Alterations in Heterochromatic Knobs in Maize Callus Culture by Breakage-Fusion-Bridge Cycle and Unequal Crossing Over.

The meiotic and mitotic behavior of regenerated plants derived from a long-term callus culture, designated 12-F, was analyzed. This culture was heterozygous for an amplification of the heterochromatic knob on the long arm of chromosome 7 (K7L). We aimed to investigate if the amplification resulted from a breakage-fusion-bridge (BFB) cycle or from unequal sister chromatid recombination. Therefore, C-banded mitotic metaphases and pachytene, diakinesis, and anaphase I of regenerated plants were analyzed. Additionally, the occurrence of alterations in K7L was investigated in C-banded metaphases from short-term callus cultures derived from lines related to the donor genotype of the 12-F culture. As a result, plants homozygous and heterozygous for the amplification were detected. Meiosis was normal with few abnormalities, such as a low frequency of univalents at diakinesis. In the callus cultures a chromosome 7 with knobs of different sizes in the sister chromatids was detected and interpreted as a result of unequal crossing over. Other chromosomal alterations were consistent with the occurrence of BFB cycles. The finding of unequal crossing over in the cultures supports the conclusion that the amplification in the culture 12-F would be derived from this mechanism. If the amplification was derived from a BFB cycle, the terminal euchromatic segment between knob and the telomere would be deleted, and possibly, homozygous plants would not be viable.

Breakage-fusion-bridge cycles and de novo telomere formation on broken chromosomes in maize callus cultures.

Breakpoints involved in chromosome alterations associated with heterochromatin have been detected in maize plants regenerated from callus culture. A cytogenetic analysis of plants regenerated from a maize callus was performed aiming to analyze the stability of a chromosome 7 bearing a deficiency-duplication (Df-Dp), which was interpreted as derived from a chromatid type breakage-fusion-bridge (BFB) cycle. The Df-Dp chromosome 7 was stable in mitotic and meiotic cells of the regenerated plants. Fluorescence in situ hybridization showed signals of telomeric sequences on the broken chromosome arm and provided evidence of de novo telomere formation. The stability of two types of altered chromosome 7 was investigated in C-banded metaphases from samples of the original callus that were collected during a period of 30-42 months after culture initiation. New alterations involving heterochromatic knobs of chromosomes 7 and 9 were observed. The aberrant chromosomes were stable in the subcultures, thus providing evidence of broken chromosome healing. The examination of anaphases showed the presence of bridges, which was consistent with the occurrence of BFB cycles. De novo telomere formation occurred in euchromatic and heterochromatic chromosome termini. The results point to events of chromosomal evolution that might occur in plants.

Karyotype variability in tropical maize sister inbred lines and hybrids compared with KYS standard line.

Maize karyotype variability has been extensively investigated. The identification of maize somatic and pachytene chromosomes has improved with the development of fluorescence in situ hybridization (FISH) using tandemly repeated DNA sequences as probes. We identified the somatic chromosomes of sister inbred lines that were derived from a tropical flint maize population (Jac Duro [JD]), and hybrids between them, using FISH probes for the 180-bp knob repeat, centromeric satellite (CentC), centromeric satellite 4 (Cent4), subtelomeric clone 4-12-1, 5S ribosomal DNA and nucleolus organizing region DNA sequences. The observations were integrated with data based on C-banded mitotic metaphases and conventional analysis of pachytene chromosomes. Heterochromatic knobs visible at pachynema were coincident with C-bands and 180-bp FISH signals on somatic chromosomes, and most of them were large. Variation in the presence of some knobs was observed among lines. Small 180-bp knob signals were invariant on the short arms of chromosomes 1, 6, and 9. The subtelomeric 4-12-1 signal was also invariant and useful for identifying some chromosomes. The centromere location of chromosomes 2 and 4 differed from previous reports on standard maize lines. Somatic chromosomes of a JD line and the commonly used KYS line were compared by FISH in a hybrid of these lines. The pairing behavior of chromosomes 2 and 4 at pachytene stage in this hybrid was investigated using FISH with chromosome-specific probes. The homologues were fully synapsed, including the 5S rDNA and CentC sites on chromosome 2, and Cent4 and subtelomeric 4-12-1 sites on chromosome 4. This suggests that homologous chromosomes could pair through differential degrees of chromatin packaging in homologous arms differing in size. The results contribute to current knowledge of maize global diversity and also raise questions concerning the meiotic pairing of homologous chromosomes possibly differing in their amounts of repetitive DNA.

Genetic diversity of carotenoid-rich bananas evaluated by Diversity Arrays Technology (DArT).

The aim of this work was to evaluate the carotenoid content and genetic variability of banana accessions from the Musa germplasm collection held at Embrapa Cassava and Tropical Fruits, Brazil. Forty-two samples were analyzed, including 21 diploids, 19 triploids and two tetraploids. The carotenoid content was analyzed spectrophotometrically and genetic variability was estimated using 653 DArT markers. The average carotenoid content was 4.73 µg.g (-1) , and ranged from 1.06 µg.g (-1) for the triploid Nanica (Cavendish group) to 19.24 µg.g (-1) for the triploid Saney. The diploids Modok Gier and NBA-14 and the triploid Saney had a carotenoid content that was, respectively, 7-fold, 6-fold and 9-fold greater than that of cultivars from the Cavendish group (2.19 µg.g (-1)). The mean similarity among the 42 accessions was 0.63 (range: 0.24 to 1.00). DArT analysis revealed extensive genetic variability in accessions from the Embrapa Musa germplasm bank.

Genetic diversity of carotenoid-rich bananas evaluated by Diversity Arrays Technology (DArT)

The aim of this work was to evaluate the carotenoid content and genetic variability of banana accessions from the Musa germplasm collection held at Embrapa Cassava and Tropical Fruits, Brazil. Forty-two samples were analyzed, including 21 diploids, 19 triploids and two tetraploids. The carotenoid content was analyzed spectrophotometrically and genetic variability was estimated using 653 DArT markers. The average carotenoid content was 4.73 µg.g-1, and ranged from 1.06 µg.g-1 for the triploid Nanica (Cavendish group) to 19.24 µg.g-1 for the triploid Saney. The diploids Modok Gier and NBA-14 and the triploid Saney had a carotenoid content that was, respectively, 7-fold, 6-fold and 9-fold greater than that of cultivars from the Cavendish group (2.19 µg.g-1). The mean similarity among the 42 accessions was 0.63 (range: 0.24 to 1.00). DArT analysis revealed extensive genetic variability in accessions from the Embrapa Musa germplasm bank.

Karyotype characterization of Crotalaria juncea (L) by chromosome banding and physical mapping of 18S-5.8S-26S and 5S rRNA gene sites

The chromosomes of Crotalaria juncea, a legume of agronomic interest with a 2n = 16 karyotype composed of metacentric chromosomes, were analyzed using several cytogenetic techniques. C-banding revealed heterochromatic regions around the centromeres in all chromosomes and adjacent to the secondary constriction on the chromosome 1 short arm. Fluorescent staining with the GC-specific chromomycin A3 (CMA) highlighted these heterochromatic regions and a tiny site on the chromosome 1 long arm while the AT-specific stain 4'-6-diamidino-2-phenylindole (DAPI) induced a reversed pattern. Staining with CMA combined with AT-specific distamycin A (DA) counterstaining quenched the pericentromeric regions of all chromosomes, but enhanced fluorescence was observed at the heterochromatic regions around the secondary constriction and on the long arms of chromosomes 1 and 4. Fluorescence in situ hybridization (FISH) revealed 18S-5.8S-26S rRNA gene sites (45S rDNA) on chromosomes 1 and 4, and one 5S rDNA locus on chromosome 1. All the rDNA sites were co-located with the positive-CMA/DA bands, suggesting they were very rich in GC. Silver staining revealed signals at the main 45S rDNA locus on chromosome 1 and, in some cells, chromosome 4 was labeled. Two small nucleoli were detected in a few interphase cells, suggesting that the minor site on chromosome 4 could be active at some stages of the cell cycle.