Lpa1-5525 : a new lpa1 mutant isolated in a mutagenized population by a novel non-disrupting screening method

Phytic acid, or myo-inositol 1,2,3,4,5,6-hexakisphosphate, is the main storage form of phosphorus in plants. It is localized in seeds, deposited as mixed salts of mineral cations in protein storage vacuoles; during germination, it is hydrolyzed by phytases to make available P together with all the other cations needed for seed germination. When seeds are used as food or feed, phytic acid and the bound cations are poorly bioavailable for human and monogastric livestock due to their lack of phytase activity. Therefore, reducing the amount of phytic acid is one strategy in breeding programs aimed to improve the nutritional properties of major crops. In this work, we present data on the isolation of a new maize (Zea mays L.) low phytic acid 1 (lpa1) mutant allele obtained by transposon tagging mutagenesis with the Ac element. We describe the generation of the mutagenized population and the screening to isolate new lpa1 mutants. In particular, we developed a fast, cheap and non-disrupting screening method based on the different density of lpa1 seed compared to the wild type. This assay allowed the isolation of the lpa1-5525 mutant characterized by a new mutation in the lpa1 locus associated with a lower amount of phytic phosphorus in the seeds in comparison with the wild type

Evolutionary trends and phylogenetic association of key morphological traits in the Italian rice varietal landscape

Efficient germplasm exploitation in crop breeding requires comprehensive knowledge of the available genetic diversity. Linking molecular data to phenotypic expression is fundamental for the profitable utilisation of genetic resources. Italian rice germplasm is an invaluable source of genes, being characterised by marked heterogeneity. A phenotypic characterisation is presented in this paper, with a focus on the evolutionary trends, and on the comparison with available molecular studies. A panel of 351 Italian rice varieties was analysed using seven key morphological traits, employing univariate and multivariate analyses. Considerable variability was found, with clear morphological trends towards reduced plant height, earliness, and spindle-shaped caryopses. Previous findings indicating that genetic diversity was maintained throughout time could not be confirmed, as small phenotypic variability was found in the most recent rice varieties. Consistency with phylogenetic data from previous studies was partial: one phylogenetic subgroup was phenotypically well distinct, while the others had overlapping characteristics and encompassed a wide range of phenotypic variation. Our study provides a quantitative ready-to-use set of information to support new breeding programs, as well as the basis to develop variety-specific calibrations of eco-physiological models, to identify promising traits in light of climate change conditions and alternative management scenarios

An ancient pointed maize rich in phlobaphenes: the "Nero Spinoso" from Valcamonica (Brescia, Italy)

The variety of the species Zea mays called \u201cNero Spinoso\u201d is a traditional crop in the Middle Valle Camonica (Northern Italy) although its cultivation was almost completely abandoned during the second half of the twentieth century due to the introduction of commercial hybrids and to a reduction in the consumption of polenta, for centuries a staple food of the rural population. This work characterized this ancient pigmented maize. A preliminary spectrophotometric analysis revealed the presence of flavonols, phenolic acids and in particular phlobaphenes, probably responsible for ear coloration. Phlobaphenes are reddish insoluble substances synthesized in maize through the flavonoids pathway by the polymerization of flavan-4-ols. In maize this pigment is due to the presence of the pericarp1 gene (p1), a transcription factor belonging to the MYB gene family driving the accumulation of the pigment in the pericarp layer. Genetic and molecular analysis confirmed the presence of the P1 gene in this landrace and histological analysis confirmed that the pigment is accumulated in the pericarp. In the meantime HPLC analyses also showed the presence of a large amount of carotenoids and, probably thanks to the high amount of pigments, this maize variety showed very high antioxidant ability. Given the chronic disease prevention properties of antioxidant molecules, this variety could be of great interest also from a nutritional point of view. Characterization will allow an accurate description of this landrace with the aim of studying and preserving maize biodiversity in Europe and in particular in Italy. We have collaborated with the municipalities of Esine and Piancogno in the application process needed to register this ancient cultivar on the National Register of conservation varieties with the aim of valorizing this ancient landrace. The "Nero Spinoso" maize was recorded in the National Register in January 2016

Genetic and chemical comparison among Camelina sativa varieties

Camelina sativa (Camelina sativa L. Crantz.) belonging to the mustard family, typically contain about 40 % oil in the seeds, 90 % of which is made up of unsaturated fatty acids: about 30\u201340% fraction of alpha linolenic acid, 15\u201325% fraction of linoleic acid, 15% fraction of oleic acid and around 15% eicosenoic acid. Genetic studies of the genome of C. sativa suggest a polyploid structure being more probably a hexaploid species. In European countries and Russia, camelina was grown as an agricultural crop before the II World War; now the renewed interest on this crop is mainly due to the search for new sources of essential fatty acids, particularly n-3(omega-3) fatty acids to be used in human food and animal feed products. In this work we compared different varieties of camelina grown in different conditions: in experimental field, greenhouse and grow chamber. We characterized the genetic material by SSRs to assess the genetic diversity to assist future breeding programs. In particular we have set up a breeding program aimed at reducing the glucosinolate content in camelina seed because of its toxic effect when present at high levels in seeds used as feed. Glucosinolate are sulphur-containing glucosides, found mainly in Brassicaceae, involved in plant defense. In the last year these molecules have been studied also because of their activities as natural pesticides and their protective effects against cancer, heart disease and chronic inflammatory disease. We are now characterizing camelina seeds for glucosinolate content in order to develop a diagnostic marker based on the analytical determination of the sulfur isotopic signature (\uf06434S). Such a marker will allow to easily select genetic materials with different glucosinolate contents without the use of complex and expensive analytical techniques

Phytic acid prevents oxidative stress in seeds : evidence from a maize (Zea mays L.) low phytic acid mutant

A maize mutant defective in the synthesis of phytic acid during seed maturation was used as a tool to study the consequences of the lack of this important reserve substance on seed survival. Data on germinability, free iron level, free radical relative abundance, protein carbonylation level, damage to DNA, degree of lipid peroxidation, \u3b1- and \u3b3-tocopherol amount and antioxidant capacity were recorded on seeds of maize B73 and of an isogenic low phytic acid mutant (lpa1-241), either unaged or incubated for 7 d in accelerated ageing conditions (46\ub0C and 100% relative humidity). The lpa1-241 mutant, compared to wild type (wt), showed a lower germination capacity, which decreased further after accelerated ageing. Whole lpa1-241 mutant kernels contained about 50% more free or weakly bound iron than wt ones and showed a higher content of free radicals, mainly concentrated in embryos; in addition, upon accelerated ageing, lpa1-241 seed proteins were more carbonylated and DNA was more damaged, whereas lipids did not appear to be more peroxidated, but the \u3b3-tocopherol content was decreased by about 50%. These findings can be interpreted in terms of previously reported but never proven antioxidant activity of phytic acid through iron complexation. Therefore, a novel role in plant seed physiology can be assigned to phytic acid, that is, protection against oxidative stress during the seed's life span. As in maize kernels the greater part of phytic acid (and thus of metal ions) is concentrated in the embryo, its antioxidant action may be of particular relevance in this crop

Genetic Variation and Breeding Potential of Phytate and Inorganic Phosphorus in a Maize Population

Seed P is predominantly bound in the organic compound phytate, which makes the bioavailability of P low for monogastric animals fed maize (Zea mays L.)-based diets. Decreasing phytate and increasing inorganic P (Pi, an available form of P) concentrations in maize grain would be desirable to help ameliorate environmental problems associated with high P in feces. Our objective was to investigate the potential of improving the P profile of maize grain through breeding and selection. Ninety S1 families from the BS31 population were evaluated at two locations for phytate, Pi, and other grain quality and agronomic traits. Phytate concentrations ranged from 1.98 to 2.46 g kg−1, and the broad-sense heritability (H) was relatively low (0.60). Both genetic variance and H (0.84) were much greater for Pi Few unfavorable genetic correlations were observed between either Pi or phytate and other key economic traits. Also, selection differentials of multiple trait indices indicated that the P profile of maize grain and grain yield and moisture could be improved simultaneously. Many cycles of selection will be needed, however, to reach desirable phytate and Pi concentrations, especially when selecting for multiple traits. Regardless, our results are encouraging given that the families evaluated were related S1 families and the number of families was relatively small

Traditional farmers’ varieties: a valuable source of genetic variability for biofortification programs

Several studies underlined the superiority from a nutritional point of view of ancient varieties. In the last years the interest for landraces has been growing, for this reason preservation and valorisation of these genetic sources is very important. In particular these varieties are source of precious genetic variability interesting from a scientific point of view to preserve biodiversity but also for biofortification programs aimed to support small rural communities, where the particular maize germplasm has been developed. In this work we characterized from the nutritional point of view 13 ancient Italian varieties and one coming from Spain (Millo Corvo). In this pre-breeding work we demonstrate the nutritional superiority of ancient varieties if compared with modern hybrids. In particular Spinato di Gandino is the best variety for milling properties and for oil, protein, and total phosphorus content; Storo is the best variety for calorific value and for carotenoids and free phosphorus content. Using these varieties in the next future we will start a bio-fortification program aimed to obtain new populations with improved yields and high nutritional value

Change of Gene Structure and Function by Non-Homologous End-Joining, Homologous Recombination, and Transposition of DNA

An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal interval exemplifies the evolution of gene regulation and allelic diversity in general. It also illustrates the evolutionary pace of changes in plants, where many of the above mechanisms are of somatic origin. In contrast to animals, somatic alterations can easily be transmitted through meiosis because the germline in plants is contiguous to somatic tissue, permitting the recovery of such chromosomal rearrangements. The analyzed region contains the P1-wr allele, a variant of the genetically well-defined p1 gene, which encodes a Myb-like transcriptional activator in maize. The P1-wr allele consists of eleven nearly perfect P1-wr 12-kb repeats that are arranged in a tandem head-to-tail array. Although a technical challenge to sequence such a structure by shotgun sequencing, we overcame this problem by subcloning each repeat and ordering them based on nucleotide variations. These polymorphisms were also critical for recombination and expression analysis in presence and absence of the trans-acting epigenetic factor Ufo1. Interestingly, chimeras of the p1 and p2 genes, p2/p1 and p1/p2, are framing the P1-wr cluster. Reconstruction of sequence amplification steps at the p locus showed the evolution from a single Myb-homolog to the multi-gene P1-wr cluster. It also demonstrates how non-homologous end-joining can create novel gene fusions. Comparisons to orthologous regions in sorghum and rice also indicate a greater instability of the maize genome, probably due to diploidization following allotetraploidization

The R2R3-MYB Transcription Factor Gene Family in Maize

MYB proteins comprise a large family of plant transcription factors, members of which perform a variety of functions in plant biological processes. To date, no genome-wide characterization of this gene family has been conducted in maize (Zea mays). In the present study, we performed a comprehensive computational analysis, to yield a complete overview of the R2R3-MYB gene family in maize, including the phylogeny, expression patterns, and also its structural and functional characteristics. The MYB gene structure in maize and Arabidopsis were highly conserved, indicating that they were originally compact in size. Subgroup-specific conserved motifs outside the MYB domain may reflect functional conservation. The genome distribution strongly supports the hypothesis that segmental and tandem duplication contribute to the expansion of maize MYB genes. We also performed an updated and comprehensive classification of the R2R3-MYB gene families in maize and other plant species. The result revealed that the functions were conserved between maize MYB genes and their putative orthologs, demonstrating the origin and evolutionary diversification of plant MYB genes. Species-specific groups/subgroups may evolve or be lost during evolution, resulting in functional divergence. Expression profile study indicated that maize R2R3-MYB genes exhibit a variety of expression patterns, suggesting diverse functions. Furthermore, computational prediction potential targets of maize microRNAs (miRNAs) revealed that miR159, miR319, and miR160 may be implicated in regulating maize R2R3-MYB genes, suggesting roles of these miRNAs in post-transcriptional regulation and transcription networks. Our comparative analysis of R2R3-MYB genes in maize confirm and extend the sequence and functional characteristics of this gene family, and will facilitate future functional analysis of the MYB gene family in maize

Maize Inbreds Exhibit High Levels of Copy Number Variation (CNV) and Presence/Absence Variation (PAV) in Genome Content

Following the domestication of maize over the past ∼10,000 years, breeders have exploited the extensive genetic diversity of this species to mold its phenotype to meet human needs. The extent of structural variation, including copy number variation (CNV) and presence/absence variation (PAV), which are thought to contribute to the extraordinary phenotypic diversity and plasticity of this important crop, have not been elucidated. Whole-genome, array-based, comparative genomic hybridization (CGH) revealed a level of structural diversity between the inbred lines B73 and Mo17 that is unprecedented among higher eukaryotes. A detailed analysis of altered segments of DNA conservatively estimates that there are several hundred CNV sequences among the two genotypes, as well as several thousand PAV sequences that are present in B73 but not Mo17. Haplotype-specific PAVs contain hundreds of single-copy, expressed genes that may contribute to heterosis and to the extraordinary phenotypic diversity of this important crop