Cytogenetics and morphological delimitation between three species of Passiflora L. (subgenus Distephana Cervi).

Several studies on cytogenetic characterisation of passion flowers are helpful to elucidate doubts about taxa relationships, delimitation and classification into more coherent groups based on karyomorphological data. Molecular and conventional cytogenetic techniques were applied to three Passiflora species with red flowers, P. coccinea, P. vitifolia and P. tholozanii, for species karyotype relationships. Additionally, for descriptive morphology, were used flowers, leaves and seeds. Results describe for the first time the karyomorphological and chromosome number (2n = 18) for P. tholozanii. anova was performed (P < 0.05) and statistical significance for average chromosome size (CV: 16.53%) between species. Genomic in situ hybridisation (GISH) proved relationships between P. coccinea and P. tholozanii, which suggests a common origin, however, we could not identify hybridisation between genomic probes from P. vitifolia in P. tholozanii chromosomes. Among the species analysed, P. tholozanii has great similarity in karyotypic and morphology to P. coccinea but not to P. vitifolia. We suggest the inclusion of P. tholozanii in the same subgenus and section as P. coccinea based on the similarity in karyomorphological and morphological traits between the species. Additionally, GISH might indicate a common or hybrid origin of P. tholozanii.

Contributions of classical and molecular cytogenetic in meiotic analysis and pollen viability for plant breeding.

The analysis of meiotic behavior has been widely used in the study of plants as they provide relevant information about the viability of a species. Meiosis boasts a host of highly conserved events and changes in genes that control these events will give rise to irregularities that can alter the normal course of meiosis and may lead to complete sterility of the plant. The recombination of genes that occur in meiosis is an important event to generate variability and has been important in studies for genetic improvement and to create viable hybrids. The use of fluorescence in situ hybridization and genomic in situ hybridization (GISH) in meiosis allows the localization of specific regions, enables to differentiate genomes in a hybrid, permits to observe the pairing of homoeologous chromosomes, and if there was a recombination between the genomes of progenitor species. Furthermore, the GISH allows us to observe the close relationship between the species involved. This article aims to report over meiosis studies on plants and hybrids, the use and importance of molecular cytogenetic in meiotic analysis and contributions of meiotic analysis in breeding programs.

Multivariate analysis of backcross progeny of Passiflora L. (Passifloraceae) for pre-breeding genotype selection.

The Ward-MLM procedure was used to evaluate genetic variation in four backcross progenies and in their parents, hybrid F1 HD13 and donor parent Passiflora sublanceolata. Sixteen quantitative descriptors and five qualitative characteristics of relevance to ornamental flower production were assessed. Using the pseudo-F and pseudo-T² criteria, we identified four groups among these plants in two evaluation periods. In both evaluations, the BC1 plants showed greater dissimilarity to their recurrent parent, but showed high genetic similarity with the P. sublanceolata parent. The first two canonical variables produced by the Ward-MLM procedure accounted for over 90% of the variation in both evaluation periods, enabling the representation of diversity through two-dimensional graphics. Groups II and IV were formed in the first assessment period. Groups I and IV formed in the second period and showed plants with selection potential. We found that it was essential to use both qualitative and quantitative variables for this analysis. Assessments of quantitative descriptors indicate that the selection of BC1 plants can be performed in any of the four progenies. Because of the similarities observed for some floral descriptors between BC1 and the P. sublanceolata parent, a second generation backcross was not recommended. However, the selection of BC1 plants for evaluation and direct use as an ornamental cultivar, or as a resource in other breeding programs, can be recommended.

Establishment of the genomic in situ hybridization (GISH) technique for analysis in interspecific hybrids of Passiflora.

The genomic in situ hybridization (GISH) technique was applied to Passiflora interspecific F1 HD13-133 hybrids (Passiflora sublanceolata x Passiflora foetida) and HD15-101 (Passiflora gardineri x Passiflora gibertii), and the backcrossed hybrids (BC1) HD18-106 and HD18-113 (Passiflora sublanceolata x HD13-133). GISH was performed using genomic probes prepared with the DNA from the paternal genitor, whereas the maternal DNA was used as blocking DNA and employed at various concentrations (20X, 40X, 60X, and 100X) in relation to the probe concentration. At the same time, GISH was applied with the use of simultaneous probes from both genomes, paternal and maternal, that were detected with avidin-FITC and anti-digoxigenin-rhodamine, respectively. Both methodologies allowed the distinguishing of the maternal and paternal genomes, thus confirming the hybrid nature of all the analyzed genotypes. Furthermore, the presence of recombinant chromosomes in BC1 hybrids revealed the occurrence of meiotic recombination in HD13 hybrids. This application of the GISH technique is an important step towards genomic analyses of Passiflora hybrids: it can broaden the phylogenetic and evolutionary studies of the genus and, at the same time, contribute to breeding programs.

ISSR markers in wild species of Passiflora L. (Passifloraceae) as a tool for taxon selection in ornamental breeding.

Inter simple sequence repeat (ISSR) molecular markers were developed and used to investigate interspecific genetic variation in 25 wild species of Passiflora preserved in an active germplasm bank (BAG-Passifloras); intraspecific diversity was also analyzed in P. cincinnata accessions. Of 31 primers tested, 20 identified polymorphic loci with a total of 331 bands, suggesting high polymorphism in the sample. Interspecific polymorphism was greater than intraspecific polymorphism. This is a common finding in studies of genetic variation using dominant markers. The ISSRs revealed species-specific amplification bands in 11 species; these bands ranged from 200 to 1000 bp, and they will be of use for developing SCAR markers for the identification of germplasm in further studies. The use of Jaccard's similarity coefficient to obtain a dendrogram by the UPGMA clustering method distributed the taxa into five major groups, with differences among grouping with respect to principal coordinate analysis. Despite the high cophenetic correlation coefficient (r = 0.94) of the dendrogram, taxonomic inconsistencies were observed; similar irregularities have been reported previously in studies using dominant markers. Intraspecific analysis of P. cincinnata accessions revealed a larger genetic distance between those from Bahia (P2) and from Minas Gerais (P2), indicating that both accessions have considerable potential as parents in a genetic improvement program for this species.

Analysis of transferability of microsatellite primers (SSR) in wild Passiflora species and intraspecific genetic diversity in Passiflora alata.

The genus Passiflora L. is the most representative of Passifloraceae, with over 500 known species, among which 150-200 originated from Brazil. In addition to the great commercial importance of this genus for the fruit market, many of the species have exotic flowers with a huge diversity of colors and can thereby be exploited as ornamental plants. This study was aimed at investigating the transferability of microsatellite primers in wild Passiflora species (P. cacao, P. cincinnata, P. glandulosa, P. gibertii, and P. mucronata) and characterizing 29 P. alata accessions using microsatellite primers that were previously developed in a library enriched with microsatellites from P. edulis f. flavicarpa for P. alata. The interspecies cross-amplification rate varied, and P. cacao exhibited the highest rate of amplification, suggesting a greater degree of proximity to P. edulis. The study of intraspecific accessions in P. alata found genetic similarity, with values ranging from 0.47 to 1.00 and an average similarity of 0.74. Hence, this study revealed the intraspecific genetic variability of P. alata in the Universidade Estadual de Santa Cruz's Active Germplasm Bank and will lead to the adoption of mating strategies between accessions; thus making their use more suitable for breeding purposes.

Karyotype variation in cultivars and spontaneous cocoa mutants (Theobroma cacao L.).

Four mutant cocoa accessions with morphological changes and a cultivar sample were karyomorphologically characterized. Slides were prepared by enzymatic digestion of the root meristem and squashed in 45% acetic acid, followed by 2% Giemsa staining. The chromosome number of 2n = 20 was seen in all accessions. The karyotype formula for Cacau Comum and Cacau Rui was 2n = 20m. Submetacentric chromosomes were observed in Cacau Pucala and Cacau Jaca, both with 2n = 18m + 2sm, but the karyotype formula for Cacau Sem Vidro was 2n = 16m + 4sm. Satellites were located on the long arm of the 1st and 2nd chromosome pairs of Cacau Comum, whereas Cacau Pucala had satellites on the 6th chromosome pair. Greater karyotypic variation in Cacau Sem Vidro was found, whose 1st and 2nd chromosome pairs had satellites on the long arm and 6th and 10th pairs had satellites on the short arm. Analysis revealed a lower average chromosome length in Cacau Comum (1.53 ± 0.026 µm) and a higher length in Cacau Sem Vidro (2.26 ± 0.038 µm). ANOVA revealed significant difference (P < 0.01) for the average chromosome length and the length of chromosome pairs within and between accessions. The average chromosome lengths of mutants of Cacau Rui and Cacau Jaca were not statistically different by the Tukey test at 5% probability. The karyotypic diversity observed in this study is not necessarily associated with the changing character of the accessions analyzed, but may reflect the genetic variation observed in Theobroma cacao.