A critical comparison of technologies for a plant genome sequencing project

BACKGROUND: A high-quality genome sequence of any model organism is an essential starting point for genetic and other studies. Older clone-based methods are slow and expensive, whereas faster, cheaper short-read-only assemblies can be incomplete and highly fragmented, which minimizes their usefulness. The last few years have seen the introduction of many new technologies for genome assembly. These new technologies and associated new algorithms are typically benchmarked on microbial genomes or, if they scale appropriately, on larger (e.g., human) genomes. However, plant genomes can be much more repetitive and larger than the human genome, and plant biochemistry often makes obtaining high-quality DNA that is free from contaminants difficult. Reflecting their challenging nature, we observe that plant genome assembly statistics are typically poorer than for vertebrates. RESULTS: Here, we compare Illumina short read, Pacific Biosciences long read, 10x Genomics linked reads, Dovetail Hi-C, and BioNano Genomics optical maps, singly and combined, in producing high-quality long-range genome assemblies of the potato species Solanum verrucosum. We benchmark the assemblies for completeness and accuracy, as well as DNA compute requirements and sequencing costs. CONCLUSIONS: The field of genome sequencing and assembly is reaching maturity, and the differences we observe between assemblies are surprisingly small. We expect that our results will be helpful to other genome projects, and that these datasets will be used in benchmarking by assembly algorithm developers.</p

A deletion affecting an LRR-RLK gene co-segregates with the fruit flat shape trait in peach

Altres ajuts: CERCA Programme/Generalitat de CatalunyaIn peach, the flat phenotype is caused by a partially dominant allele in heterozygosis (Ss), fruits from homozygous trees (SS) abort a few weeks after fruit setting. Previous research has identified a SSR marker (UDP98-412) highly associated with the trait, found suitable for marker assisted selection (MAS). Here we report a ∼10 Kb deletion affecting the gene PRUPE.6G281100, 400 Kb upstream of UDP98-412, co-segregating with the trait. This gene is a leucine-rich repeat receptor-like kinase (LRRRLK) orthologous to the Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) group. PCR markers suitable for MAS confirmed its strong association with the trait in a collection of 246 cultivars. They were used to evaluate the DNA from a round fruit derived from a somatic mutation of the flat variety 'UFO-4', revealing that the mutation affected the flat associated allele (S). Protein BLAST alignment identified significant hits with genes involved in different biological processes. Best protein hit occurred with AtRLP12, which may functionally complement CLAVATA2, a key regulator that controls the stem cell population size. RT-PCR analysis revealed the absence of transcription of the partially deleted allele. The data support PRUPE.6G281100 as a candidate gene for flat shape in peach

A peach germplasm collection for increasing the genetic diversity in European breeding programs

Trabajo presentado al VIII International Peach Symposium, celebrado en Matera (Italia) del 17 al 20 de junio de 2013.European breeding programs are hampered by the low intraspecific genetic diversity, which is due to the self-compatibility of this homozygous species along with the low number of genotypes introduced and thus used for breeding. In 2009, four research institutions which carried out peach breeding programs in Aragon, Catalonia, Valencia and Murcia, started a new peach germplasm collection worldwide aimed at enlarging the peach genetic diversity available for breeding. The plant material was introduced from germplasm collections located in China, Central Asia, Iran and the USA (National Germplasm Repository of Davis). Sanitary status was assessed by molecular diagnosis of known diseases caused by virus, viroid, bacteria and phytoplasm pathogens. Healthy plant material was grafted and maintained in quarantine conditions. The new germplasm collection was established in two places: Zaragoza as high chilling and Murcia as low chilling requirements. Pomological and molecular data were gathered and a public database constructed. The descriptors used were from the National Center for Genetic Resources from the INIA. Introduced budwood and seeds resulted in more than 250 new genotypes from 15 countries. The molecular analysis of a subset of the collection with 21 SSR markers evenly distributed in the genome resulted in a high number of alleles per SSR (mean A=9.5) and low observed heterozygosity (mean Ho=0.38). Variability was further assessed by geographic origin. Population structure analysis revealed the existence of 8 subpopulations explained, in some cases, by the geographic origin of the genotypes. As a result of the project a new database containing 95 accessions and 38 variables is available.Peer reviewe

La disCAPACIDAD desde la mirada de la comunidad universitaria: Enfoques y visiones

La diversidad que caracteriza el entono académico actual requiere proveer al colectivo que presenta Necesidades Específicas de Apoyo Educativo –NEAE- los medios adecuados que faciliten su desarrollo académico. Este propósito nos lleva no sólo a seguir enriqueciendo la biblioteca virtual de contenido audiovisual especializado en materias docentes e investigadoras, dentro del marco “Speaking Library”, sino también a conocer con profundidad las necesidades de este colectivo, bien sean estudiantes o docentes. De hecho, desde la Red I+Do+i (Investigación Docencia e Innovación) de concepción multidisciplinar y en la que han participado profesorado y estudiantes de diferentes centros, entendemos que resulta primordial conocer la percepción que se tiene de la discapacidad en el medio académico en el que se desenvuelve. Precisamente, esta visión de 360º nos ha llevado a analizar esta problemática desde cuatro ángulos diferentes, profesores y estudiantes, con y sin discapacidad. Sólo así podremos facilitar a este colectivo, de acuerdo a la naturaleza de la discapacidad que presentan, los recursos que requieren para que se sientan integrados de la misma manera que aquellas personas que no tienen dichas necesidades

Relationship between tobacco, cagA and vacA i1 virulence factors and bacterial load in patients infected by Helicobacter pylori

Background and Aim Several biological and epidemiological studies support a relationship between smoking and Helicobacter pylori (H. pylori) to increase the risk of pathology. However, there have been few studies on the potential synergistic association between specific cagA and vacA virulence factors and smoking in patients infected by Helicobacter pylori. We studied the relationship between smoking and cagA, vacA i1 virulence factors and bacterial load in H. pylori infected patients. Methods Biopsies of the gastric corpus and antrum from 155 consecutive patients in whom there was clinical suspicion of infection by H. pylori were processed. In 106 patients H. pylori infection was detected. Molecular methods were used to quantify the number of microorganisms and presence of cagA and vacA i1 genes. A standardized questionnaire was used to obtain patients’ clinical data and lifestyle variables, including tobacco and alcohol consumption. Adjusted Odds Ratios (ORadjusted) were estimated by unconditional logistic regression. Results cagA was significantly associated with active-smoking at endoscope: ORadjusted 4.52. Evidence of association was found for vacA i1 (ORadjusted 3.15). Bacterial load was higher in active-smokers, although these differences did not yield statistical significance (median of 262.2 versus 79.4 copies of H. pylori per cell). Conclusions The association between smoking and a higher risk of being infected by a virulent bacterial population and with higher bacterial load, support a complex interaction between H. pylori infection and environmental factors

Stakeholders' perspectives on the operationalisation of the ecosystem service concept : Results from 27 case studies

The ecosystem service (ES) concept is becoming mainstream in policy and planning, but operational influence on practice is seldom reported. Here, we report the practitioners' perspectives on the practical implementation of the ES concept in 27 case studies. A standardised anonymous survey (n = 246), was used, focusing on the science-practice interaction process, perceived impact and expected use of the case study assessments. Operationalisation of the concept was shown to achieve a gradual change in practices: 13% of the case studies reported a change in action (e.g. management or policy change), and a further 40% anticipated that a change would result from the work. To a large extent the impact was attributed to a well conducted science-practice interaction process (>70%). The main reported advantages of the concept included: increased concept awareness and communication; enhanced participation and collaboration; production of comprehensive science-based knowledge; and production of spatially referenced knowledge for input to planning (91% indicated they had acquired new knowledge). The limitations were mostly case-specific and centred on methodology, data, and challenges with result implementation. The survey highlighted the crucial role of communication, participation and collaboration across different stakeholders, to implement the ES concept and enhance the democratisation of nature and landscape planning. (C) 2017 Published by Elsevier B.V.Peer reviewe

The Gaia mission

Gaia is a cornerstone mission in the science programme of the EuropeanSpace Agency (ESA). The spacecraft construction was approved in 2006, following a study in which the original interferometric concept was changed to a direct-imaging approach. Both the spacecraft and the payload were built by European industry. The involvement of the scientific community focusses on data processing for which the international Gaia Data Processing and Analysis Consortium (DPAC) was selected in 2007. Gaia was launched on 19 December 2013 and arrived at its operating point, the second Lagrange point of the Sun-Earth-Moon system, a few weeks later. The commissioning of the spacecraft and payload was completed on 19 July 2014. The nominal five-year mission started with four weeks of special, ecliptic-pole scanning and subsequently transferred into full-sky scanning mode. We recall the scientific goals of Gaia and give a description of the as-built spacecraft that is currently (mid-2016) being operated to achieve these goals. We pay special attention to the payload module, the performance of which is closely related to the scientific performance of the mission. We provide a summary of the commissioning activities and findings, followed by a description of the routine operational mode. We summarise scientific performance estimates on the basis of in-orbit operations. Several intermediate Gaia data releases are planned and the data can be retrieved from the Gaia Archive, which is available through the Gaia home page. http://www.cosmos.esa.int/gai

Genetic architecture of agronomic traits in peach [Prunus persica (L.) Batsch]: subacid, flat shape and nectarine

L'objectiu dels programes de millora genètica del préssec és generar varietats adaptats a les condicions agronòmiques locals i satisfer els requeriments del consumidor. Això últim implica millorar la qualitat del fruit. L'estratègia seguida per molts milloradors es basa en la selección de descendents de creuaments on s’espera segregació per a caràcters fenotípics. Es tracta d’un mètode costós tant en temps com en diners a causa del período de juvenilitad del presseguer (2-3 anys) i també als recursos que suposa el mantener les plàntules en el camp durant el procés d’avaluació i selecció. L'objectiu d'aquesta tesi va ser el desenvolupament de marcadors moleculars per a la seva aplicació en la selecció assistida per marcadors (SAM) de tres caràcters del fruit: baixa acidesa, fruit pla i pell glabra. En els dos primers capítols estudiem el locus del carácter subàcido (D) i el fruit pla (S) i hem realitzat l’anàlisis d’associació en les seves respectives regions genòmiques. Per a ambdós caràcters s'han generat i validat marcadors moleculars (SSRs i SNPs) que poden ser directament aplicats a SAM. L'estudi de l’haplotipus subàcid ens va permetre identificar diversos gens candidats. Aquestes resultats ens suggereix l'existència d'un únic origen de l’al·lel subàcido. L'anàlisi del locus S ens va permetre identificar dues INDELs altament associats amb el fruit pla. Aquests polimorfismes es van observar en regió codificant del gen ppa025511m, concretament en el segon exò del gen. Aquesta associació va ser avaluada en un ampli panell de varietats i en una població obtinguda a partir del creuament de dues parentals de fruits plans. L'anàlisi de la seqüència completa d'aquest gen va permetre la identificació de la supressió d'un fragment de 9Kb que afecta la regió 5’UTR del gen així com al primer exò, a l’intró i a una petita part del segon exò. La funció d'aquest gen va ser validada en un mutant tipus “sport” generat espontàneament en un arbre de la varietat plana ‘UFO4’. Es tracta d'un mutante quimèric mb una mutació que només afecta a les cèl·lules de la segona capa meristemàtica (LII), que genera la polpa del fruit. L'amplificació per PCR de l’INDEL d'aquest gen en la polpa del mutant rodó va revelar un canvi a l’al·lel pla. Malgrat que l‘alelo pla és dominant, els fruits plans han de presentar-lo en hetericigosis per a ser viables. Aquest fet amb la reversió a la forma rodona del mutant d’UFO4 suggereixen que aquest al·lel pot actuar com dominant negatiu. En el tercer capítol seqüenciem el genoma de 5 varietats de préssec i els seus respectius mutantes nectarina. Es van a estimar la variabilitat somática i la mutació causal de la pell glabra. La metodología empleada per al processament de les lectures i la identificació dels petis polimorfismes va generar un excés de falsos polimorfismes, possiblement causats per alineaments erronis de les seqüències repetitives del genoma. Mitjançant l'ús d'un filtrat més restrictiu es va reduir aquest valor. Els valors de diversitat nucleotídica i d’heterozigositat van ser similars als observats per Aranzana et al., (2012) i Verd et al., (2013). L’analisi del locus G va mostrar una menor π i una major Ho. Sota dues possibles hipótesis per a l’estudi l’al·lel causal del caràcter nectarina vam identificar diversos gens candidats amb funcions relacionades amb el desenvolupament de la paret cel·lular. No obstant això cap d'ells va resultar ser PpeMYB25 on recentment s’ha descrit una inserció de 7Kb asssociada al carácter nectarina (Vendramin et al., 2014). La insuficient cobertura de seqüenciació en la regió genómica del locus G pot haver estat la causa de la no identificació d’aquest polimorfisme en les nostres seqüències.The aim of current breeding programs is to provide new fruit varieties adapted to the local agronomic conditions and to satisfy the requirements of the consumers. This last fact implies to improve the fruit quality. The strategy followed by most breeding programs is based on performing controlled crosses to select those individuals showing the target traits. Although this approach has succeed in the production of most of the varieties available today, it is time consuming and costly due the time required to obtain fruits (2-3 years) and the resources needed to keep the seedlings in the field during the evaluation and selection processes. The objective of this thesis was to develop molecular markers useful in marker assisted selection (MAS) for three important agronomical traits in peach fruits: low acidity, flat shape and glabrous skin (nectarine trait). In the first two chapters of this document we have used region-based association analysis to study the architecture of the locus responsible for either sub-acid (D) and flat fruits (S). In both cases the study has provided markers (SSR and SNPs) ready to be applied for MAS in peach breeding programs. The study of the length of the sub-acid haplotype, which is maintained more than 24Kbp long, allowed to hypothesize about a unique origin of this trait and to identify candidate genes. Similarly, the analysis of the S locus allowed the identification of two linked INDELs in the second exon of the gene ppa025511m highly associated with the flat shape of the fruit. The association was tested in a broad panel of varieties and in the offspring of a crossing population between two flat peaches. The sequencing analysis of the whole gene allowed the identification of a big deletion, of about 9Kbp, affecting its 5’ UTR, its first exon and its intron. The function of the gene was validated in a round sport mutant from a flat peach (UFO-4). This mutant was chimeric; the mutation only affected cells of the second layer (LII) of the meristerm, which generates the flesh of the fruit. A PCR amplification of the gene and the use of specific primers for the INDEL revealed a mutation in the flat allele in the flesh of the chimeric mutant, which produced the reversion to the round shape. The obligated heterozygosis of the flat allele and the reversion to the wild shape suggest a dominant negative (DN) mechanism. In the third chapter we sequenced the whole genome of 5 peach varieties and 6 sport nectarines derived from them. We estimated the overall somatic variability and studied the causal mutation from hairy fruit to glabrous. The reads processing and SNP calling revealed an excess of false variants that was especially evident in the analysis of the sport mutants. One of the main causes for the false variants was the misalignments of repetitive regions. The use of more restrictive SNP calling filters reduced the excess of false variants. The nucleotide diversity and heterozygosity of the varieties was similar to the one reported for peach (Aranzana et al., 2012; Verde et al., 2013). The analysis of the variations in the G locus region showed lower and higher Ho. To look for the causal allele for the nectarine trait we postulated two possible causes for the new mutation. These data provided several candidate genes involved in the cell wall development, however none of them was the gene PpeMYB25, where a big insertion of 7Kb in its second exon has recently been described as linked with the trait (Vendramin et al., 2014). This was probably due to an insufficient sequencing coverage in this genomic region

Region-wide association analysis and high-throughput resequencing strategies in peach to develop molecular markers for flat fruit marker-assisted selection

IV International Symposium on Molecular Markers in Horticulture.Commercial interest in flat-fruit peaches is increasing. Many genetic studies have aimed to unravel the genetic basis of fruit shape, especially in model species. However, no candidate genes have been reported to date for peach. This trait in peach is determined by a single dominant gene S mapped on chromosome 6. The allele conferring the flat appearance is partly dominant: s/s fruits are round and S/s flat, while S/S fruits abort a few weeks after anthesis. Several markers have been identified around the S locus through the analysis of mapping populations and germplasm, and one of them works efficiently in marker-assisted selection (MAS). By conducting an association analysis of the flat shape candidate region and by resequencing five flat and five round peach cultivars, we have identified size polymorphisms as well as single nucleotide polymorphisms (SNPs) that co-segregate with the trait. These polymorphisms affect coding regions of an LRR-RL kinase. Associated polymorphisms have been evaluated in a panel of peach commercial cultivars (including flat and round fruits) demonstrating their efficiency for MAS.Peer reviewe

An efcient CRISPR-Cas9 enrichment sequencing strategy for characterizing complex and highly duplicated genomic regions. A case study in the Prunus salicina LG3-MYB10 genes cluster

Background: Genome complexity is largely linked to diversifcation and crop innovation. Examples of regions with duplicated genes with relevant roles in agricultural traits are found in many crops. In both duplicated and non‑dupli‑cated genes, much of the variability in agronomic traits is caused by large as well as small and middle scale structural variants (SVs), which highlights the relevance of the identifcation and characterization of complex variability between genomes for plant breeding. Results: Here we improve and demonstrate the use of CRISPR‑Cas9 enrichment combined with long‑read sequenc-ing technology to resolve the MYB10 region in the linkage group 3 (LG3) of Japanese plum (Prunus salicina). This region, which has a length from 90 to 271 kb according to the P. salicina genomes available, is associated with fruit color variability in Prunus species. We demonstrate the high complexity of this region, with homology levels between Japanese plum varieties comparable to those between Prunus species. We cleaved MYB10 genes in fve plum varieties using the Cas9 enzyme guided by a pool of crRNAs. The barcoded fragments were then pooled and sequenced in a single MinION Oxford Nanopore Technologies (ONT) run, yielding 194 Mb of sequence. The enrichment was confrmed by aligning the long reads to the plum reference genomes, with a mean read on‑target value of 4.5% and a depth per sample of 11.9x. From the alignment, 3261 SNPs and 287 SVs were called and phased. A de novo assembly was constructed for each variety, which also allowed detection, at the haplotype level, of the variability in this region. Conclusions: CRISPR‑Cas9 enrichment is a versatile and powerful tool for long‑read targeted sequencing even on highly duplicated and/or polymorphic genomic regions, being especially useful when a reference genome is not available. Potential uses of this methodology as well as its limitations are further discussed. Keywords: Cas9 enrichment, Complex regions, Long‑read, Targeted sequencing, Variability, Gene duplication, MYB10info:eu-repo/semantics/publishedVersio