment rates were over 90%, and the genic regions were well represented in the capture array. After stringent filtering, 6.5 million high-quality variants were called, and the data set was used to assess the interspecific relationships within the genus Lens. Discussion: The developed exome capture array provides large amounts of genomic data to be used in many downstream analyses. The method will have useful applications in marker-assisted breeding programs aiming to improve the quality of cultivated lentil

A Distinct Genetic Cluster in Cultivated Chickpea as Revealed by Genome-wide Marker Discovery and Genotyping

The accurate description of plant biodiversity is of utmost importance to efficiently address efforts in conservation genetics and breeding. Herein, we report the successful application of a genotyping-by-sequencing (GBS) approach in chickpea ( L.), resulting in the characterization of a cultivated germplasm collection with 3187 high-quality single nucleotide polymorphism (SNP) markers. Genetic structure inference, principal component analysis, and hierarchical clustering all indicated the identification of a genetic cluster corresponding to black-seeded genotypes traditionally cultivated in Southern Italy. Remarkably, this cluster was clearly distinct at both genetic and phenotypic levels from germplasm groups reflecting commercial chickpea classification into and seed types. Fixation index estimates for individual polymorphisms pointed out loci and genomic regions that might be of significance for the diversification of agronomic and commercial traits. Overall, our findings provide information on genetic relationships within cultivated chickpea and highlight a gene pool of great interest for the scientific community and chickpea breeding, which is limited by the low genetic diversity available in the primary gene pool

Detection of adulterations with different grains in wheat products based on the hyperspectral image technique: The specific cases of flour and bread

[EN] The objective of this study was to test the capability of a SW-NIR hyperspectral image technique to detect adulterations in wheat flour and bread with cheap grains, such us sorghum, oats and corn, and to compare the hyperspectral information with the physicochemical alterations in the properties of products. Wheat flour was adulterated at four different degrees (2.5, 5, 7.5 and 10%) with sorghum, oat and corn flours. Flours were prepared and used to make bread. Flours and breads were characterized according to several physicochemical parameters (pasting properties, water activity, mass loss during the baking process and texture profile analysis). Crumbs were extracted from breads and conditioned. Hyperspectral image captures were taken of both flours and conditioned crumbs. The data analysis was based on multivariate statistical process control method (MSPC), where the differentiation of adulterated samples was observed in all cases for both flours and crumbs. Finally, in order to relate the image analysis results and the adulterated sample properties, a correlation significance map was created between the physicochemical properties of samples and the multivariate statistical parameters. The SW-NIR image technique was capable of detecting adulterations in each case and high correlation significances were observed (alpha = 0.01) between wavelengths from specific spectra zones and the physicochemical properties of samples.Verdú Amat, S.; Vásquez, F.; Grau Meló, R.; Ivorra Martínez, E.; Sánchez Salmerón, AJ.; Barat Baviera, JM. (2016). Detection of adulterations with different grains in wheat products based on the hyperspectral image technique: The specific cases of flour and bread. Food Control. 62:373-380. doi:10.1016/j.foodcont.2015.11.002S3733806

Development of a multiplex DNA-based traceability tool for crop plant materials

The authenticity of food is of increasing importance for producers, retailers and consumers. All groups benefit from the correct labelling of the contents of food products. Producers and retailers want to guarantee the origin of their products and check for adulteration with cheaper or inferior ingredients. Consumers are also more demanding about the origin of their food for various socioeconomic reasons. In contrast to this increasing demand, correct labelling has become much more complex because of global transportation networks of raw materials and processed food products. Within the European integrated research project ‘Tracing the origin of food’ (TRACE), a DNA-based multiplex detection tool was developed—the padlock probe ligation and microarray detection (PPLMD) tool. In this paper, this method is extended to a 15-plex traceability tool with a focus on products of commercial importance such as the emmer wheat Farro della Garfagnana (FdG) and Basmati rice. The specificity of 14 plant-related padlock probes was determined and initially validated in mixtures comprising seven or nine plant species/varieties. One nucleotide difference in target sequence was sufficient for the distinction between the presence or absence of a specific target. At least 5% FdG or Basmati rice was detected in mixtures with cheaper bread wheat or non-fragrant rice, respectively. The results suggested that even lower levels of (un-)intentional adulteration could be detected. PPLMD has been shown to be a useful tool for the detection of fraudulent/intentional admixtures in premium foods and is ready for the monitoring of correct labelling of premium foods worldwide