Plant Genetic Bases Associated With Microbiota Descriptors Shed Light Into a Novel Holobiont Generalist Genes Theory

Plants as animals are associated with a cortege of microbes influencing their health, fitness and evolution. Scientists refer to all living organisms as holobionts, complex genetic units that coevolve simultaneously. This is what has been recently proposed as the hologenome theory. This exciting theory has important implications on animal and plant health; however, it still needs consistent proof to be validated. Indeed, holobionts are still poorly studied in their natural habitats where coevolution processes occur. Compared to animals, wild plant populations are an excellent model to explore the hologenome theory. These sessile holobionts have coevolved with their microbiota for decades, and natural selection and adaptive processes acting on wild plants are likely to regulate the plant-microbe interactions. Here, we conducted a microbiota survey, plant genome sequencing and genome-environmental analysis (GEA) of 26 natural populations of the plant species Brassica rapa. We collected plants over two seasons in Italy and France and analysed the root and rhizosphere microbiota. When conducting GEA, we evidenced neat peaks of association correlating with both fungal and bacterial microbiota. Surprisingly, we found 13 common genes between fungal and bacterial diversity descriptors that we referred to under the name of holobiont generalist genes (HGGs)

Combined cytogenetic and molecular methods for taxonomic verification and description of Brassica populations deriving from different origins

Agriculture faces great challenges to overcome global warming and improve system sustainability, requiring access to novel genetic diversity. So far, wild populations and local landraces remain poorly explored. This is notably the case for the two diploid species, Brassica oleracea L. (CC, 2n=2x=18) and B. rapa L. (AA, 2n=2x=20). In order to explore the genetic diversity in both species, we have collected populations in their centre of origin, the Mediterranean basin, on a large contrasting climatic and soil gradient from northern Europe to southern sub-Saharan regions. In these areas, we also collected 14 populations belonging to five B. oleracea closely related species. Our objective was to ensure the absence of species misidentification at the seedling stage among the populations collected and to describe thereafter their origins. We combined flow cytometry, sequencing of a species-specific chloroplast genomic region, as well as cytogenetic analyses in case of unexpected results for taxonomic verification. Out of the 112 B. oleracea and 154 B. rapa populations collected, 103 and 146, respectively, presented a good germination rate and eighteen populations were misidentified. The most frequent mistake was the confusion of these diploid species with B. napus. Additionally for B. rapa, two autotetraploid populations were observed. Habitats of the collected and confirmed wild populations and landraces are described in this study. The unique plant material described here will serve to investigate the genomic regions involved in adaptation to climate and microbiota within the framework of the H2020 Prima project ‘BrasExplor’

Positional Cloning of “Lisch-like”, a Candidate Modifier of Susceptibility to Type 2 Diabetes in Mice

In 404 Lepob/ob F2 progeny of a C57BL/6J (B6) x DBA/2J (DBA) intercross, we mapped a DBA-related quantitative trait locus (QTL) to distal Chr1 at 169.6 Mb, centered about D1Mit110, for diabetes-related phenotypes that included blood glucose, HbA1c, and pancreatic islet histology. The interval was refined to 1.8 Mb in a series of B6.DBA congenic/subcongenic lines also segregating for Lepob. The phenotypes of B6.DBA congenic mice include reduced β-cell replication rates accompanied by reduced β-cell mass, reduced insulin/glucose ratio in blood, reduced glucose tolerance, and persistent mild hypoinsulinemic hyperglycemia. Nucleotide sequence and expression analysis of 14 genes in this interval identified a predicted gene that we have designated “Lisch-like” (Ll) as the most likely candidate. The gene spans 62.7 kb on Chr1qH2.3, encoding a 10-exon, 646–amino acid polypeptide, homologous to Lsr on Chr7qB1 and to Ildr1 on Chr16qB3. The largest isoform of Ll is predicted to be a transmembrane molecule with an immunoglobulin-like extracellular domain and a serine/threonine-rich intracellular domain that contains a 14-3-3 binding domain. Morpholino knockdown of the zebrafish paralog of Ll resulted in a generalized delay in endodermal development in the gut region and dispersion of insulin-positive cells. Mice segregating for an ENU-induced null allele of Ll have phenotypes comparable to the B.D congenic lines. The human ortholog, C1orf32, is in the middle of a 30-Mb region of Chr1q23-25 that has been repeatedly associated with type 2 diabetes

Effects of temperature on R1m6-mediated resistance to Leptosphaeria maculans in Brassica napus

Near-isogenic lines of Brassica napus lines with/without the resistance gene Rlm6 were used to investigate the effects of temperature on Rlm6-mediated resistance to Leptosphaeria maculans. Leaves were inoculated with ascospores of L. maculans carrying the corresponding avirulence gene AvrLm6. Inoculated plants were incubated at 15°C or 25°C. DarmorMX (with Rlm6) was resistant to L. maculans at 15°C but susceptible at 25°C, and Darmor (without Rlm6) was susceptible at both temperatures. On Darmor, large grey leaf lesions developed at both 15 and 25°C. On DarmorMX, small dark necrotic spots were produced at 15°C but large grey lesions were produced at 25°C. The incubation period of L. maculans (from inoculation to the appearance of first lesion) was longer on DarmorMX than on Darmor. The infection efficiency (number of lesions resulting from inoculation with 100 ascospores) was greater on Darmor than on DarmorMX at 15 and 25°C. Further characterisation of the Rlm6-mediated resistance showed that the resistance was effective at 25°C if inoculated plants were pre-incubated at 15°C for 5 days before moving them to 25°C, but not if inoculated plants were pre-incubated at 15°C for only 2 days before moving them to 25°C. We conclude that temperature affects Rlm6-mediated resistance to L. maculans in B. napus leaves

Effects of temperature on R1m6-mediated resistance to Leptosphaeria maculans in Brassica napus

Near-isogenic lines of Brassica napus lines with/without the resistance gene Rlm6 were used to investigate the effects of temperature on Rlm6-mediated resistance to Leptosphaeria maculans. Leaves were inoculated with ascospores of L. maculans carrying the corresponding avirulence gene AvrLm6. Inoculated plants were incubated at 15°C or 25°C. DarmorMX (with Rlm6) was resistant to L. maculans at 15°C but susceptible at 25°C, and Darmor (without Rlm6) was susceptible at both temperatures. On Darmor, large grey leaf lesions developed at both 15 and 25°C. On DarmorMX, small dark necrotic spots were produced at 15°C but large grey lesions were produced at 25°C. The incubation period of L. maculans (from inoculation to the appearance of first lesion) was longer on DarmorMX than on Darmor. The infection efficiency (number of lesions resulting from inoculation with 100 ascospores) was greater on Darmor than on DarmorMX at 15 and 25°C. Further characterisation of the Rlm6-mediated resistance showed that the resistance was effective at 25°C if inoculated plants were pre-incubated at 15°C for 5 days before moving them to 25°C, but not if inoculated plants were pre-incubated at 15°C for only 2 days before moving them to 25°C. We conclude that temperature affects Rlm6-mediated resistance to L. maculans in B. napus leaves