31 research outputs found
Microarray-based uncovering reference genes for quantitative real time PCR in grapevine under abiotic stress
Research ArticleBackground: Quantitative real time polymerase chain reaction is becoming the primary tool for detecting mRNA
and transcription data analysis as it shows to have advantages over other more commonly used techniques.
Nevertheless, it also presents a few shortcomings, with the most import being the need for data normalisation,
usually with a reference gene. Therefore the choice of the reference gene(s) is of great importance for correct data
analysis. Microarray data, when available, can be of great assistance when choosing reference genes. Grapevine was
submitted to water stress and heat stress as well as a combination of both to test the stability of the possible
reference genes.
Results: Using the analysis of microarray data available for grapevine, six possible reference genes were selected for
RT-qPCR validation: PADCP, ubiq, TIF, TIF-GTP, VH1-IK, aladin-related. Two additional genes that are commonly used as
reference genes were included: act and L2. The stability of those genes was tested in leaves of grapevine in both
field plants and in greenhouse plants under water or heat stress or a combination of both. Gene stability was
analyzed with the softwares GeNorm, NormFinder and the ΔCq method resulting in several combinations of
reference genes suitable for data normalisation. In order to assess the best combination, the reference genes were
tested in putative stress marker genes (PCO, Galsynt, BKCoAS and HSP17) also chosen from the same microarray, in
water stress, heat stress and the combination of both.
Conclusions: Each method selected different gene combinations (PADCP + act, TIF + TIF-GTP and ubiq + act).
However, as none of the combinations diverged significantly from the others used to normalize the expression of
the putative stress marker genes, then any combination is suitable for data normalisation under the conditions
tested. Here we prove the accuracy of choosing grapevine reference genes for RT-qPCR through a microarray
analysi
PROGRAMA DE PORTUGUÊS DO ENSINO BÁSICO: UMA REVISÃO
Revisitação crítica do Programa de Português para o Ensino Básico em vigor, homologado em 2009 e coordenado por Carlos Reis. Com base numa reflexão sobre os desafios colocados a alunos e professores de língua materna numa sociedade em mudança acelerada, caracterizada pela rápida desatualização do conhecimento, argumenta-se a favor da presença da literatura portuguesa e também das literaturas de língua portuguesa nas listas curriculares, destacando-se a natureza da leitura literária e as suas funções na educação de crianças e adolescentes
Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster)
Somatic embryogenesis (SE) is a propagation
tool of particular interest for accelerating the deployment
of new high-performance planting stock in multivarietal
forestry. However, genetic conformity in in vitro propagated
plants should be assessed as early as possible,
especially in long-living trees such as conifers. The main
objective of this work was to study such conformity based
on genetic stability at simple sequence repeat (SSR) loci
during somatic embryogenesis in maritime pine (Pinus
pinaster Ait.). Embryogenic cell lines (ECLs) subjected to
tissue proliferation during 6, 14 or 22 months, as well as
emblings regenerated from several ECLs, were analyzed.
Genetic variation at seven SSR loci was detected in ECLs
under proliferation conditions for all time points, and in 5
out of 52 emblings recovered from somatic embryos. Three
of these five emblings showed an abnormal phenotype
consisting mainly of plagiotropism and loss of apical
dominance. Despite the variation found in somatic
embryogenesis-derived plant material, no correlation was
established between genetic stability at the analyzed loci
and abnormal embling phenotype, present in 64% of the
emblings. The use of microsatellites in this work was
efficient for monitoring mutation events during the somatic
embryogenesis in P. pinaster. These molecular markers
should be useful in the implementation of new breeding
and deployment strategies for improved trees using SE
Flower development and sex specification in wild grapevine
Background: Wild plants of Vitis closely related to the cultivated grapevine (V. v. vinifera) are believed to have been first domesticated 10,000 years BC around the Caspian Sea. V. v. vinifera is hermaphrodite whereas V. v. sylvestris is a dioecious species. Male flowers show a reduced pistil without style or stigma and female flowers present reflexed stamens with infertile pollen. V. vinifera produce perfect flowers with all functional structures. The mechanism for flower sex determination and specification in grapevine is still unknown.Results: To understand which genes are involved during the establishment of male, female and complete flowers, we analysed and compared the transcription profiles of four developmental stages of the three genders. We showed that sex determination is a late event during flower development and that the expression of genes from the ABCDE model is not directly correlated with the establishment of sexual dimorphism. We propose a temporal comprehensive model in which two mutations in two linked genes could be players in sex determination and indirectly establish the Vitis domestication process. Additionally, we also found clusters of genes differentially expressed between genders and between developmental stages that suggest a role involved in sex differentiation. Also, the detection of differentially transcribed regions that extended existing gene models (intergenic regions) between sexes suggests that they may account for some of the variation between the subspecies.Conclusions: There is no evidence of differences of expression levels in genes from the ABCDE model that could explain the shift from hermaphroditism to dioecy. We propose that sex specification occurs after floral organ identity has been established and therefore, sex determination genes might be having an effect downstream of the ABCDE model genes. For the first time a full transcriptomic analysis was performed in different flower developmental stages in the same individual. Our experimental approach enabled us to create a comprehensive catalogue of transcribed genes across
developmental stages and genders that will contribute for future work in sex determination in seed plants.This work was supported by the funded project PTDC/AGR-GPL/119298/2010 from Fundacao para a Ciencia e Tecnologia and MRocheta, JLCoito and JCunha by the fellowships FRH/BPD/64905/2009, SFRH/BD/85824/2012 and SFRH/BPD/74895/2010, respectively. We are also grateful to Eng. Eiras- Dias, curator from Portuguese Ampelographic Collection (property of Instituto Nacional de Investigacao Agraria e Veterinaria, Dois Portos), for the collaboration in this work allowing the access to the Vitis collection
Portuguese wild grapevine genome re-sequencing (Vitis vinifera sylvestris)
Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-76012-6.The first genome of Vitis vinifera vinifera (PN40024), published in 2007, boosted grapevine related studies. While this reference genome is a suitable tool for the overall studies in the field, it lacks the ability to unveil changes accumulated during V. v. vinifera domestication. The subspecies V. v. sylvestris preserves wild characteristics, making it a good material to provide insights into V. v. vinifera domestication. The difference in the reproductive strategy between both subspecies is one of the characteristics that set them apart. While V. v. vinifera flowers are hermaphrodite, V. v. sylvestris is mostly dioecious. In this paper, we compare the re-sequencing of the genomes from a male and a female individual of the wild sylvestris, against the reference vinifera genome (PN40024). Variant analysis reveals a low number but with high impact modifications in coding regions, essentially non-synonymous single nucleotide polymorphisms and frame shifts caused by insertions and deletions. The sex-locus was manually inspected, and the results obtained are in line with the most recent works related with wild grapevine sex. In this paper we also describe for the first time RNA editing in transcripts of 14 genes in the sex-determining region, including VviYABBY and VviPLATZ.Tis work was supported by Fundação para a Ciência e Tecnologia (FCT) through the Research Center LEAF
(UIDP/04129/2020). Authors JLCoito, MJNRamos, MRocheta, were funded by FCT fellowships SFRH/
BD/85824/2012 and CEECIND2017, SFRH/BD/110274/2015, SFRH/BPD/64905/2009, respectively
RNA editing in inflorescences of wild grapevine unveils association to sex and development
RNA editing challenges the central dogma of molecular biology, by modifying the genetic
information at the transcription level. Recent reports, suggesting increased levels of RNA
editing in plants, raised questions on the nature and dynamics of such events during
development. We here report the occurrence of distinct RNA editing patterns in wild Vitis
flowers during development, with twelve possible RNA editing modifications observed
for the first time in plants. RNA editing events are gender and developmental stage
specific, identical in subsequent years of this perennial species and with distinct
nucleotide frequencies neighboring editing sites on the 5' and 3' flanks. The
transcriptome dynamics unveils a new regulatory layer responsible for gender plasticity
enhancement or underling dioecy evolution in Vitis
The dynamics of flower development in Castanea sativa Mill
The sweet chestnut tree (Castanea sativa Mill.) is one of the most significant Mediterranean tree species, being an important natural resource for the wood and fruit industries. It is a monoecious species, presenting unisexual male catkins and bisexual catkins, with the latter having distinct male and female flowers. Despite the importance of the sweet chestnut tree, little is known regarding the molecular mechanisms involved in the determination of sexual organ identity. Thus, the study of how the different flowers of C. sativa develop is fundamental to understand the reproductive success of this species and the impact of flower phenology on its productivity. In this study, a C. sativa de novo transcriptome was assembled and the homologous genes to those of the ABCDE model for floral organ identity were identified. Expression analysis showed that the C. sativa B- and C-class genes are differentially expressed in the male flowers and female flowers. Yeast two-hybrid analysis also suggested that changes in the canonical ABCDE protein–protein interactions may underlie the mechanisms necessary to the development of separate male and female flowers, as reported for the monoecious Fagaceae Quercus suber. The results here depicted constitute a step towards the understanding of the molecular mechanisms involved in unisexual flower development in C. sativa, also suggesting that the ABCDE model for flower organ identity may be molecularly conserved in the predominantly monoecious Fagaceae family.This work was funded by FCT/COMPETE/FEDER with the project grant POCI-01-0145-
FEDER-027980/PTDC/ASP-SIL/27980/2017—“FlowerCAST—Characterisation of genetic and environmental determinants involved in reproductive development of Castanea sativa”. A.T.A. and S.A.
were supported by FCT with PhD grants (ref. SFRH/BD/136834/2018 and SFRH/BD/146660/2019,
respectively)
Polyploidization as a Retraction Force in Plant Genome Evolution: Sequence Rearrangements in Triticale
BACKGROUND: Polyploidization is a major evolutionary process in plants where hybridization and chromosome doubling induce enormous genomic stress and can generate genetic and epigenetic modifications. However, proper evaluation of DNA sequence restructuring events and the precise characterization of sequences involved are still sparse. METHODOLOGY/PRINCIPAL FINDINGS: Inter Retrotransposons Amplified Polymorphism (IRAP), Retrotransposons Microsatellite Amplified Polymorphism (REMAP) and Inter Simple Sequence Repeat (ISSR) largely confirmed the absence of any intraspecific variation in wheat, rye and triticale. The comparative analysis of banding profiles between wheat and rye inbred lines revealed 34% of monomorphic (common to both parental species) bands for the ten different primer combinations used. The analysis of triticale plants uncovered nearly 51% of rearranged bands in the polyploid, being the majority of these modifications, due to the loss of rye bands (83%). Sequence analysis of rye fragments absent in triticale revealed for instance homology with hydroxyproline-rich glycoproteins (HRGP), a protein that belongs to a major family of inducible defence response proteins. Conversely, a wheat-specific band absent in triticale comprises a nested structure of copia-like retrotransposons elements, namely Claudia and Barbara. Sequencing of a polyploid-specific band (absent in both parents) revealed a microsatellite related sequence. Cytological studies using Fluorescent In Situ Hybridization (FISH) with REMAP products revealed a widespread distribution of retrotransposon and/or microsatellite flanking sequences on rye chromosomes, with a preferential accumulation in heterochromatic sub-telomeric domains. CONCLUSIONS/SIGNIFICANCE: Here, we used PCR-based molecular marker techniques involving retrotransposons and microsatellites to uncover polyploidization induced genetic restructuring in triticale. Sequence analysis of rearranged genomic fragments either from rye or wheat origin showed these to be retrotransposon-related as well as coding sequences. Further FISH analysis revealed possible chromosome hotspots for sequence rearrangements. The role of chromatin condensation on the origin of genomic rearrangements mediated by polyploidization in triticale is also discussed