Corrigendum: The Conservation of VIT1-Dependent Iron Distribution in Seeds.

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Genome wide association study of 5 agronomic traits in olive (Olea europaea L.).

Olive (Olea europaea L.) is one of the most economically and historically important fruit crops worldwide. Genetic progress for valuable agronomic traits has been slow in olive despite its importance and benefits. Advances in next generation sequencing technologies provide inexpensive and highly reproducible genotyping approaches such as Genotyping by Sequencing, enabling genome wide association study (GWAS). Here we present the first comprehensive GWAS study on olive using GBS. A total of 183 accessions (FULL panel) were genotyped using GBS, 94 from the Turkish Olive GenBank Resource (TOGR panel) and 89 from the USDA-ARS National Clonal Germplasm Repository (NCGR panel) in the USA. After filtering low quality and redundant markers, GWAS was conducted using 24,977 SNPs in FULL, TOGR and NCGR panels. In total, 52 significant associations were detected for leaf length, fruit weight, stone weight and fruit flesh to pit ratio using the MLM_K. Significant GWAS hits were mapped to their positions and 19 candidate genes were identified within a 10-kb distance of the most significant SNP. Our findings provide a framework for the development of markers and identification of candidate genes that could be used in olive breeding programs.

The Conservation of VIT1-Dependent Iron Distribution in Seeds.

One third of people suffer from anemia, with iron (Fe) deficiency being the most common reason. The human diet includes seeds of staple crops, which contain Fe that is poorly bioavailable. One reason for low bioavailability is that these seeds store Fe in cellular compartments that also contain antinutrients, such as phytate. Thus, several studies have focused on decreasing phytate concentrations. In theory, as an alternative approach, Fe reserves might be directed to cellular compartments that are free of phytate, such as plastids. However, it is not known if seed plastid can represent a major Fe storage compartment in nature. To discover distinct types of Fe storage in nature, we investigated metal localizations in the seeds of more than twenty species using histochemical or X-ray based techniques. Results showed that in Rosids, the largest clade of eudicots, Fe reserves were primarily confined to the embryo of the seeds. Furthermore, inside the embryos, Fe accumulated specifically in the endodermal cell layer, a well-known feature that is mediated by VACUOLAR IRON TRANSPORTER1 (VIT1) in model plant Arabidopsis thaliana. In rice, Fe enrichment is lost around the provasculature in the mutants of VIT1 orthologs. Finally, in Carica papaya, Fe accumulated in numerous organelles resembling plastids; however, these organelles accumulated reserve proteins but not ferritin, failing to prove to be plastids. By investigating Fe distribution in distinct plant lineages, this study failed to discover distinct Fe storage patterns that can be useful for biofortification. However, it revealed Fe enrichment is widely conserved in the endodermal cell layer in a VIT1-dependent manner in the plant kingdom.

Post-transcriptional adaptation of the aquatic plant Spirodela polyrhiza under stress and hormonal stimuli.

The Lemnaceae family comprises aquatic plants of angiosperms gaining attention due to their utility in wastewater treatment, and rapid production of biomass that can be used as feed, fuel, or food. Moreover, it can serve as a model species for neotenous growth and environmental adaptation. The latter properties are subject to post-transcriptional regulation of gene expression, meriting investigation of how miRNAs in Spirodela polyrhiza, the most basal and most thoroughly sequenced member of the family, are expressed under different growth conditions. To further scientific understanding of its capacity to adapt to environmental cues, we measured miRNA expression and processing of their target sequences under different temperatures, and in the presence of abscisic acid, copper, kinetin, nitrate, and sucrose. Using two small RNA sequencing experiments and one degradome sequencing experiment, we provide evidence for 108 miRNAs. Sequencing cleaved mRNAs validated 42 conserved miRNAs with 83 targets and 24 novel miRNAs regulating 66 targets and created a list of 575 predicted and verified targets. These analyses revealed condition-induced changes in miRNA expression and cleavage activity, and resulted in the addition of stringently reviewed miRNAs to miRBase. This combination of small RNA and degradome sequencing provided not only high confidence predictions of conserved and novel miRNAs and targets, but also a view of the post-transcriptional regulation of adaptations. A unique aspect is the role of miR156 and miR172 expression and activity in its clonal propagation and neoteny. Additionally, low levels of 24 nt sRNAs were observed, despite the lack of recent retrotransposition.

Association mapping of loci linked to copper, phosphorus, and potassium concentrations in the seeds of C. arietinum and C. reticulatum.

Due to its high nutritional value, chickpea is one of the most important and cost-effective legumes for human diet. Nutrient elements, such as Cu, P, K have numerous essential functions for the human metabolism. In this study, association mapping of loci linked to the seed Cu, P and K concentrations were performed on a population consisting of 107 Cicer reticulatum and 73 Cicer arietinum individuals in four environments (two locations x two years). A total of 121,840 SNPs were genotyped across 180 individuals by GBS analysis. The association mapping between the SNP markers and the seed Cu, P, K concentrations were identified and eight SNPs were found to be significantly associated with variations in three nutrient elements in more than two environments This research suggests that association mapping is a useful methodology for the identification of loci controlling the Cu, P and K uptake in chickpea seeds for further association mapping, molecular breeding, and marker-assisted selection and plant breeding studies and provides a broader understanding of the relationship between the investigated Cicer species and the effects of environmental conditions.

A consensus linkage map of lentil based on DArT markers from three RIL mapping populations.

BACKGROUND: Lentil (Lens culinaris ssp. culinaris Medikus) is a diploid (2n = 2x = 14), self-pollinating grain legume with a haploid genome size of about 4 Gbp and is grown throughout the world with current annual production of 4.9 million tonnes. MATERIALS AND METHODS: A consensus map of lentil (Lens culinaris ssp. culinaris Medikus) was constructed using three different lentils recombinant inbred line (RIL) populations, including "CDC Redberry" x "ILL7502" (LR8), "ILL8006" x "CDC Milestone" (LR11) and "PI320937" x "Eston" (LR39). RESULTS: The lentil consensus map was composed of 9,793 DArT markers, covered a total of 977.47 cM with an average distance of 0.10 cM between adjacent markers and constructed 7 linkage groups representing 7 chromosomes of the lentil genome. The consensus map had no gap larger than 12.67 cM and only 5 gaps were found to be between 12.67 cM and 6.0 cM (on LG3 and LG4). The localization of the SNP markers on the lentil consensus map were in general consistent with their localization on the three individual genetic linkage maps and the lentil consensus map has longer map length, higher marker density and shorter average distance between the adjacent markers compared to the component linkage maps. CONCLUSION: This high-density consensus map could provide insight into the lentil genome. The consensus map could also help to construct a physical map using a Bacterial Artificial Chromosome library and map based cloning studies. Sequence information of DArT may help localization of orientation scaffolds from Next Generation Sequencing data.

Association Mapping in Turkish Olive Cultivars Revealed Significant Markers Related to Some Important Agronomic Traits.

Olive (Olea europaea L.) is one of the most important fruit trees especially in the Mediterranean countries due to high consumption of table olive and olive oil. In olive breeding, the phenotypic traits associated to fruit are the key factors that determine productivity. Association mapping has been used in some tree species and a lot of crop plant species, and here, we perform an initial effort to detect marker-trait associations in olive tree. In the current study, a total of 96 olive genotypes, including both oil and table olive genotypes from Turkish Olive GenBank Resources, were used to examine marker-trait associations. For olive genotyping, SNP, AFLP, and SSR marker data were selected from previously published study and association analysis was performed between these markers and 5 yield-related traits. Three different approaches were used to check for false-positive results in association tests, and association results obtained from these models were compared. Using the model utilizing both population structure and relative kinship, eleven associations were significant with FDR ≤ 0.05. The largest number of significant associations was detected for fruit weight and stone weight. Our results suggested that association mapping could be an effective approach for identifying marker-trait associations in olive genotypes, without the development of mapping populations. This study shows for the first time the use of association mapping for identifying molecular markers linked to important traits in olive tree.

Identification QTLs Controlling Genes for Se Uptake in Lentil Seeds.

Lentil (Lens culinaris Medik.) is an excellent source of protein and carbohydrates and is also rich in essential trace elements for the human diet. Selenium (Se) is an essential micronutrient for human health and nutrition, providing protection against several diseases and regulating important biological systems. Dietary intake of 55 µg of Se per day is recommended for adults, with inadequate Se intake causing significant health problems. The objective of this study was to identify and map quantitative trait loci (QTL) of genes controlling Se accumulation in lentil seeds using a population of 96 recombinant inbred lines (RILs) developed from the cross "PI 320937" × "Eston" grown in three different environments for two years (2012 and 2013). Se concentration in seed varied between 119 and 883 µg/kg. A linkage map consisting of 1,784 markers (4 SSRs, and 1,780 SNPs) was developed. The map spanned a total length of 4,060.6 cM, consisting of 7 linkage groups (LGs) with an average distance of 2.3 cM between adjacent markers. Four QTL regions and 36 putative QTL markers, with LOD scores ranging from 3.00 to 4.97, distributed across two linkage groups (LG2 and LG5) were associated with seed Se concentration, explaining 6.3-16.9% of the phenotypic variation.

Association mapping for five agronomic traits in the common bean (Phaseolus vulgaris L.).

BACKGROUND: The common bean is the most important grain legume and a major source of protein in many developing countries. We analysed the following traits: pod fibre (PF), seeds per pod (SPP), plant type (PT), growth habit (GH), and days to flowering (DF) for a set of diverse common bean accessions and determined whether such traits were associated with amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. RESULTS: In this study, 66 common bean genotypes were used and genotyped with 233 AFLP, 105 SNP and 80 SSR markers. The association analysis between markers and five traits was performed using a General Linear Model (GLM) in Trait Analysis by aSSociation, Evolution and Linkage (TASSEL). The population structure was determined using the STRUCTURE software, and seven groups (K = 7) were identified among genotypes. The associations for such traits were identified and quantified; 62 markers were associated with the five traits. CONCLUSION: This study demonstrated that association mapping using a reasonable number of markers, distributed across the genome and with the appropriate number of individuals harboured to detect DNA markers linked to the traits of PF, SPP, PT, GH and DF in common bean.

Genetic assessment of common bean (Phaseolus vulgaris L.) accessions by peroxidase gene-based markers.

BACKGROUND: Peroxidase, a plant-specific oxidoreductase, is a heme-containing glycoprotein encoded by a large multigenic family in plants. Plant peroxidases (POXs, EC 1.11.1.7) play important roles in many self-defense interactions in plants. Here, 67 common bean (Phaseolus vulgaris L.) genotypes were studied using a POX gene-based marker method. Comparison of POX genes could resolve evolutionary relationships in common bean. RESULTS: Eighty fragments were obtained with 20 primer pairs that amplified one (POX8c) to eight (ATP29) bands, with a mean of four bands per primer pair. The average (polymorphic information content) PIC value for the POX products was 0.40. The maximum variation (93%) was found between Turkey (#33) and India (#52) and between Antalya (#33) and India (#53). The minimum variation (0%) was found among four pairs: Bozdag (#2) and Karadeniz (#38), Kirklareli (#11) and Turkey (#15, 16, 43), Bandirma (#13) and Turkey (#15, 16, 43), and Kirklareli (#10) and Bandirma (#22). UPGMA was used to discriminate the common bean genotypes into five clusters, while STRUCTURE software was used to investigate the genetic population structure. CONCLUSION: The results showed that POX gene family markers can be used to study genotypic diversity and provide new information for breeding programs and common bean improvement practices.

SNP discovery by illumina-based transcriptome sequencing of the olive and the genetic characterization of Turkish olive genotypes revealed by AFLP, SSR and SNP markers.

BACKGROUND: The olive tree (Olea europaea L.) is a diploid (2n = 2x = 46) outcrossing species mainly grown in the Mediterranean area, where it is the most important oil-producing crop. Because of its economic, cultural and ecological importance, various DNA markers have been used in the olive to characterize and elucidate homonyms, synonyms and unknown accessions. However, a comprehensive characterization and a full sequence of its transcriptome are unavailable, leading to the importance of an efficient large-scale single nucleotide polymorphism (SNP) discovery in olive. The objectives of this study were (1) to discover olive SNPs using next-generation sequencing and to identify SNP primers for cultivar identification and (2) to characterize 96 olive genotypes originating from different regions of Turkey. METHODOLOGY/PRINCIPAL FINDINGS: Next-generation sequencing technology was used with five distinct olive genotypes and generated cDNA, producing 126,542,413 reads using an Illumina Genome Analyzer IIx. Following quality and size trimming, the high-quality reads were assembled into 22,052 contigs with an average length of 1,321 bases and 45 singletons. The SNPs were filtered and 2,987 high-quality putative SNP primers were identified. The assembled sequences and singletons were subjected to BLAST similarity searches and annotated with a Gene Ontology identifier. To identify the 96 olive genotypes, these SNP primers were applied to the genotypes in combination with amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) markers. CONCLUSIONS/SIGNIFICANCE: This study marks the highest number of SNP markers discovered to date from olive genotypes using transcriptome sequencing. The developed SNP markers will provide a useful source for molecular genetic studies, such as genetic diversity and characterization, high density quantitative trait locus (QTL) analysis, association mapping and map-based gene cloning in the olive. High levels of genetic variation among Turkish olive genotypes revealed by SNPs, AFLPs and SSRs allowed us to characterize the Turkish olive genotype.

Altered level of apurinic/apyrimidinic endonuclease/redox factor-1 (APE/REF-1) mRNA in the hippocampus of ovariectomized rats treated by raloxifene against kainic acid.

1. Accumulated clinical evidence suggests that selective oestrogen receptor modulators (SERM), such as raloxifene, may be neuroprotective. Oxidative stress is a likely molecular mechanism in the neurotoxicity of kainic acid (KA), an excitotoxic substance. The expression levels of the apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) gene seem to correlate with cellular sensitivity to reactive oxygen species (ROS) and a reduction in the expression of APE/Ref-1 may cause oxidative DNA damage. 2. The aim of the present study was to assess the effects of KA and raloxifene on the level of APE/Ref-1 mRNA in the hippocampus of ovariectomized rats. The expression of the APE/Ref-1 gene was quantified using reverse transcription followed by real-time polymerase chain reaction. 3. The results show that the level of APE/Ref-1 mRNA increased significantly in raloxifene-treated rats. However, raloxifene treatment did not affect the seizure severity induced by KA. We also observed that raloxifene treatment against simultaneous KA injection maintained the increased level of APE/Ref-1 mRNA in the hippocampus. 4. Therefore, the results of the present study seem to support previous data suggesting the potential significance of raloxifene in neuroprotection.

Gene loss and movement in the maize genome.

Maize (Zea mays L. ssp. mays), one of the most important agricultural crops in the world, originated by hybridization of two closely related progenitors. To investigate the fate of its genes after tetraploidization, we analyzed the sequence of five duplicated regions from different chromosomal locations. We also compared corresponding regions from sorghum and rice, two important crops that have largely collinear maps with maize. The split of sorghum and maize progenitors was recently estimated to be 11.9 Mya, whereas rice diverged from the common ancestor of maize and sorghum approximately 50 Mya. A data set of roughly 4 Mb yielded 206 predicted genes from the three species, excluding any transposon-related genes, but including eight gene remnants. On average, 14% of the genes within the aligned regions are noncollinear between any two species. However, scoring each maize region separately, the set of noncollinear genes between all four regions jumps to 68%. This is largely because at least 50% of the duplicated genes from the two progenitors of maize have been lost over a very short period of time, possibly as short as 5 million years. Using the nearly completed rice sequence, we found noncollinear genes in other chromosomal positions, frequently in more than one. This demonstrates that many genes in these species have moved to new chromosomal locations in the last 50 million years or less, most as single gene events that did not dramatically alter gene structure.

fs3.1: a major fruit shape QTL conserved in Capsicum.

fs3.1 is a major fruit shape (defined as the ratio of fruit length to fruit width) quantitative trait locus (QTL) originally detected in an intraspecific cross of Capsicum annuum between the blocky and elongated-fruited inbreds 'Maor' and 'Perennial', respectively. In addition to increasing fruit shape index, the 'Perennial' allele at fs3.1 increased fruit elongation and decreased fruit width and pericarp thickness. We verified the effect of fs3.1 in backcross inbred lines (BILs) derived from crossing 'Perennial' with 'Maor' and with a second blocky-type inbred line of C. annuum. To determine the effect of the fs3.1 region in additional Capsicum species, we constructed an advanced backcross population from the cross of 'Maor' and the oval-fruited Capsicum frutescens BG 2816 and an F2 of the introgression line IL 152 that contains an introgression of the fs3.1 region from Capsicum chinense PI 152225. QTLs for fruit shape, fruit width, and pericarp thickness, but not for fruit length, were detected in both crosses, indicating the conservation of the fs3.1 region as a QTL affecting fruit shape in pepper. We also tested tomato (Lycopersicon spp.) introgression lines containing the corresponding fs3.1 region from L. pennellii and L. hirsutum, but we did not detect a significant fruit shape QTL in these lines. The effect of fs3.1 on the growth of fruit dimensions varied with the genetic background. By measuring the length and width of ovaries and fruits of near-isogenic C. annuum lines that differ in fs3.1 during fruit development, we determined that fs3.1 controls shape predominantly by increasing the growth rate of the longitudinal axis in the first 2 weeks after pollination. However, in the crosses of C. annuum with C. frutescens and C. chinense, fs3.1 predominantly exerted its effect on the width dimension.