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Translocation of a parthenogenesis gene candidate to an alternate carrier chromosome in apomictic Brachiaria humidicola.

ABSTRACT: BACKGROUND:The apomictic reproductive mode of Brachiaria (syn. Urochloa) forage species allows breeders to faithfully propagate heterozygous genotypes through seed over multiple generations. In Brachiaria, reproductive mode segregates as single dominant locus, the apospory-specific genomic region (ASGR). The AGSR has been mapped to an area of reduced recombination on Brachiaria decumbens chromosome 5. A primer pair designed within ASGR-BABY BOOM-like (BBML), the candidate gene for the parthenogenesis component of apomixis in Pennisetum squamulatum, was diagnostic for reproductive mode in the closely related species B. ruziziensis, B. brizantha, and B. decumbens. In this study, we used a mapping population of the distantly related commercial species B. humidicola to map the ASGR and test for conservation of ASGR-BBML sequences across Brachiaria species. RESULTS:Dense genetic maps were constructed for the maternal and paternal genomes of a hexaploid (2n?=?6x?=?36) B. humidicola F1 mapping population (n?=?102) using genotyping-by-sequencing, simple sequence repeat, amplified fragment length polymorphism, and transcriptome derived single nucleotide polymorphism markers. Comparative genomics with Setaria italica provided confirmation for x?=?6 as the base chromosome number of B. humidicola. High resolution molecular karyotyping indicated that the six homologous chromosomes of the sexual female parent paired at random, whereas preferential pairing of subgenomes was observed in the apomictic male parent. Furthermore, evidence for compensated aneuploidy was found in the apomictic parent, with only five homologous linkage groups identified for chromosome 5 and seven homologous linkage groups of chromosome 6. The ASGR mapped to B. humidicola chromosome 1, a region syntenic with chromosomes 1 and 7 of S. italica. The ASGR-BBML specific PCR product cosegregated with the ASGR in the F1 mapping population, despite its location on a different carrier chromosome than B. decumbens. CONCLUSIONS:The first dense molecular maps of B. humidicola provide strong support for cytogenetic evidence indicating a base chromosome number of six in this species. Furthermore, these results show conservation of the ASGR across the Paniceae in different chromosomal backgrounds and support postulation of the ASGR-BBML as candidate genes for the parthenogenesis component of apomixis.

SUBMITTER: Worthington M

PROVIDER: S-EPMC6332668 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Translocation of a parthenogenesis gene candidate to an alternate carrier chromosome in apomictic Brachiaria humidicola.

Worthington Margaret M Ebina Masumi M Yamanaka Naoki N Heffelfinger Christopher C Quintero Constanza C Zapata Yeny Patricia YP Perez Juan Guillermo JG Selvaraj Michael M Ishitani Manabu M Duitama Jorge J de la Hoz Juan Fernando JF Rao Idupulapati I Dellaporta Stephen S Tohme Joe J Arango Jacobo J

BMC genomics 20190114 1

<h4>Background</h4>The apomictic reproductive mode of Brachiaria (syn. Urochloa) forage species allows breeders to faithfully propagate heterozygous genotypes through seed over multiple generations. In Brachiaria, reproductive mode segregates as single dominant locus, the apospory-specific genomic region (ASGR). The AGSR has been mapped to an area of reduced recombination on Brachiaria decumbens chromosome 5. A primer pair designed within ASGR-BABY BOOM-like (BBML), the candidate gene for the pa ...[more]

PMID: 30642244

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Project description:Introduction: Biological Nitrification Inhibition (BNI) is defined as the plant-mediated control of soil nitrification via the release of nitrification inhibitors. BNI of Brachiaria humidicola (syn. Urochloa humidicola) has been mainly attributed to root-exuded fusicoccane-type diterpenes, e.g., 3-epi-brachialactone. We hypothesized, however, that BNI of B. humidicola is caused by an assemblage of bioactive secondary metabolites. Methods: B. humidicola root exudates were collected hydroponically, and metabolites were isolated by semi-preparative HPLC. Chemical structures were elucidated by HRMS as well as 1D and 2D NMR spectroscopy. Nitrification inhibiting potential of isolated metabolites was evaluated by a Nitrosomonas europaea based bioassay. Results and discussion: Besides previously described brachialactone isomers and derivatives, five phenol and cinnamic acid derivatives were identified in the root exudates of B. humidicola: 2-hydroxy-3-(hydroxymethyl)benzaldehyde, vanillin, umbelliferone and both trans- and cis-2,6-dimethoxycinnamic acid. Notably, vanillin revealed a substantially higher nitrification inhibiting activity than 3-epi-brachialactone (ED50 ∼ 12.5 μg·ml-1, ED80 ∼ 20 μg·ml-1), identifying this phenolic aldehyde as novel nitrification inhibitor (NI). Furthermore, vanillin exudation rates were in the same range as 3-epi-brachialactone (1-4 μg·h-1·g-1 root DM), suggesting a substantial contribution to the overall inhibitory activity of B. humidicola root exudates. In relation to the verification of the encountered effects within soils and considering the exclusion of any detrimental impact on the soil microbiome, the biosynthetic pathway of vanillin via the precursor phenylalanine and the intermediates p-coumaric acid/ferulic acid (precursors of further phenolic NI) might constitute a promising BNI breeding target. This applies not only to Brachiaria spp., but also to crops in general, owing to the highly conserved nature of these metabolites.

Project description:BACKGROUND: Brachiaria brizantha is an important forage grass. The occurrence of both apomictic and sexual reproduction within Brachiaria makes it an interesting system for understanding the molecular pathways involved in both modes of reproduction. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to compare expression profile of target genes and, in order to obtain reliable results, it is important to have suitable reference genes. In this work, we evaluated eight potential reference genes for B. brizantha qRT-PCR experiments, isolated from cDNA ovary libraries. Vegetative and reproductive tissues of apomictic and sexual B. brizantha were tested to validate the reference genes, including the female gametophyte, where differences in the expression profile between sexual and apomictic plants must occur. RESULTS: Eight genes were selected from a cDNA library of ovaries of B. brizantha considering the similarity to reference genes: EF1 (elongation factor 1 alpha), E1F4A (eukaryotic initiation factor 4A), GAPDH (glucose-6-phosphate dehydrogenase), GDP (glyceroldehyde-3-phosphate dehydrogenase), SUCOA (succinyl-CoA ligase), TUB (tubulin), UBCE (ubiquitin conjugating enzyme), UBI (ubiquitin). For the analysis, total RNA was extracted from 22 samples and raw Ct data after qRT-PCR reaction was analyzed for primer efficiency and for an overall analysis of Ct range among the different samples. Elongation factor 1 alpha showed the highest expression levels, whereas succinyl-CoA ligase showed the lowest within the chosen set of samples. GeNorm application was used for evaluation of the best reference genes, and according to that, the least stable genes, with the highest M values were tubulin and succinyl-CoA ligase and the most stable ones, with the lowest M values were elongation factor 1 alpha and ubiquitin conjugating enzyme, when both reproductive and vegetative samples were tested. For ovaries and spikelets of both sexual and apomictic B. brizantha the genes with the lowest M values were BbrizUBCE, BbrizE1F4A and BbrizEF1. CONCLUSION: In total, eight genes belonging to different cellular processes were tested. Out of them, BbrizTUB was the less stable while BbrizEF1 followed by BbrizUBCE were the more stable genes considering male and female reproductive tissues, spikelets, roots and leaves. Regarding the best reference genes for ovary tissues, where apomictic and sexual reproduction must occur, the best reference genes were BbrizUBCE, BbrizE1F4A and BbrizEF1. Our results provide crucial information for transcriptional analysis in the Brachiaria ssp, helping to improve the quality of gene expression data in these species, which constitute an excellent plant system for the study of apomixis.

Project description:Some flowering plants exhibit a phenomenon called apomixis, defined as the ability to produce seeds asexually and with the same genetic constitution as the mother plant. The existence of a genetic basis for apomixis is evident, but the molecular mechanisms that underlie it remain unclear. The search for M-bM-^@M-^\the master apomixis geneM-bM-^@M-^] had led to the isolation of diverse candidate transcripts. However, neither of them could be clearly confirmed. The techniques employed so far have been based on differential-display PCR and have not allowed detecting transcripts with low levels of expression or low differential expression. For all these reasons, in this study we employed cDNA microarrays combined with subtractive libraries in order to isolate transcripts with differential expression between sexual and apomictic plants of the genus Brachiaria. We used ovaries corresponding to two developmental stages: i) stage I, appearing of initial aposporic cell in apomictic plants; and ii) stage II, degeneration of meiotic-derived tetrad and establishment of diploid embryo sac in apomictic plants. Subtractive libraries revealed some differential bands, which were spotted over glass slides together with clones from non-subtractive libraries, and with amplicons associated to apomixis obtained using degenerate PCR. A total of 26 distinct sequences showed differential expression, only 1 of them was overexpressed in apomictic plants, and the majority was related to stage I. Bioinformatic analyses confirmed meiosis-related roles for some of the genes, a result consistent with a higher level of expression in sexual ovaries. Nevertheless, for some sequences a clear function could not be assigned. Some of them appear to be related to the polyploid stage of apomictic plants, in one hand, and to putative transcription factors, on the other hand. The implications of these results under a molecular model of apomeiosis are discussed. We employed RNA from ovaries from sexual plants as a control, and RNA from ovaries of apomictic plants as experiment. Four different sexual and four apomictic genotypes were employed. A sexual genotype was compared against a given apomictic genotype, for a total of 4 comparisions. Each comparision were performed ifor two developmental stages, for a total of eight comparisions. In each comparision, a dye swap was done.

Project description:The tropical forage grass Brachiaria humidicola (Bh) suppresses the activity of soil nitrifiers through biological nitrification inhibition (BNI). As a result, nitrate ( NO 3 - ) formation and leaching are reduced which is also expected to tighten the soil nitrogen (N) cycle. However, the beneficial relationship between reduced NO 3 - losses and enhanced N uptake due to BNI has not been experimentally demonstrated yet. Nitrification discriminates against the 15N isotope and leads to 15N depleted NO 3 - , but 15N enriched NH 4 + in soils. Leaching of 15N depleted NO 3 - enriches the residual N pool in the soil with 15N. We hypothesized that altered nitrification and NO 3 - leaching due to diverging BNI magnitudes in contrasting Bh genotypes influence soil 15N natural abundance (δ15N), which in turn is reflected in distinct δ15N in Bh shoot biomass. Consequently, high BNI was expected to be reflected in low plant δ15N of Bh. It was our objective to investigate under controlled conditions the link between shoot value of δ15N in several Bh genotypes and leached NO 3 - amounts and shoot N uptake. Additionally, plant 15N and N% was monitored among a wide range of Bh genotypes with contrasting BNI potentials in field plots for 3 years. We measured leaf δ15N of young leaves (regrown after cutback) of Bh and combined it with nitrification rates (NRs) of incubated soil to test whether there is a direct relationship between plant δ15N and BNI. Increased leached NO 3 - was positively correlated with higher δ15N in Bh, whereas the correlation between shoot N uptake and shoot δ15N was inverse. Field cultivation of a wide range of Bh genotypes over 3 years decreased NRs in incubated soil, while shoot δ15N declined and shoot N% increased over time. Leaf δ15N of Bh genotypes correlated positively with NRs of incubated soil. It was concluded that decreasing plant δ15N of Bh genotypes over time reflects the long-term effect of BNI as linked to lower NO 3 - formation and reduced NO 3 - leaching. Accordingly, a low δ15N in Bh shoot tissue verified its potential as indicator of high BNI activity of Bh genotypes.

Dataset Information

Translocation of a parthenogenesis gene candidate to an alternate carrier chromosome in apomictic Brachiaria humidicola.

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Translocation of a parthenogenesis gene candidate to an alternate carrier chromosome in apomictic Brachiaria humidicola.

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