Vera Tavares de Campos Carneiro

Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha

BackgroundBrachiaria 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.ResultsEight 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.ConclusionIn 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.

Duplication of the chromosome number of diploid Brachiaria brizantha plants using colchicine

Abstract Some species of Brachiaria, generally tetraploid apomictic varieties, have become important forage grasses in the tropics. Breeding of Brachiaria depends on compatibility with the available apomitic tretraploid cultivars. This paper describes a procedure for chromosome duplication of two Bracharia brizantha diploid sexual accessions, using colchicine treatment of basal segments of in-vitro-grown plants. Explants were cultured on a medium containing 1 mg/l naphthaleneacetic acid, 3 mg/l kinetin and 0.01% colchicine for 48 h and transferred to the same medium without colchicine until shoot regeneration occurred. Regenerated plants were screened by flow cytometry, and chromosome number duplication was confirmed by cytological analysis of root tips.

Implication of 5′-flanking sequence elements in expression of a plant tRNALeugene

A comparison of 5′-flanking sequences from 68 different nuclear plant tRNA genes was analyzed to find consensus sequences. Three conserved features stood out, all of which are present in the tRNALeu gene used in this study: (1) a high proportion of A and T residues upstream of all tRNA genes; (2) a region of low duplex stability about 30–35 bp before the coding sequence, often containing a TATA-box like motif; (3) a CAA triplet in the region of the presumed transcription start. The effect of replacement of the AT-rich upstream sequences with GC-rich sequences or unrelated AT-rich sequences was tested by progressive deletions and by inserting randomly cloned sequences upstream of the tRNA gene. GC-rich 5′-flanking sequences were found to be generally incompatible with high levels of expression. The TATA-box like motifs and the CAA triplet were removed or altered by deletion or directed mutagenesis. Mutation of the CAA triplet significantly decreased expression of the tRNALeu gene, suggesting that this CAA triplet is important for transcription efficiency, but mutation or elimination of the TATA-box like motifs generally had little effect. The presence or absence of each of these features in tRNA genes from other organisms is discussed; there are clear and interesting differences between plant tRNA genes and those of yeast and mammals.

Expressed sequence-tag analysis of ovaries of Brachiaria brizantha reveals genes associated with the early steps of embryo sac differentiation of apomictic plants

In apomixis, asexual mode of plant reproduction through seeds, an unreduced megagametophyte is formed due to circumvented or altered meiosis. The embryo develops autonomously from the unreduced egg cell, independently of fertilization. Brachiaria is a genus of tropical forage grasses that reproduces sexually or by apomixis. A limited number of studies have reported the sequencing of apomixis-related genes and a few Brachiaria sequences have been deposited at genebank databases. This work shows sequencing and expression analyses of expressed sequence-tags (ESTs) of Brachiaria genus and points to transcripts from ovaries with preferential expression at megasporogenesis in apomictic plants. From the 11 differentially expressed sequences from immature ovaries of sexual and apomictic Brachiaria brizantha obtained from macroarray analysis, 9 were preferentially detected in ovaries of apomicts, as confirmed by RT-qPCR. A putative involvement in early steps of Panicum-type embryo sac differentiation of four sequences from B. brizantha ovaries: BbrizHelic, BbrizRan, BbrizSec13 and BbrizSti1 is suggested. Two of these, BbrizSti1 and BbrizHelic, with similarity to a gene coding to stress induced protein and a helicase, respectively, are preferentially expressed in the early stages of apomictic ovaries development, especially in the nucellus, in a stage previous to the differentiation of aposporous initials, as verified by in situ hybridization.

Evidence of sexuality in induced tetraploids of Brachiaria brizantha (Poaceae)

Difficulties in obtaining new breeding lines of Brachiaria (Trin.) Griseb., an important forage grass in Brazil, are mostly related to differences in ploidy among the accessions, and to apomixis, an asexual mode of reproduction. Usually, sexual accessions are diploid while apomicts are polyploid. Induced tetraploids of Brachiaria brizantha (A. Rich.) Stapf have been successfully obtained and this paper presents the results of a study of their reproductive modes and fertility. Despite frequent meiotic aberrations during microspore development, the induced tetraploids produced viable pollen and produced progeny after controlled self-pollination. Similarly to the original diploid sexual progenitor, embryo sacs of the Polygonum type with confirmed meiotic origin were present in the induced tetraploids suggesting chromosome doubling did not alter the reproductive mode. The embryo sac of the Polygonum type was also observed in progenies obtained after self and open pollination. Nevertheless, embryo sacs of the Polygonum and the Panicum types within the same ovule were observed in some progenies obtained after open pollination, probably having resulted from hybridization with tetraploid, apomictic plants. Indeed, the compatibility of the progeny with tetraploid, apomictic B. brizantha was confirmed by the formation of mature caryopses after controlled pollination. Evidence is presented that the induced tetraploids and their progeny are sexual plants and that they are compatible with natural tetraploid B. brizantha. The induced tetraploids will be useful for analyses of apomictic inheritance as well as in the development of sexual tetraploid lines in Brachiaria breeding programs.

Physical mapping of rDNA genes corroborates allopolyploid origin in apomictic Brachiaria brizantha

Brachiaria (Trin.) Griseb belongs to the family Poaceae, and within the genus, apomixis or sexuality is present in different accessions of the same species. The majority of Brachiaria species are polyploid and apomictic, making strategies for crop improvement by breeding very intricate. In spite of the high frequency of apomictic polyploids, the relationship of polyploidy and hybridization with apomixis in Brachiaria is still unclear. Further analysis requires detailed knowledge regarding the genomic composition of the polyploids. The present work introduces the use of fluorescent in situ hybridization (FISH) into cytogenetic analysis of Brachiaria. Physical mapping of heterologous rDNA sequences, associated with conventional karyotyping of the B. brizantha diploid sexual (BRA 002747) and the tetraploid apomictic (BRA000591) accessions, provided evidence of the latter being of allopolyploid origin. Based on our results and on previous knowledge on apomixis in B. brizantha, we suggest that the origin of apomixis was probably a consequence of hybridization.

Plant regeneration from embryogenic callus and cell suspensions of Brachiaria brizantha

In monocots, in vitro plant regeneration can be obtained through somatic embryogenesis (Vasil and Vasil 1980, 1982; Vasil 2005). Evidence of more than one morphogenic response in the same explant, under the control of different auxin/cytokinin ratios and concentrations, was observed in species of the Poaceae family such as sorghum (Sorghum bicolor, (L.) Moench.) , minor mil le t (Paspalum scrobiculatum L.), sugar cane (Saccharum officinarum L.), and baby bamboo (Pogonatherum paniceum Lam. Hack.) (reviewed by Wang et al. 2008). Brachiaria callus has been induced using seeds as explants from Brachiaria brizantha (Hochst. ex A. Rich.) Stapf, Brachiaria decumbens Stapf, Brachiaria ruziziensis Germain et Evrard and Brachiaria dictyoneura [Brachiaria humidicola (Rendle) Schweick vr. Lanero, ex B. dictyoneura] (Tohme et al. 1996). Subsequently, using the same methodology, calli were induced from 76% of isolated embryos from seeds of B. brizantha (Lenis-Manzano 1998). The formation of somatic embryos and multiple shoots from seedling apical meristems was demonstrated for B. ruziziensis (Ishigaki et al. 2009). In this system, mature seeds were used as source of explants and cultured on a medium containing 4 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Ishigaki et al. (2012) reported that embryogenic calli were more suitable than shoot apex explants for genetic transformation of ruzigrass. B. brizantha cv. Marandu is the most important and cultivated forage grass in Brazil. It is tolerant to spittlebugs (Homoptera: Cercopidae complex, mainly genera Deois and Zulia), an economically important pest for Brachiaria (Felismino et al. 2012). This cultivar is considered as the key forage in beef cattle production in Brazil. Breeding is hindered by its apomictic reproductive mode (the production of clones of the mother plant), which reduces the possibility of being used for hybridizations (Valle and Savidan 1996). Alternatives to conventional breeding of B. brizantha would include genetic modification via transformation. However, methods of transformation and tissue culture of these plants are not yet established. The development of embryogenic calli has been reported from 73% of the B. brizantha cv. Marandu isolated seed embryos in culture and 67% of calli regenerated plants (Silveira et al. 2003). More recently, the histology of embryos isolated from the in vitro cultivated seed was shown (Lenis-Manzano et al. 2010). The formation of multiple shoots was reported in cultured basal segments from micropropagated plantlets of this cultivar (Pinheiro et al. 2000), a system that was efficient for in vitro chromosome duplication in Brachiaria. Somatic embryogenesis and organogenesis in B. brizantha is influenced by several factors such as genotype, explant type, and culture conditions (Cabral et al. 2011). A high rate of cell division in explants used for genetic transformation ofmonocots is a prerequisite for the integration of exogenous DNA (Hiei et al. 1997; Vasil 2005). Rapid cell proliferation is a characteristic of embryogenic tissue. Cell suspension cultures (CS) are well known for their cell division capacity and the possibility of developmental-stage Electronic supplementary material The online version of this article (doi:10.1007/s11627-015-9690-0) contains supplementary material, which is available to authorized users. G. B. Cabral (*) :V. T. C. Carneiro : J. P. da Silva :D. M. A. Dusi Embrapa Genetic Resources and Biotechnology, Brasília, Brazil e-mail: glaucia.cabral@embrapa.br

The MAP3K-coding QUI-GON JINN (QGJ) Gene Is Essential To The Formation of Unreduced Embryo Sacs In Paspalum

Apomixis is a clonal mode of reproduction via seeds, which results from the failure of meiosis and fertilization in the sexual female reproductive pathway. In previous transcriptomic surveys, we identified a mitogen-activated protein kinase kinase kinase (N46) displaying differential representation in florets of sexual and apomictic Paspalum notatum genotypes. Here, we retrieved and characterized the N46 full cDNA sequence from sexual and apomictic floral transcriptomes. Phylogenetic analyses showed that N46 was a member of the YODA family, which was re-named QUI-GON JINN (QGJ). Differential expression in florets of sexual and apomictic plants was confirmed by qPCR. In situ hybridization experiments revealed expression in the nucellus of aposporous plants’ ovules, which was absent in sexual plants. RNAi inhibition of QGJ expression in two apomictic genotypes resulted in significantly reduced rates of aposporous embryo sac formation, with respect to the level detected in wild type aposporous plants and transformation controls. The QGJ locus segregated independently of apospory. However, a probe derived from a related long non-coding RNA sequence (PN_LNC_QGJ) revealed RFLP bands cosegregating with the Paspalum apospory-controlling region (ACR). PN_LNC_QGJ is expressed in florets of apomictic plants only. Our results indicate that the activity of QGJ in the nucellus of apomictic plants is necessary to form non-reduced embryo sacs and that a long non-coding sequence with regulatory potential is similar to sequences located within the ACR.

Genetic transformation of Brachiaria brizantha cv. Marandu by biolistics.

Brachiaria brizantha is a forage grass well adapted to tropical areas and cultivated in millions of hectares in Brazil. The apomictic mode of reproduction in this species, in addition to differences in ploidy between sexual and apomictic plants, impairs crossbreeding. The development of a methodology to transform apomictic cultivars will provide an option to introduce agronomic important traits to B. brizantha cv. Marandu. In addition, it will open the possibility to study in vivo the function of candidate genes involved in the apomictic reproduction. The objective of this work was to evaluate peeled seeds, isolated embryo from mature seeds, embryogenic calluses and embryogenic cell suspensions, as target explant for genetic transformation via biolistics. Plasmids bearing the marker genes gus and hptII under the control of the rice actin 1 promoter (pAct1-Os) or the maize ubiquitin 1 promoter (pUbi1Zm) were used. All the target-explants used were suitable for transient gene expression after bombardment, showing gus expression and resistance to hygromycin. Using embryogenic calluses and cell suspensions as target tissues, transgenic plants were regenerated and transgenes detected.

Somatic Embryogenesis and Plant Regeneration of Brachiaria brizantha

The genus Brachiaria (Trin.) Griseb. belongs to the family Poaceae, order Poales, class Monocotyledonae. In Brachiaria brizantha (Hochst. ex A. Rich.) Stapf., embryogenic callus can be induced from seeds from apomictic plants, which results in high frequency somatic embryo development and plant regeneration. We report here a detailed protocol for callus induction from apomictic seed; followed by in vitro morphogenesis (somatic embryo and bud differentiation), plant regeneration, and acclimatization in the greenhouse. Important details regarding the positioning of seeds for callus induction and precautions to avoid endophytic contamination and the occurrence of albino plants are presented.