Epigenetic Marks Associated to the Study of Nucleolar Dominance in Urochloa P. Beauv.

The genus Urochloa P. Beauv. presents a prominent role in the tropical agricultural scenario being composed of species with different ploidy levels. Studies on the genomic relationship within this genus as well as specific analysis involving epigenetic marks are limited. The aim of the present study was to identify the cytosine methylation (5-mCyt) and histone H3 lysine 9 dimethylation (H3k9me2) in the different modulations of 45S ribosomal DNA (rDNA) sites in interphase nuclei and to associate these results with gene expression analysis in Urochloa ruziziensis (2n = 4x = 36), Urochloa brizantha cv. Marandu (2n = 4x = 36), and their respective interspecific hybrid H1863 (2n = 4x = 36). Immunolocalization techniques were performed in combination with Fluorescence in situ hybridization (FISH) for the location of the 45S rDNA sites. Predominantly, we observed intra- and perinucleolar sites, mostly hypomethylated and/or hyper/hypomethylated, decondensed or partially condensed. The gene expression analysis was performed qualitatively through the conventional PCR using complementary DNA and confirmed by the RT-qPCR technique and primers designed for the ITS-1 region of U. brizantha and U. ruziziensis. The molecular analyses performed on leaves showed that there is dominance of U. brizantha 45S rDNA gene expression on U. ruziziensis in the H1863 hybrid. In roots, the analyses showed that the 45S rDNA genes of the two parents are expressed in the hybrid genome. Thus, it is plausible to infer a tissue-specific nuclear dominance model in which the pattern of hypermethylated cytosine sites with heterochromatic marks and, therefore, silenced were mostly inherited from U. ruziziensis, whereas the rDNA originated from U. brizantha was characterized by cytosine and H3k9 hypomethylation.

     

Aluminum tolerance measured by root growth and mucilage protection in Urochloa brizantha and Urochloa decumbens

Aluminum toxicity on root systems was analyzed through comparing root growth and evaluating the protective function of mucilage in Urochloa decumbens and Urochloa brizantha. Seedlings were grown in a solution with different concentrations of AlCl₃ and with mucilage removed or present. The root elongation rate, total length, number of roots and presence of aluminum at the root apex were measured. Root development was inhibited by aluminum and the elongation rate was maintained without any difference between the two species. A significant reduction in root length was found in U. brizantha. Aluminum did not influence root branching in either species and accumulated mostly in the rhizosphere of U. brizantha, where the mucilage has less of a protective function. The greatest aluminum tolerance was found in U. decumbens, observed through maintenance of total root system growth from lower to higher aluminum toxicity.     

Diverged subpopulations in tropical Urochloa (Brachiaria) forage species indicate a role for facultative apomixis and varying ploidy in their population structure and evolution

Background Urochloa (syn. Brachiaria) is a genus of tropical grasses sown as forage feedstock, particularly in marginal soils. Here we aimed to clarify the genetic diversity and population structure in Urochloa species to understand better how population evolution relates to ploidy level and occurrence of apomictic reproduction.MethodsWe explored the genetic diversity of 111 accessions from the five Urochloa species used to develop commercial cultivars. These accessions were conserved from wild materials collected at their centre of origin in Africa, and they tentatively represent the complete Urochloa gene pool used in breeding programmes. We used RNA-sequencing to generate 1.1 million single nucleotide polymorphism loci. We employed genetic admixture, principal component and phylogenetic analyses to define subpopulations.ResultsWe observed three highly differentiated subpopulations in U. brizantha, which were unrelated to ploidy: one intermixed with U. decumbens, and two diverged from the former and the other species in the complex. We also observed two subpopulations in U. humidicola, unrelated to ploidy; one subpopulation had fewer accessions but included the only characterized sexual accession in the species. Our results also supported a division of U. decumbens between diploids and polyploids, and no subpopulations within U. ruziziensis and U. maxima.ConclusionsPolyploid U. decumbens are more closely related to polyploid U. brizantha than to diploid U. decumbens, which supports the divergence of both polyploid groups from a common tetraploid ancestor and provides evidence for the hybridization barrier of ploidy. The three differentiated subpopulations of apomictic polyploid U. brizantha accessions constitute diverged ecotypes, which can probably be utilized in hybrid breeding. Subpopulations were not observed in non-apomictic U. ruziziensis. Sexual Urochloa polyploids were not found (U. brizantha, U. decumbens) or were limited to small subpopulations (U. humidicola). The subpopulation structure observed in the Urochloa sexual–apomictic multiploidy complexes supports geographical parthenogenesis, where the polyploid genotypes exploit the evolutionary advantage of apomixis, i.e. uniparental reproduction and clonality, to occupy extensive geographical areas.     

Identification of genome constitution of Oryza malampuzhaensis, O. minuta,and O. punctata by multicolor genomic in situ hybridization

Multicolor genomic in situ hybridization (McGISH) was applied to identify the genomic constitution of three tetraploid species (2n = 4x = 48) in the Oryza officinalis complex of the genus Oryza, i.e. Oryza malam-puzhaensis, Oryza minuta, and Oryza punctata. The genomic probes used were from three diploids, i.e. Oryza officinalis (CC), Oryza eichingeri (CC) and Oryza punctata (BB), respectively. The results indicated that all three tetraploids are allotetraploid with the genomic constitution of BBCC, and among them the genome constitution of O. malampuzhaensis was verified for the first time. Restoration of the independent taxonomic status of O. malampuzhaensis is suggested. One pair of satellite chromosomes belonging to the B genome was identified in O. malampuzhaensis, but no such satellite chromosomes were found in either O. minuta or the tetraploid O. punctata. The average chromosome length of the C genome was found to be slightly larger than that of the B-genome chromosomes of O. minuta, but not in the tetraploids O. punctata and O. malampuzhaensis. McGISH also revealed that the B genome of O. minuta and the B genome of diploid O. punctata showed clear differentiation from each other. Therefore, the suggestion was proposed that the B genome in diploid O. punctata was not the source of the B genome of O. minuta. The present results proved that multicolor GISH had high resolution in identifying the genomic constitution of polyploid Oryza species. Received: 14 February 2000 / Accepted: 13 November 2000     

Molecular characterisation and origin of the Coffea arabica L. genome

Restriction fragment length polymorphism (RFLP) markers were used in combination with genomic in situ hybridisation (GISH) to investigate the origin of the allotetraploid species Coffea arabica (2n = 44). By comparing the RFLP patterns of potential diploid progenitor species with those of C. arabica, the sources of the two sets of chromosomes, or genomes, combined in C. arabica were identified. The genome organisation of C. arabica was confirmed by GISH using simultaneously labelled total genomic DNA from the two putative genome donor species as probes. These results clearly suggest that C. arabica is an amphidiploid formed by hybridisation between C. eugenioides and C. canephora, or ecotypes related to these diploid species. Our results also indicate low divergence between the two constituent genomes of C. arabica and those of its progenitor species, suggesting that the speciation of C. arabica took place relatively recently. Precise localisation in Central Africa of the site of the speciation of C. arabica, based on the present distribution of the coffee species, appears difficult, since the constitution and extent of tropical forest has varied considerably during the late Quaternary period. Received: 6 June 1998 / Accepted: 10 November 1998     

Complex polyploid and hybrid species in an apomictic and sexual tropical forage grass group: genomic composition and evolution in Urochloa (Brachiaria) species

Background and AimsDiploid and polyploid Urochloa (including Brachiaria, Panicum and Megathyrsus species) C₄ tropical forage grasses originating from Africa are important for food security and the environment, often being planted in marginal lands worldwide. We aimed to characterize the nature of their genomes, the repetitive DNA and the genome composition of polyploids, leading to a model of the evolutionary pathways within the group including many apomictic species.MethodsSome 362 forage grass accessions from international germplasm collections were studied, and ploidy was determined using an optimized flow cytometry method. Whole-genome survey sequencing and molecular cytogenetic analysis were used to identify chromosomes and genomes in Urochloa accessions belonging to the ‘brizantha’ and ‘humidicola’ agamic complexes and U. maxima.Key ResultsGenome structures are complex and variable, with multiple ploidies and genome compositions within the species, and no clear geographical patterns. Sequence analysis of nine diploid and polyploid accessions enabled identification of abundant genome-specific repetitive DNA motifs. In situ hybridization with a combination of repetitive DNA and genomic DNA probes identified evolutionary divergence and allowed us to discriminate the different genomes present in polyploids.ConclusionsWe suggest a new coherent nomenclature for the genomes present. We develop a model of evolution at the whole-genome level in diploid and polyploid accessions showing processes of grass evolution. We support the retention of narrow species concepts for Urochloa brizantha, U. decumbens and U. ruziziensis, and do not consider diploids and polyploids of single species as cytotypes. The results and model will be valuable in making rational choices of parents for new hybrids, assist in use of the germplasm for breeding and selection of Urochloa with improved sustainability and agronomic potential, and assist in measuring and conserving biodiversity in grasslands.     

Genomic in situ hybridization identifies genome donor of finger millet (Eleusine coracana)

Eleusine coracana, commonly called finger millet, is an important cereal of semi-arid regions, cultivated in parts of Africa and India for its grain. It is reported to be an allotetraploid with a chromosome number 2n = 4x = 36, and diploid species E. indica, with chromosome number 2n = 2x = 18, is considered to be one of its genome donors. In situ hybridization of the E. coracana genome with the genomic DNA of various diploid species of the genus confirmed that E. indica is one of the genome donors to E. coracana and that E. floccifolia is another genome contributor to this allotetraploid species. In situ hybridization also showed a close genomic relationship between 4 diploid species, E. indica, E. floccifolia, E. tristachya and E. intermedia, and also between these and tetraploid species E. coracana. The common genomic in situ hybridization (GISH) signals of the genomic DNA of E. indica and E. tristachya on 15–18 chromosomes of E. coracana clearly indicated that these 2 species have a close genomic similarity. GISH on 25–27 chromosomes of E. coracana withthe genomic DNA of E. intermedia and cross in situ hybridization signals on the chromosomes of E. coracana with genomic DNA of E. intermedia and E. indica or E. intermedia and E. floccifolia has showed that E. intermedia may be an intermediate species of E. indica and E. floccifolia. Received: 15 May 2000 / Accepted: 4 September 2000     

Differential aluminum resistance in Brachiaria species

Brachiaria are increasingly cropped in the tropics because these species combine good fodder quality and yield with high resistance to aluminum (Al) toxicity, an important stress factor in acidic soils. The mechanisms for the extraordinarily high resistance to Al toxicity in Brachiaria decumbens remain unclear. It has been suggested that the presence of a multiseriate exodermis might contribute to efficient Al exclusion in B. decumbens. However, no data concerning the root structure of less Al-resistant Brachiaria species have been reported. The aim of the present study was determine whether the exodermis is a distinctive feature of Al hyper-resistant B. decumbens compared with Brachiaria species with lower Al resistance. B. decumbens, B. brizantha, and B. ruziziensis were grown in nutrient solution without (control) or with 200 μM Al (32 μM Al³⁺ activity) for 96 h. Differences in the Al resistance were assessed using various indicators: Al-induced inhibition of root elongation, membrane damage, and the maintenance of nutrient homeostasis. Transversal root sections were examined using fluorescence microscopy to reveal the presence of an exodermis through auto-fluorescence. Aluminum resistance decreased in the order B. decumbens > B. brizantha > B. ruziziensis. Both the hyper-resistant B. decumbens and the moderately resistant B. brizantha were more efficient in Al-exclusion than the sensitive B. ruziziensis. Apoplastic barriers, in the form of a multiseriate exodermis, were constitutively present in B. decumbens, but not in Al-sensitive B. ruziziensis. Under control conditions, B. brizantha exhibited slightly auto-fluorescent epidermal cell walls, while under Al exposure auto-fluorescent deposits were observed in the intercellular spaces between the epidermal and sub-epidermal cell layers. The results provide circumstantial evidence of a role for apoplastic barriers in the Al resistance of B. decumbens and, to a lesser extent, in B. brizantha. Nonetheless, additional research is required to determine a causal relationship between the exodermal barrier and Al resistance.     

Allopolyploid speciation of the Mexican tetraploid potato species Solanum stoloniferum and S. hjertingii revealed by genomic in situ hybridization

Thirty-six percent of the wild potato (Solanum L. section Petota Dumort.) species are polyploid, and about half of the polyploids are tetraploid species (2n = 4x = 48). Determination of the type of polyploidy and development of the genome concept for members of section Petota traditionally has been based on the analysis of chromosome pairing in species and their hybrids and, most recently, DNA sequence phylogenetics. Based on these data, the genome designation AABB was proposed for Mexican tetraploid species of series Longipedicellata Buk. We investigated this hypothesis with genomic in situ hybridization (GISH) for both representatives of the series, S. stoloniferum Schltdl. and S. hjertingii Hawkes. GISH analysis supports an AABB genome constitution for these species, with S. verrucosum Schltdl. (or its progenitor) supported as the A genome donor and another North or Central American diploid species (S. cardiophyllum Lindl., S. ehrenbergii (Bitter) Rydb., or S. jamesii Torrey) as the B genome donor. GISH analysis of chromosome pairing of S. stoloniferum also confirms the strict allopolyploid nature of this species. In addition, fluorescence in situ hybridization data suggest that 45S rDNA regions of the two genomes of S. stoloniferum were changed during coevolution of A and B genomes of this allotetraploid species.     

The genome composition of hexaploid Psammopyrum athericum and octoploid Psammopyrum pungens (Poaceae: Triticeae).

The genomic constitution of two species in the genus Psammopyrum, i.e., Ps. athericum (2n = 6x = 42) and Ps. pungens (2n = 8x = 56), was studied by genomic in situ hybridization (GISH). In Ps. athericum, one diploid chromosome set hybridized to a genomic probe from Pseudoroegneria ferganensis (St genome), one diploid set to a probe from Agropyron cristatum (P genome), and one diploid set to a probe from Thinopyrum junceiforme (EbEe genomes) or Th. bessarabicum (Eb genome). Substituting the St-genome probe with an L-genome probe from Festucopsis serpentinii resulted in exactly the same hybridization pattern, suggesting a genomic constitution of EStP or ELP for Ps. athericum. The same probes used on Ps. pungens showed two diploid sets of chromosomes hybridizing to the St-genome probe, one diploid set hybridizing to the P-genome probe, and one diploid set hybridizing to the EbEe-genome probe. The L-genome probe hybridized to approximately 14 of the chromosomes that were labeled by the St-genome probe. Hence the genomic constitution for Ps. pungens is proposed to be EStStP or EStLP.     

A fertile amphiploid between durum wheat (Triticum turgidum) and the x Agroticum amphiploid (Agropyron cristatum x T. tauschii)

Agropyron (Gaertn) is a genus of Triticeae which includes the crested wheatgrass complex, i.e. A. cristatum (L.) as representative species containing the P genome. This species is an important source for increase the genetic variability of both durum and bread wheat. Among the possible interesting features to be introgressed into wheat are resistance to wheat streak mosaic virus, rust diseases, and tolerance to drought, cold and moderate salinity. By crossing tetraploid wheat (Triticum turgidum conv durum, 2n = 4x = 28; AABB) with a fertile allotetraploid (2n = 4x = 28; DDPP) between diploid wheat (T. tauschii) and crested wheatgrass (A. cristatum L.), amphiploid plants were obtained. Fluorescence in situ hybridization (FISH) using both genomic DNA from A. cristatum and the repetitive probe pAs1, proved that the plants were true amphiploids with a chromosome number 2n = 8x = 56 and genomic constitution AABBDDPP. Using total genomic in situ hybridization (GISH) to study meiotic metaphase I, data on allosyndetic and autosyndetic chromosome pairing were obtained. The amphiploids were perennial like the male parent but their morphology was close to that of the wheat parent. They were resistant to wheat leaf rust and powdery mildew under field conditions.     

Cytogenetic evidence of mixed disomic and polysomic inheritance in an allotetraploid (AABB) Musa genotype

Background and Aims

Edible bananas originated mainly from two wild species, Musa acuminata Colla (AA) and Musa balbisiana Colla (BB), and triploid cultivars with an AAA, AAB or ABB genome are the most widely used. In the present study, chromosome pairing affinities are investigated in a sterile AB Indian variety and in its fertile colchicine-induced allotetraploid (AABB) derivative to determine the inheritance pattern of the tetraploid genotype. The potential implications of interspecific recombination and chromosomal composition of diploid gametes for Musa improvement are presented.

Methods

The pairing of different chromosome sets at diploid and tetraploid levels was investigated through a combination of conventional cytogenetic and genomic in-situ hybridization (GISH) analyses of meiotic chromosomes, leading to a likelihood model of the pairing behaviour. GISH analysis of mitotic chromosomes was also conducted to reveal the chromosome constitution of hybrids derived from crosses involving the allotetraploid genotype.

Key Results

Analysis of chromosome associations at both ploidy levels suggested that the newly formed allotetraploid behaves as a ‘segmental allotetraploid’ with three chromosome sets in a tetrasomic pattern, three sets in a likely disomic pattern and the five remaining sets in an intermediate pattern. Balanced and unbalanced diploid gametes were detected in progenies, with the chromosome constitution appearing to be more homogenous in pollen than in ovules.

Conclusions

Colchicine-induced allotetraploids in Musa provide access to the genetic background of natural AB varieties. The segmental inheritance pattern exhibited by the AABB allotetraploid genotype implies chromosome exchanges between M. acuminata and M. balbisiana species and opens new horizons for reciprocal transfer of valuable alleles.     

Genomic relationships between the cultivated peanut (Arachis hypogaea, Leguminosae) and its close relatives revealed by double GISH

Arachis hypogaea is a natural, well-established allotetraploid (AABB) with 2n = 40. However, researchers disagree on the diploid genome donor species and on whether peanut originated by a single or multiple events of polyploidization. Here we provide evidence on the genetic origin of peanut and on the involved wild relatives using double GISH (genomic in situ hybridization). Seven wild diploid species (2n = 20), harboring either the A or B genome, were tested. Of all genomic DNA probe combinations assayed, A. duranensis (A genome) and A. ipaensis (B genome) appeared to be the best candidates for the genome donors because they yielded the most intense and uniform hybridization pattern when tested against the corresponding chromosome subsets of A. hypogaea. A similar GISH pattern was observed for all varieties of the cultigen and also for A. monticola. These results suggest that all presently known subspecies and varieties of A. hypogaea have arisen from a unique allotetraploid plant population, or alternatively, from different allotetraploid populations that originated from the same two diploid species. Furthermore, the bulk of the data demonstrated a close genomic relationship between both tetraploids and strongly supports the hypothesis that A. monticola is the immediate wild antecessor of A. hypogaea.     

Allele mining in diverse accessions of tropical grasses to improve forage quality and reduce environmental impact

Background and AimsThe C₄Urochloa species (syn. Brachiaria) and Megathyrsus maximus (syn. Panicum maximum) are used as pasture for cattle across vast areas in tropical agriculture systems in Africa and South America. A key target for variety improvement is forage quality: enhanced digestibility could decrease the amount of land required per unit production, and enhanced lipid content could decrease methane emissions from cattle. For these traits, loss-of-function (LOF) alleles in known gene targets are predicted to improve them, making a reverse genetics approach of allele mining feasible. We therefore set out to look for such alleles in diverse accessions of Urochloa species and Megathyrsus maximus from the genebank collection held at the CIAT.MethodsWe studied allelic diversity of 20 target genes (11 for digestibility, nine for lipid content) in 104 accessions selected to represent genetic diversity and ploidy levels of U. brizantha, U. decumbens, U. humidicola, U. ruziziensis and M. maximum. We used RNA sequencing and then bait capture DNA sequencing to improve gene models in a U. ruziziensis reference genome to assign polymorphisms with high confidence.Key ResultsWe found 953 non-synonymous polymorphisms across all genes and accessions; within these, we identified seven putative LOF alleles with high confidence, including those in the non-redundant SDP1 and BAHD01 genes present in diploid and tetraploid accessions. These LOF alleles could respectively confer increased lipid content and digestibility if incorporated into a breeding programme.ConclusionsWe demonstrated a novel, effective approach to allele discovery in diverse accessions using a draft reference genome from a single species. We used this to find gene variants in a collection of tropical grasses that could help reduce the environmental impact of cattle production.     

Chromosomal distribution of H3K4me2, H3K9me2 and 5-methylcytosine: variations associated with polyploidy and hybridization in <Emphasis Type="Italic">Brachiaria</Emphasis> (Poaceae)

Key message

Assessment of chromosomal distribution of modified histones and 5-methylcytosine shown that there are diversification of chromosomal types among species of Brachiaria and its interspecific hybrids.

Abstract

Histone post-translational modifications and DNA methylation are epigenetic processes that are involved in structural and functional organization of the genome. This study compared the chromosomal distribution of modified histones and 5-methylcytosine (5-mCyt) in species and interspecific hybrids of Brachiaria with different ploidy levels and reproduction modes. The relation between H3K9me2 and 5-mCyt was observed in the nucleolus organizer region, centromeric central domain and pericentromeric region. H3K4me2 was detected in euchromatic domains, mainly in the terminal chromosomal regions. Comparison of chromosomal distribution among species and hybrids showed greater variation of chromosomal types for the H3K9me2 in B. decumbens (tetraploid and apomictic species) and the 963 hybrid, while, for the H3K4me2, the variation was higher in B. brizantha and B. decumbens (tetraploid and apomictic species) and 963 hybrid. The chromosome distribution of 5-mCyt was similar between B. brizantha and B. decumbens, which differ from the distribution observed in B. ruziziensis (diploid and sexual species). Significant alterations in DNA methylation were observed in the artificially tetraploidized B. ruziziensis and in the interspecific hybrids, possibly as result of hybridization and polyploidization processes. The monitoring of histone modifications and DNA methylation allowed categorizing nuclear and chromosomal distribution of these epigenetic marks, thus contributing to the knowledge of composition and structure of the genome/epigenome of Brachiaria species and hybrids. These data can be useful for speciation and genome evolution studies in genus Brachiaria, and represent important markers to explore relationships between genomes.

     

Evidence of Intergenomic Relationships in Triploid Hybrids of Coffee (<Emphasis Type="Italic">Coffea sp.)</Emphasis> as Revealed by Meiotic Behavior and Genomic in Situ Hybridization

Interspecific hybrids involving the cultivated C. arabica (2n = 4x = 44, E^aE^aC^aC^a) and two related diploid species (2n = 2x = 22), C. eugenioides (EE) and C. liberica (LL), were produced and analyzed for their relative genome affinity using different complementary approaches, including chromosome association analysis, genomic in situ hybridization (GISH) and pollen fertility. The mean arm pairing frequency (c) and the relative affinity index (x) of triploid hybrids with known genome combinations were used as a measure of chromosome homology. Triploid hybrids were highly sterile as a result of meiotic abnormalities (fertility ranged from 1 to 15 %). Nevertheless, all hybrids exhibited a significant occurrence of genome affinities (x = 0.96 for E^aC^aE and 0.81 for E^aC^aL). Further analysis using the GISH approach revealed that C. eugenioides was more closely related to C. arabica than to C. liberica, which was in agreement with the ancestral history of the allotetraploid C. arabica. The absence of incompatibility barriers at the stylar level in the flowers of the triploid hybrids indicates the possibility of desirable gene transfer through breeding strategies.     

Genomic in situ hybridization (GISH) discriminates between the A and the B genomes in diploid and tetraploid Setaria species.

Genomic in situ hybridization (GISH) was used to investigate genomic relationships between different Setaria species of the foxtail millet gene pool (S. italica) and one interspecific F1 hybrid. The GISH patterns obtained on the two diploid species S. viridis (genome A) and S. adhaerans (genome B), and on their F1 hybrid showed clear differentiation between these two genomes except at the nucleolar organizing regions. Similar GISH patterns allowed differentiation of S. italica from S. adhaerans. However, GISH patterns did not distinguish between the genomes of S. italica and its putative wild ancestor S. viridis. GISH was also applied to polyploid Setaria species and enabled confirmation of the assumed allotetraploid nature of S. faberii and demonstration that both S. verticillata and S. verticillata var. ambigua were also allotetraploids. All these tetraploid species contained two sets of 18 chromosomes each, one from genome A and the other from genome B. Only one polyploid species, S. pumila, was shown to bear an unknown genomic composition that is not closely related either to genome A or to genome B.     

Coevolution of A and B genomes in allotetraploid Triticum dicoccoides.

Data is presented on the coevolution of A and B genomes in allotetraploid wheat Triticum dicoccoides (2n = 4x = 28, genome AABB) obtained by genomic in situ hybridization (GISH). Probing chromosomes of T. dicoccoides with DNA from the proposed A/B diploid genome ancestors shows evidence of enriching A-genome with repetitive sequences of B-genome type. Thus, ancestral S-genome sequences have spread throughout the AB polyploid genome to a greater extent than have ancestral A-genome sequences. The substitution of part of the A-genome heterochromatin clusters by satellite DNA of the B genome is detected by using the molecular banding technique. The cause may be interlocus concerted evolution and (or) colonization. We propose that the detected high level of intergenomic invasion in old polyploids might reflect general tendencies in speciation and stabilization of the allopolyploid genome.     

Responses to drought of five Brachiaria species. I. Biomass production,leaf growth,root distribution,water use and forage quality

The introduction of African grasses in Neotropical savannas has been a key factor to improve pasture productivity. We compared the response of five Brachiaria species to controlled drought (DT) in terms of biomass yield and allocation, pattern of root distribution, plant water use, leaf growth, nutrient concentration and dry matter digestibility. The perennial C₄ forage grasses studied were B. brizantha (CIAT 6780), B. decumbens (CIAT 606), B. dictyoneura (CIAT 6133), B. humidicola (CIAT 679) and B. mutica. Two DT periods, which mimic short dry spells frequent in the rainy season, were imposed by suspending irrigation until wilting symptoms appeared. They appeared after 14 days in B. brizantha, B. decumbens and B. mutica, and after 28 days in B. humidicola and B. dictyoneura. The impossed drought stress was mild and only the largest grass, B. brizantha, showed reduced (23%) plant yield. The other grasses were able to adjust growth and biomass allocation in response to DT leaving total plant yield relatively unaffected. Brachiaria mutica, had a homogeneous root distribution throughout the soil profile. In the other species more than 80% of root biomass was allocated within the first 30 cm of the soil profile. Brachiaria brizantha and B. decumbens had the lowest proportion of roots below 50 cm. Drought caused a general reduction in root biomass. The shoot:root ratio in B. mutica and B. humidicola increased in response to DT at the expense of a reduction in root yield down to 50 cm depth. Although the total water volume utilized under DT was similar among grasses, the rate of water use was highest (0.25 l day^–1) in B. brizantha, B. decumbens and B. mutica and lowest (0.13 l day^–1) in B. humidicola and B. dictyoneura. In all species leaf expansion was reduced by DT but it was rapidly reassumed after rewatering. Drought increased specific leaf mass (SLM) only in B. brizantha compensating for leaf area reduction, but leaf area ratio (LAR) was unaffected in all species. In almost all grasses DT increased leaf N and K concentration and in vitro dry matter digestibility. The results indicate that B. brizantha, B. decumbens and to a lesser extent, B. mutica are better adapted to short dry periods, whereas B. humidicola and B. dictyoneura are better adapted to longer dry periods.     

New microsatellite markers developed from <Emphasis Type="Italic">Urochloa humidicola</Emphasis> (Poaceae) and cross amplification in different <Emphasis Type="Italic">Urochloa</Emphasis> species

Background

Urochloa humidicola is a forage grass that grows in tropical regions and is recognized for its tolerance to seasonal flooding. It is a polyploid and apomictic species with high phenotypic plasticity. As molecular tools are important in facilitating the development of new cultivars and in the classification of related species, the objectives of this study were to develop new polymorphic microsatellite markers from an enriched library constructed from U. humidicola and to evaluate their transferability to other Urochloa species.

Findings

Microsatellite sequences were identified from a previously constructed enriched library, and specific primers were designed for 40 loci. Isolated di-nucleotide repeat motifs were the most abundant followed by tetra-nucleotide repeats. Of the tested loci, 38 displayed polymorphism when screened across 34 polyploid Urochloa sp. genotypes, including 20 accessions and six hybrids of U. humidicola and two accessions each from U. brizantha, U. dictyoneura, U. decumbens and U. ruziziensis. The number of bands per Simple Sequence Repeat (SSR) locus ranged from one to 29 with a mean of 11.5 bands per locus. The mean Polymorphism Information Content (PIC) of all loci was 0.7136, and the mean Discrimination Power (DP) was 0.7873. Six loci amplified in all species tested. STRUCTURE analysis revealed six different allelic pools, and the genetic similarity values analyzed using Jaccard's coefficient ranged from 0.000 to 0.913.

Conclusions

This work reports new polymorphic microsatellite markers that will be useful for breeding programs for Urochloa humidicola and other Urochloa species as well as for genetic map development, germplasm characterization, evolutionary and taxonomic studies and marker-assisted trait selection.