Citation: dos Reis GB, Mesquita AT, Torres GA, Andrade-Vieira LF, Pereira AV, Davide LC (2014) Genomic homeology between Pennisetum purpureum and Pennisetum glaucum (Poaceae). Comparative Cytogenetics 8(3): 199-209. doi: 10.3897/CompCytogen.v8i3.7732
The genus Pennisetum (Richard, 1805) includes two economically important tropical forage plants: Pennisetum purpureum (Schumacher, 1827) (elephant grass), with 2n = 4x = 28 chromosomes and genomes A'A'BB, and Pennisetum glaucum (Linnaeus, 1753) (pearl millet), with 2n = 2x = 14 chromosomes and genomes AA. The genetic proximity between them allows hybrids to be obtained (2n = 3x = 21) that yield forage of higher quality in relation to the parents. The study of genomic relationships provides subsidies for the knowledge about phylogenetic relations and evolution, and is useful in breeding programs seeking gene introgression. Concerning elephant grass and pearl millet, the homeology between the genomes A and A', and between these and the genome B, has been reported by conventional cytogenetic techniques. The objective of the present study was to demonstrate the degree of homeology between these genomes by means of genomic in situ hybridization (GISH). The results confirmed the homeology between the genomes A of pearl millet and A'B of elephant grass, and showed that there are differences in the distribution and proportion of homologous regions after hybridization. Discussion regarding the evolutionary origin of P. purpureum and P. glaucum was also included.
Homeology, Pennisetum purpureum, Pennisetum glaucum, Genomic in situ hybridization
The genus Pennisetum (Richard, 1805) is one of the most important in family Poaceae family. It comprises about 140 species, distributed in five sections (Penicillaria, Brevivalvula, Gymnothrix, Heterostachya and Eu-Pennisetum) based on morphological characteristics (
Molecular analyses based on mitochondrial DNA (
In spite of their integrating distinct genic groups and differing as to ploidy level, the genetic proximity between these two species becomes evident when the occurrence of natural hybridization is observed. This sexual compatibility is partial, and results in sterile triploid hybrids (2n = 3x = 21, genome AA’B) (
In this sense, despite evidence for a common evolutionary origin between Pennisetum glaucum and Pennisetum purpureum and the economic importance of these species, there are no studies providing more conclusive data with respect to the homeology among genomes A, A’ and B. Thus, the objective of this work is to describe the proportion and distribution of the homologous regions present in genomes A of Pennisetum glaucum and A’B of Pennisetum purpureum, by cytomolecular analyses using genomic in situ hybridization (GISH).
The evaluations were carried out in mitotic metaphases of the parental Pennisetum purpureum (access BAG 65) and Pennisetum glaucum (access BN2), and of the triploid hybrid originating from this crossing (BAG 65 × BN2). The plant material and genomic DNAs were provided by the Active Germplasm Bank of Elephant Grass (BAGCE) from EMBRAPA Dairy Cattle (Brazilian Research Institute) and elephant grass breeding program, experimental field José Henrique Bruschi, municipality of Coronel Pacheco, Minas Gerais State, Brazil.
Roots from seeds or cuttings of BAG 65, BN2 and triploid hybrid accession were collected and pretreated with a 12.5 mg.L-1 cycloheximide: 150 mg.L-1 8-hydroxyquinoline solution for 2 h 45 min, at 4 °C, and fixed in ethanol: acetic acid solution (3:1), as proposed by
Genomic DNAs of Pennisetum glaucum and Pennisetum purpureum were labeling with biotin-16-dUTP through nick-translation reaction method, thus yielding the genomic probes.
The hybridization technique was carried out according to
In order to evaluate the level of homeology between genomes A, A’ and B, the chromosomes of five metaphases from each genome were measured, as well as the proportion occupied by the genomic probe, using the Image Tool 3.0 program. The obtained data were used to create karyograms for comparison of the evaluated genomes.
Previous analyses of meiotic pairing in the triploid hybrid have showed that the genomes A and A´ are more related. On the other side, between both and the genome B there are affinity/homeology reduced (
The higher level of homeology between genomes A and A’ was confirmed because the 14 chromosomes belonging to genome A’ of Pennisetum purpureum were strongly marked and distinguished from the 14 chromosomes from genome B using the genomic DNA of Pennisetum glaucum (genome A) as probe in metaphases of Pennisetum purpureum (Fig. 1a). The chromosomes of genome A’ presented marks in along almost role chromosome length, whereas genome B presented small marks dispersed over the length of its chromosomes (Fig. 2a). Moreover, approximately 29% of Pennisetum purpureum genome (A’B) was hybridized by the genome A of Pennisetum glaucum (Table 1). This percentual represents only the A’ genome since the markers on genome B chromosomes were not record because it were dispersed on chromosomes. The observed homeology was only quantified in the genome A’ of Pennisetum purpureum, due to the difficulty in measuring the small and dispersed marks found in genome B (Fig. 2a).
The homeology between genomes A and A’ was confirmed by the extensive marking of Pennisetum glaucum chromosomes by the probe A’B of Pennisetum purpureum. All 14 chromosomes from genome A of Pennisetum glaucum were almost completely marked, with large blocks of probe signals observed on the chromosomes (Fig. 1b). The markings by the probe of genome A’B observed in the centromeric and pericentric regions represented 63% of the genome of Pennisetum glaucum (Table 1 and Fig. 2b). These marked portions result from hybridization, both between the genomes A and A’ and, in smaller proportion, genomes A and B, observed both in karyograms of Pennisetum purpureum and triploid hybrids (Fig. 2a, c).
In the triploid hybrid (AA’B) the hybridized portion of genome A (Pennisetum glaucum) corresponded to 54%, and the signal of probe A’B (Pennisetum purpureum) to 49% of its total genome (Table 1). Despite the similarity in proportion, the distribution pattern for the probes from the parental individuals was different in the hybrid (Fig. 1c). The seven chromosome of the hybrid were entirely marked with the probe of genomic DNA from Pennisetum glaucum. The remaining chromosomes from Pennisetum purpureum parental (genome A´B) presented marks only in the centromeric and pericentromeric regions (Fig. 2c). However, when the probe with DNA of Pennisetum purpureum was used in chromosomes of the triploid hybrid the marks were observed mainly in centromeric and pericentromeric regions, but some chromosomes appearing almost totally marked (Fig. 1d).
The differences in marking pattern observed in the triploid hybrid, mainly between genomes A and A’, could be explained by the presence of two genomes (A’ and B) in the same probe. The observations evidence the changes arising from interspecific hybridization in Pennisetum purpureum genomes. Once combined in a polyploid hybrid nucleus, extensive reorganization may rapidly occur in the parental diploid genomes, both intra and intergenomically (
Besides the existing homeology among genomes A, A’ and B, the utilization of GISH in the genomes of Pennisetum glaucum, Pennisetum purpureum and interspecific hybrid enable to verify the differences in chromosomes size, and also chromosomes number of these species. Analyzing cells of the interspecific hybrid, the difference in size between the parental chromosomes becomes evident, with those of Pennisetum glaucum being larger (Fig. 2c). It can also be observed that the total length of Pennisetum purpureum chromosomes did not increase proportionally in relation to those of Pennisetum glaucum (Table 1 and Fig. 2a and b), and that the chromosomes of genome B do not differ significantly in size in relation to genome A’ (Table 2 and Fig. 2a). These differences in size and chromosome number between the two species reflect their evolutionary history.
The evolutionary tendency among true grasses, which have a common and recent origin, is that the most derived species have emerged after reduction of the number and increase of the size of chromosomes in relation to the ancestors (
This hypothesis, presented for the evolution and divergence of Pennisetum purpureum and Pennisetum glaucum from the common ancestor, may be further reinforced by the differences observed in relation to the size of chromosomes from genomes A, A’ and B, as shown in Table 2. Analyzing the size of the monoploid complement of genome A, it can be verified that it is 24% larger in relation to the length of the chromosomes of genome A’ of Pennisetum purpureum. Considering that the genomes A and A’ have evolved from an ancestor genome A, the difference in chromosome size could be related to genic duplication in Pennisetum glaucum and to genomic rearrangements observed in the allotetraploid hybrid Pennisetum purpureum. Rearrangements and loss of genomic sequences are common events after hybridization (
In this work, GISH confirmed the homeology among genomes A, A’ and B and enabled the identification and distribution of the homeologous regions in the chromosomes. Moreover, the GISH markings were able to separate the different genomes, leading the comparison on the size of the chromosomes in each of these three genomes. This distinction of the different genomes confirmed the occurrence of rearrangements after interspecific hybridization, especially when the synthetic triploid hybrid was analyzed, and prove the allotetraploid origin of Pennisetum purpureum It also show that genomes A’ and B have chromosomes similar in size. In evolutionary terms, the results reinforce that the genomes A and A’ have diverged from an ancestral genome A by increase of chromosome size in Pennisetum glaucum and rearrangements and/or deletions in Pennisetum purpureum. The reorganizations occurring in the ancestral genome A during evolution have generated the subgenome A’ of Pennisetum purpureum.
Metaphases of Pennisetum purpureum (A), Pennisetum glaucum (B), and triploid hybrid (C and D). Chromosomes stained with DAPI (A, B, C, D) and probe markings in chromosomes indicated by green fluorescence (A1, B1, C1, D1). (A1) chromosomes of Pennisetum purpureum hybridized with genomic probe of Pennisetum glaucum (genome A), (B1) chromosome of Pennisetum glaucum hybridized with genomic probe of Pennisetum purpureum (genomes A'B), (C1) chromosomes of the triploid hybrid hybridized with genomic probe of Pennisetum glaucum (genome A), (D1) chromosomes of the triploid hybrid hybridized with genomic probe of Pennisetum purpureum (genomes A'B). Bar = 10 μm (A); Bar = 20 μm (B, C and D).
Karyograms of Pennisetum purpureum (A), Pennisetum glaucum (B) and triploid hybrid (C) identifying the chromosomes of genomes A, A 'and B in each genotype. Note that in (A) using genome A probe (Pennisetum glaucum), the chromosomes of genome A’ were differed from chromosomes of genome B by the staining pattern. Genome A’ chromosomes showed more apparent probe markings in green than genome B chromosomes. In (B), using the genome A'B probe (Pennisetum purpureum), all chromosomes were strongly labelled (markings in green). In (C), using the genome A probe (Pennisetum glaucum), the chromosomes of the A genome were fully labeled by the probe (markings in green), the genome A’ were strongly marked in the centromeric region and the genome B, poorly marked. It also could be note the difference in the labeling pattern between the genome A probe on the chromosomes of genome A’ in interspecific hybrid and parental Pennisetum purpureum. Bar = 10 μm.
Proportion of markings of genomic probes (A and A’B) on chromosomes of Pennisetum purpureum, Pennisetum glaucum and triploid hybrid.
Genotype | Total length of the chromosomes | Total length of the probe Pennisetum glaucum (A) | Total length of the probe Pennisetum pupureum (A’B) |
---|---|---|---|
Pennisetum purpureum | 64, 41 | 18, 42 (28, 60%)* | - |
Pennisetum glaucum | 59, 01 | - | 37, 29 (63, 19%) |
Triploid hybrid | 73, 95 | 40, 06 (54, 19%) | 36, 32 (49, 13%) |
* Proportion occupied by the probe for each genotype
Total length (µm) of monoploid complement in genomes A, A' and B for each genotype.
Genotype | Total length of the genome A | Total length of the genome A' | Total length of the genome B |
---|---|---|---|
Pennisetum purpureum | - | 23, 8 | 23, 95 |
Pennisetum glaucum | 29, 51 | - | - |
Triploid hybrid | 17, 63 | 10, 54 | 12, 44 |
The authors would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, the Fundação de Amparo à Pesquisa do Estado de Minas Gerais – FAPEMIG, and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES, for financial support to the research and granting of scholarship.