Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Bulk segregant sequencing (Bulk-Seq) analysis to identify paternal suppressors of medea in Arabidopsis thaliana

ABSTRACT: Arabidopsis thaliana seeds that maternally inherit a medea (mea) mutant allele abort before completing embryogenesis. However, mea seeds can be rescued by pollen from several natural ecotypes of A. thaliana, including the Cape Verdian accession Cvi-0. We developed a method for the mapping of parent-of-origin effects using whole-genome sequencing of segregant bulks. The strategy is to create an F2 population that contains one set of chromosomes from the maternal parent (mea, in a Ler background) but inherits two segregating sets (Ler and Cvi-0) from the other parent. The two paternally segregating sets have opposite effects in mea penetrance: Ler fathers allow full mea seed abortion, while Cvi fathers rescue 90% of medea seeds. Therefore, the two segregating paternal sets are not equally transmitted to the next generation. DNA extracted from pools of viable F2 seedlings was sequenced on an Illumina HiSeq 2000 platform, mapped to the reference TAIR10 A. thaliana genome and the ratio between Ler and Cvi-0 SNPs used to identify chromosomal regions enriched in Cvi-0 sequences. As a control, DNA pools extracted from crosses between a wild-type Ler mother and a hybrid Cvi-0:Ler father were also sequenced. Three biological replicates were made for each pool. Ler-1 x Ler-1:Cvi-0 hybrid viable seedlings pools (each is a biological replicate): WT_pool_1, WT_pool_2, WT_pool_3 mea-1/mea-1; MEA-GR x Ler-1:Cvi-0 hybrid viable seedlings pool (each is a biological replicate): mea_pool_1, mea_pool_2, mea_pool_3

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Nuno Pires

PROVIDER: E-MTAB-5196 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Quantitative Genetics Identifies Cryptic Genetic Variation Involved in the Paternal Regulation of Seed Development.

Pires Nuno D ND Bemer Marian M Müller Lena M LM Baroux Célia C Spillane Charles C Grossniklaus Ueli U

PLoS genetics 20160126 1

Embryonic development requires a correct balancing of maternal and paternal genetic information. This balance is mediated by genomic imprinting, an epigenetic mechanism that leads to parent-of-origin-dependent gene expression. The parental conflict (or kinship) theory proposes that imprinting can evolve due to a conflict between maternal and paternal alleles over resource allocation during seed development. One assumption of this theory is that paternal alleles can regulate seed growth; however, ...[more]

PMID: 26811909

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Project description:We carried out a five-generation selection experiment in which two Arabidopsis accessions (Ler and Cvi) and 17 Recombinant Inbred Lines (RILs) derived from them were subjected in 24 replicated populations to four levels of micro-landscape fragmentation and two levels of disturbance, factorially crossed with each other to yield 8 experimental selection regimes. We used 19 lines (including the two parental Ler and Cvi) in generation 1 of our experiment, nine of which contained the erecta mutation and ten of which did not. Lines expressing the erecta mutation are short and compact and are likely to disperse seeds over shorter distances. Both the non-erecta and the erecta lines spanned the full available range of seed sizes from the original 162 RILs in the collection. We set up 24 experimental landscapes consisting of 2, 4, 8, or 16 patches where the total area was constrained to be equal. Plants that germinated outside the designated suitable patches were removed. In half of the landscapes which we termed static the top 2-3 mm of seeds and soil were removed from the surface of the suitable patches after 8 weeks of growth and reproduction. In the other half of the landscapes which we termed dynamic a number of Petri dishes, equivalent in size and number to the existing suitable patches, were distributed around the landscape at random, to catch dispersing seeds. The seeds collected by these two methods were cold-treated for one week while all patches were replenished with fresh substrate. Seeds were then returned to the surface of the new patches at the beginning of the next generation. Thus, in dynamic landscapes, seeds must disperse to have any chance of entering the next generation, while in static landscapes, only seeds that do not disperse have any chance of further propagation. After five generations of such selection, a single pod from approximately 70 individuals from each landscape was removed. Single individuals derived from such pods were reared under standardized conditions to measure several phenotypic characters, such as seed mass, height, presence of the erecta mutation, flowering time etc. Clear phenotypic differences are apparent between the different landscapes. However, we have no information as to the underlying genes, which might have been selected or the epigenetic modifications that might have occurred. Material for DNA extraction from each population (? 50 plants/population) was collected for subsequent genetic analyses. The allele frequencies of Ler and Cvi alleles was determined at the genome-wide level using ATH1 Affymetrix GeneChips® (each in quadruplicate = 8 parental microarrays). 26638 single feature polymorphisms (SFPs) between these two accessions could be detected. The relative contribution of Ler and Cvi alleles in a population at each locus, for which a single feature polymorphism can be detected, was assessed. We then analyzed the DNA isolated from the experimental populations (24 landscapes of generation 5 = 24 microarrays & original populations in triplicate = 3 microarrays) and determine the relative contributions of Ler and Cvi alleles for the 26638 identified SFPs (i.e. allele frequency shifts).

Project description:F1 hybrids can outperform their parents in yield and vegetative biomass, features of hybrid vigor which form the basis of the hybrid seed industry. The yield advantage of the F1 is lost in the F2 and subsequent generations. In Arabidopsis, from F2 plants which have a F1like phenotype, we have by recurrent selection produced pure breeding F5/F6 linesHybrid Mimics, in which the characteristics of the F1 Hybrid are stabilized. These Hybrid Mimic lines, like the F1 Hybrid, have larger leaves than the parent plant, the leaves having increased photosynthetic cell numbers, and in some lines increased size of cells, suggesting an increased supply of photosynthate. A comparison of the differentially expressed genes in the F1 Hybrid with those of eight Hybrid Mimic lines has identified metabolic pathways altered in both; these pathways include down regulation of defense response pathways and altered abiotic response pathways. F6 Hybrid Mimic lines are mostly homozygous at each locus in the genome yet retain the large F1-like phenotype. Many alleles in the F6 plants, when they are homozygous, have expression levels different to the level in the parent. We consider this altered expression to be a consequence of trans-regulation of genes from one parent by genes from the other parent. Transregulation could also arise from epigenetic modifications in the F1. The pure breeding Hybrid Mimics have been valuable in probing the mechanisms of hybrid vigor and may also prove to be useful hybrid vigor equivalents in agriculture. Plant Materials: Seeds were sterilized and sown onto plates containing MSN medium (MS salts supplemented with 1% (w/v) sucrose, pH7 with KOH, 0.6% wt/vol Noble agar). After 2 days at 4°C, the plates were transferred to a growth room with conditions of 22°C/18°C (day/night) and 16h light/8h dark cycle under Philips Cool Daylight TLD 58W/840 fluorescent tubes providing a photosynthetic photon flux density of 130 - 150 umol photons m-2·s-1 as measured using a quantum meter (Model MQ-200 calibrated for electric light source, Apogee). At 18 days after sowing (DAS), the plants were transferred to soil and grown to the reproductive stage in a growth room with controlled light conditions (OSRAM L 36W/865 LumLux Daylight, 130 - 150 umol photons m-2·s-1). To minimize the edge effects, the positioning of both plates and trays on the shelves was rotated every two days. All the plants were grown under the condition described above unless specified. Plant sample preparation and RNA extraction: For the transcriptomes of 15 DAS plants, the aerial tissues of 15 day seedlings were sampled. For the transcriptomes of plants at 28 DAS, the four largest leaves of 28 day plants from the parental lines (C24, Ler), the reciprocal Hybrids and eight F4 Hybrid Mimic lines were harvested. Each sample comprised a pool of five plants. Two biological replicates of each sample were sequenced. Total RNA was isolated using QIAGEN RNeasy MiniKitTM following the product instructions. To eliminate variation in experimental conditions in different positions in the growth room, nine F4 plant lines were divided into two batches to ensure plants were grown on the same shelves in each experiment. Parental lines C24 and Ler and the F1 Hybrid from which the F4 lines derived from were grown under the same conditions in each experiment as controls. The first batch including samples C24 (RepA and RepB), Ler (RepA and RepB), C24 x Ler F1, F4-L1-1, F4-L1-2, F4-L2-1, F4-L2-2 and F4-S-1 were grown under the condition describe above, we also sampled the five smallest C24 seedlings and five smallest Ler seedlings among the population (n = 50); the mRNA sequencing was performed by the Australian Genome Research Facility (AGRF). The second batch including C24 (RepC and RepD), Ler (RepC and RepD), Ler x C24 F1 Hybrid, F4-L3-1, F4-L3-2, F4-L4-1 and F4-L4-2 were grown in the same light intensity as above but under different light tubes (SYLVANIA PREMIUM extra FL36W/865 Super Daylight deluxe light tube). For the transcriptome of the two F6 Hybrid Mimic lines at 15 DAS, C24, Ler, Ler x C24 F1 Hybrids and three siblings from each F6 line (L3-1-1-2 and L4-2-1-2) were grown on MS medium (MS salts supplemented with 1% (w/v) sucrose, pH 5.7 with KOH, add 0.6% wt/vol agar) with controlled light conditions (SYLVANIA PREMIUM extra FL36W/865 Super Daylight deluxe, 130 - 150 umol photons m-2Â·s-1). The mRNA sequencing service was provided by AGRF on the Illumina platform, 100bp paired ends.

Dataset Information

Bulk segregant sequencing (Bulk-Seq) analysis to identify paternal suppressors of medea in Arabidopsis thaliana

Publications

Quantitative Genetics Identifies Cryptic Genetic Variation Involved in the Paternal Regulation of Seed Development.

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