QTLs mapping for primary metabolites responsible of the organoleptic and nutritional characteristics of strawberry (Fragaria x ananassa)

The cultivated strawberry (Fragaria x ananassa) is the berry fruit most consumed worldwide and is well-known for its delicate flavour and nutritional properties. However, fruit quality attributes have been lost or reduced after years of traditional breeding focusing mainly on agronomical traits. To face the obstacles encountered in the improvement of cultivated crops, new technological tools, such as genomics and high throughput metabolomics, are becoming essential for the identification of genetic factors responsible of organoleptic and nutritive traits. Integration of “omics” data will allow a better understanding of the molecular and genetic mechanisms underlying the accumulation of metabolites involved in the flavour and nutritional value of the fruit. To identify genetic components affecting/controlling? fruit metabolic composition, here we present a quantitative trait loci (QTL) analysis using a 95 F1 segregating population derived from genotypes ‘1392’, selected for its superior flavour, and ‘232’ selected based in high yield (Zorrilla-Fontanesi et al., 2011; Zorrilla-Fontanesi et al., 2012). Metabolite profiling was performed on red stage strawberry fruits using gas chromatography hyphenated to time-of-flight mass spectrometry, which is a rapid and highly sensitive approach, allowing a good coverage of the central pathways of primary metabolism. Around 50 primary metabolites, including sugars, sugars derivatives, amino and organic acids, were detected and quantified after analysis in each individual of the population. QTL mapping was performed on the ‘232’ x ‘1392’ population separately over two successive years, based on the integrated linkage map (Sánchez-Sevilla et al., 2015). First, significant associations between metabolite content and molecular markers were identified by the non-parametric test of Kruskal-Wallis. Then, interval mapping (IM), as well as the multiple QTL method (MQM) allowed the identification of QTLs in octoploid strawberry. A permutation test established LOD thresholds for each metabolite and year. A total of 132 QTLs were detected in all the linkage groups over the two years for 42 metabolites out of 50. Among them, 4 (9.8%) QTLs for sugars, 9 (25%) for acids and 7 (12.7%) for amino acids were stable and detected in the two successive years. We are now studying the QTLs regions in order to find candidate genes to explain differences in metabolite content in the different individuals of the population, and we expect to identify associations between genes and metabolites which will help us to understand their role in quality traits of strawberry fruit.

Influências genéticas nas características da carcaça e carne em bovinos

Tese de Doutoramento em Ciências Veterinárias - Especialidade de Produção Animal

Connectivity patterns inferred from the genetic structure of white seabream (Diplodus sargus L.)

Themarine environment seems, at first sight, to be a homogeneousmediumlacking barriers to species dispersal. Nevertheless, populations of marine species show varying levels of gene flow and population differentiation, so barriers to gene flow can often be detected. Weaimto elucidate the role of oceanographical factors ingenerating connectivity among populations shaping the phylogeographical patterns in the marine realm, which is not only a topic of considerable interest for understanding the evolution ofmarine biodiversity but also formanagement and conservation of marine life. For this proposal,we investigate the genetic structure and connectivity between continental and insular populations ofwhite seabreamin North East Atlantic (NEA) and Mediterranean Sea (MS) aswell as the influence of historical and contemporary factors in this scenario using mitochondrial (cytochrome b) and nuclear (a set of 9 microsatellite) molecular markers. Azores population appeared genetically differentiated in a single cluster using Structure analysis. This result was corroborated by Principal Component Analysis (PCA) and Monmonier algorithm which suggested a boundary to gene flow, isolating this locality. Azorean population also shows the highest significant values of FST and genetic distances for both molecular markers (microsatellites and mtDNA). We suggest that the breakdown of effective genetic exchange between Azores and the others' samples could be explained simultaneously by hydrographic (deep water) and hydrodynamic (isolating current regimes) factors acting as barriers to the free dispersal of white seabream(adults and larvae) and by historical factors which could be favoured for the survival of Azorean white seabream population at the last glaciation. Mediterranean islands show similar genetic diversity to the neighbouring continental samples and nonsignificant genetic differences. Proximity to continental coasts and the current system could promote an optimal larval dispersion among Mediterranean islands (Mallorca and Castellamare) and coasts with high gene flow.

Genome selection in fruit breeding: application to table grapes.

Genomic selection (GS) has recently been proposed as a new selection strategy which represents an innovative paradigm in crop improvement, now widely adopted in animal breeding. Genomic selection relies on phenotyping and high-density genotyping of a sufficiently large and representative sample of the target breeding population, so that the majority of loci that regulate a quantitative trait are in linkage disequilibrium with one or more molecular markers and can thus be captured by selection. In this study we address genomic selection in a practical fruit breeding context applying it to a breeding population of table grape obtained from a cross between the hybrid genotype D8909-15 (Vitis rupestris × Vitis arizonica/girdiana), which is resistant to dagger nematode and Pierce?s disease (PD), and ?B90-116?, a susceptible Vitis vinifera cultivar with desirable fruit characteristics. Our aim was to enhance the knowledge on the genomic variation of agronomical traits in table grape populations for future use in marker-assisted selection (MAS) and GS, by discovering a set of molecular markers associated with genomic regions involved in this variation. A number of Quantitative Trait Loci (QTL) were discovered but this method is inaccurate and the genetic architecture of the studied population was better captured by the BLasso method of genomic selection, which allowed for efficient inference about the genetic contribution of the various marker loci. The technology of genomic selection afforded greater efficiency than QTL analysis and can be very useful in speeding up the selection procedures for agronomic traits in table grapes.

Marker-assisted selection integrated to the embrapa common bean breeding program.

The establishment of a specific Marker-Assisted Selection Facility at the Embrapa Rice and Beans Biotechnology Laboratory, in 2014, has better supported the routine analysis with molecular markers demanded by the Embrapa Common Bean Breeding Program. In addition, it has also supported other Embrapa plant breeding programs, such as rice and cotton.

Identification of novel drought-tolerant-associated SNPs in common bean (Phaseolus vulgaris)

Common bean (Phaseolus vulgaris L.) is a leguminous in high demand for human nutrition and a very important agricultural product. Production of common bean is constrained by environmental stresses such as drought. Although conventional plant selection has been used to increase production yield and stress tolerance, drought tolerance selection based on phenotype is complicated by associated physiological, anatomical, cellular, biochemical, and molecular changes. These changes are modulated by differential gene expression. A common method to identify genes associated with phenotypes of interest is the characterization of Single Nucleotide Polymorphims (SNPs) to link them to specific functions. In this work, we selected two drought-tolerant parental lines from Mesoamerica, Pinto Villa, and Pinto Saltillo. The parental lines were used to generate a population of 282 families (F3:5) and characterized by 169 SNPs. We associated the segregation of the molecular markers in our population with phenotypes including flowering time, physiological maturity, reproductive period, plant, seed and total biomass, reuse index, seed yield, weight of 100 seeds, and harvest index in three cultivation cycles. We observed 83 SNPs with significant association (p < 0.0003 after Bonferroni correction) with our quantified phenotypes. Phenotypes most associated were days to flowering and seed biomass with 58 and 44 associated SNPs, respectively. Thirty-seven out of the 83 SNPs were annotated to a gene with a potential function related to drought tolerance or relevant molecular/biochemical functions. Some SNPs such as SNP28 and SNP128 are related to starch biosynthesis, a common osmotic protector; and SNP18 is related to proline biosynthesis, another well-known osmotic protector.

Expression of Oct-4 in oncogenic miR-155-positive oral squamous carcinoma cells

Purpose: To identify effective molecular diagnostic methods for oral squamous cell carcinoma (OSCC) to facilitate treatment of the disease in its initial stages. Methods: To identify molecular markers, OSCC tissue samples were collected from cancer patients and healthy controls. CD44+ cells were sorted using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemistry and immunostaining experiments were performed to identify markers for OSCC. Results: The qRT-PCR data confirmed the presence of oncogenic miR-155 in the OSCC samples. The immunohistochemical and immunostaining results confirmed the expression of Oct-4, an important target for the early diagnosis of OSCC, in oncogenic miR-155-positive OSCCs. Conclusion: Detection of the expression of miR-155 and Oct-4, which are key molecular markers, may be useful in improving the early diagnosis of OSCC.

Microsatellite analysis of pacu broodstocks used in the stocking program of Paranapanema River, Brazil

O monitoramento da diversidade genética é fundamental em um programa de repovoamento. Avaliouse a diversidade genética de pacu Piaractus mesopotamicus (Holmberg, 1887) em duas estações de piscicultura em Andirá -Paraná, Brasil, utilizadas no programa de repovoamento do Rio Paranapanema. Foram amplificados seis loci microssatélite para avaliar 60 amostras de nadadeira. O estoque de reprodutores B apresentou maior número de alelos e heterozigose (alelos: 22 e H O: 0,628) que o estoque de reprodutores A (alelos: 21 e H O: 0,600). Alelos com baixos níveis de frequência foram observados nos dois estoques. Os coeficientes positivos de endogamia no locus Pme2 (estoque A: F IS = 0,30 e estoque B: F IS = 0,20), Pme5 (estoque B: F IS = 0,15), Pme14 (estoque A: F IS = 0,07) e Pme28 (estoque A: F IS = 0,24 e estoque B: F IS = 0,20), indicaram deficiência de heterozigotos. Foi detectada a presença de um alelo nulo no lócus Pme2. As estimativas negativas nos loci Pme4 (estoque A: F IS = -0,43 e estoque B: F IS= -0,37), Pme5 (estoque A: F IS = - 0,11), Pme14 (estoque B: F IS = - 0,15) e Pme32 (estoque A: F IS = - 0,93 e estoque B: F IS = - 0,60) foram indicativas de excesso de heterozigotos. Foi evidenciado desequilíbrio de ligação e riqueza alélica baixa só no estoque A. A diversidade genética de Nei foi alta nos dois estoques. A distância (0,085) e identidade (0,918) genética mostraram similaridade entre os estoques, o qual reflete uma possível origem comum. 6,05% da variância genética total foi devida a diferenças entre os estoques. Foi observado um recente efeito gargalo nos dois estoques. Os resultados indicaram uma alta diversidade genética nos estoques de reprodutores e baixa diferenciação genética entre eles, o que foi causado pelo manejo reprodutivo das pisciculturas, redução do tamanho populacional e intercâmbio genético entre as pisciculturas.

ANALYSIS OF POPULUS TREMULOIDES CLONAL VARIATION AND DELINEATION IN THE OTTAWA NATIONAL FOREST

The technique of delineating Populus tremuloides (Michx.) clonal colonies based on morphology and phenology has been utilized in many studies and forestry applications since the 1950s. Recently, the availability and robustness of molecular markers has challenged the validity of such approaches for accurate clonal identification. However, genetically sampling an entire stand is largely impractical or impossible. For that reason, it is often necessary to delineate putative genet boundaries for a more selective approach when genetically analyzing a clonal population. Here I re-evaluated the usefulness of phenotypic delineation by: (1) genetically identifying clonal colonies using nuclear microsatellite markers, (2) assessing phenotypic inter- and intraclonal agreement, and (3) determining the accuracy of visible characters to correctly assign ramets to their respective genets. The long-term soil productivity study plot 28 was chosen for analysis and is located in the Ottawa National Forest, MI (46° 37'60.0" N, 89° 12'42.7" W). In total, 32 genets were identified from 181 stems using seven microsatellite markers. The average genet size was 5.5 ramets and six of the largest were selected for phenotypic analyses. Phenotypic analyses included budbreak timing, DBH, bark thickness, bark color or brightness, leaf senescence, leaf serrations, and leaf length ratio. All phenotypic characters, except for DBH, were useful for the analysis of inter- and intraclonal variation and phenotypic delineation. Generally, phenotypic expression was related to genotype with multiple response permutation procedure (MRPP) intraclonal distance values ranging from 0.148 and 0.427 and an observed MRPP delta value=0.221 when the expected delta=0.5. The phenotypic traits, though, overlapped significantly among some clones. When stems were assigned into phenotypic groups, six phenotypic groups were identified with each group containing a dominant genotype or clonal colony. All phenotypic groups contained stems from at least two clonal colonies and no clonal colony was entirely contained within one phenotypic group. These results demonstrate that phenotype varies with genotype and stand clonality can be determined using phenotypic characters, but phenotypic delineation is less precise. I therefore recommend that some genetic identification follow any phenotypic delineation. The amount of genetic identification required for clonal confirmation is likely to vary based on stand and environmental conditions. Further analysis, however, is needed to test these findings in other forest stands and populations.

Species delimitation in sharpnose sharks (genus Rhizoprionodon) in the western Atlantic Ocean using mitochondrial DNA

Despite Springer’s (1964) revision of the sharpnose sharks (genus Rhizoprionodon), the taxonomic definition and ranges of Rhizoprionodon in the western Atlantic Ocean remains problematic. In particular, the distinction between Rhizoprionodon terraenovae and R. porosus, and the occurrence of R. terraenovae in South American waters are unresolved issues involving common and ecologically important species in need of fishery management in Caribbean and southwest Atlantic waters. In recent years, molecular markers have been used as efficient tools for the detection of cryptic species and to address controversial taxonomic issues. In this study 415 samples of the genus Rhizoprionodon captured in the western Atlantic Ocean from Florida to southern Brazil were examined for sequences of the COI gene and the D-loop and evaluated for nucleotide differences. The results on nucleotide composition, AMOVA tests, and relationship distances using Bayesian-likelihood method and haplotypes network, corroborates Springer’s (1964) morphometric and meristic finding and provide strong evidence that supports consideration of R. terraenovae and R. porosus as distinct species.

A single nucleotide polymorphism in NEUROD1 is associated with production traits in nelore beef cattle.

Feed efficiency and carcass characteristics are late-measured traits. The detection of molecular markers associated with them can help breeding programs to select animals early in life, and to predict breeding values with high accuracy. The objective of this study was to identify polymorphisms in the functional and positional candidate gene NEUROD1 (neurogenic differentiation 1), and investigate their associations with production traits in reference families of Nelore cattle. A total of 585 steers were used, from 34 sires chosen to represent the variability of this breed. By sequencing 14 animals with extreme residual feed intake (RFI) values, seven single nucleotide polymorphisms (SNPs) in NEUROD1 were identified. The investigation of marker effects on the target traits RFI, backfat thickness (BFT), ribeye area (REA), average body weight (ABW), and metabolic body weight (MBW) was performed with a mixed model using the restricted maximum likelihood method. SNP1062, which changes cytosine for guanine, had no significant association with RFI or REA. However, we found an additive effect on ABW (P ≤ 0.05) and MBW (P ≤ 0.05), with an estimated allele substitution effect of -1.59 and -0.93 kg0.75, respectively. A dominant effect of this SNP for BFT was also found (P ≤ 0.010). Our results are the first that identify NEUROD1 as a candidate that affects BFT, ABW, and MBW. Once confirmed, the inclusion of this SNP in dense panels may improve the accuracy of genomic selection for these traits in Nelore beef cattle as this SNP is not currently represented on SNP chips.

Molecular Characterization of Sclerospora graminicola, the Incitant of Pearl Millet Downy Mildew Using ISSR Markers

The DNA polymorphism among 22 isolates of Sclerospora graminicola, the causal agent of downy mildew disease of pearl millet was assessed using 20 inter simple sequence repeats (ISSR) primers. The objective of the study was to examine the effectiveness of using ISSR markers for unravelling the extent and pattern of genetic diversity in 22 S. graminicola isolates collected from different host cultivars in different states of India. The 19 functional ISSR primers generated 410 polymorphic bands and revealed 89% polymorphism and were able to distinguish all the 22 isolates. Polymorphic bands used to construct an unweighted pair group method of averages (UPGMA) dendrogram based on Jaccard's co-efficient of similarity and principal coordinate analysis resulted in the formation of four major clusters of 22 isolates. The standardized Nei genetic distance among the 22 isolates ranged from 0.0050 to 0.0206. The UPGMA clustering using the standardized genetic distance matrix resulted in the identification of four clusters of the 22 isolates with bootstrap values ranging from 15 to 100. The 3D-scale data supported the UPGMA results, which resulted into four clusters amounting to 70% variation among each other. However, comparing the two methods show that sub clustering by dendrogram and multi dimensional scaling plot is slightly different. All the S. graminicola isolates had distinct ISSR genotypes and cluster analysis origin. The results of ISSR fingerprints revealed significant level of genetic diversity among the isolates and that ISSR markers could be a powerful tool for fingerprinting and diversity analysis in fungal pathogens.

Molecular identification of 16 Porphyra lines using sequence-related amplified polymorphism markers

Sequence-related amplified polymorphism (SRAP) is a novel molecular marker technique designed to amplify open reading frames (ORFs). The SRAP analytic system was set up and applied to Porphyra germplasm identification in this study for the first time. Sixteen Porphyra lines were screened by SRAP technique with 30 primer combinations. In the analysis, 14 primer combinations produced stable and reproducible amplification patterns in three repetitive experiments. Among the total 533 amplified fragments, 522 (98%) were polymorphic, with an average of 38 fragments for each primer combination, ranging in size from 50 to 500 bp. The 533 fragments were visually scored one by one and then used to develop a dendrogram with Unweighted Pair-Group Method Arithmetic Average (UPGMA), and the 16 Porphyra lines were divided into two major groups at the 0.68 similarity level. From the total 533 fragments, I I amplified by two primer combinations, ME1/EM1 and ME4/EM6, were used to develop the DNA fingerprints of the 16 Porphyra lines. The DNA fingerprints were then converted into binary codes, with I and 0 representing presence and absence of the corresponding amplified fragment, respectively. In the DNA fingerprints, each of the 16 Porphyra lines has its unique binary code and can be easily distinguished from the others. This is the first report on the development of SRAP technique and its utilization in germplasm identification of seaweeds. The results demonstrated that SRAP is a simple, stable, polymorphic and reproducible molecular marker technique for the classification and identification of Porphyra lines. (c) 2007 Elsevier B.V. All rights reserved.