Stanisława Maria Rogalska

Cytomixis-like chromosomes/chromatin elimination from pollen mother cells (PMCs) in wheat-rye allopolyploids

Polyploidy is an important event and major force in plant speciation. Amongst the polyploids, allopolyploids have attracted special attention to investigate genetic and epigenetic mechanisms. Also, they are the means for the development of new genotypes and genomic combinations to facilitate genetic enhancement and agricultural productivity. Whereas natural allopolyploids are genetically stable and well adapted, the newly synthesized ones are highly unstable. This instability is manifested into alterations at genomic and/or phenotypic level. Here we present the phenomenon of direct chromosome/chromatin elimination from pollen mother cells (PMCs) in wheat-rye hybrids as one aspect of instability leading to irregular meiosis and disturbances in meiotic process. One of the prominent irregularities noticed is peripherally separated uncondensed or pycnotic masses of chromatin in all meiotic stages. We have observed that this chromatin undergoes elimination by budding-like way, whereby a “mini-cell” is created. It was also found that nucleoli are the first to be eliminated along with a small mass of chromatin. By means of GISH we have shown that both rye and wheat chromatin might be eliminated. In the separated groups of chromosomes/chromatin neither DNaseI nor DNase II activity was detected. Immunolocalization of tubulin allowed for differentiation between chromatin elimination from microspores and elimination from earlier stages of meiosis. It was noticeable, that in microspores special cytoskeleton structure pushing micronuclei out from the cells was created. Elimination occurred before and after meiosis as well as in each stage of meiotic division, but its intensity varied, depending on the PMC. The basis of the elimination mechanism might be the same as in cytomixis, because both phenomena share common symptoms, although cytomixis per se was rare in the analyzed hybrids.

RAPD polymorphism in the prebreeding material for cultivation of synthetic variations of lucerne (Medicago sativa L.)

Genetic diversity between synthetic cultivars (Syn5, Syn7), inbred lines (D3, D5, E1/2, G1/1, G1/2) and ecotypes (E16, E51, E182, E231) of lucerne (Medicago sativa L.) was studied using the RAPD-PCR method. The plants differed in the efficiency of seed set and in the yield of green mass. The ecotypes E182 and E231 and the synthetic population Syn5 showed the highest fertility. Additionally, Syn5 also showed the highest efficiency of seed set and the yield of green mass. Among the inbred lines, D3 was characterised by the highest yield of green mass and E1/2 by the highest fertility. An optimal combination of yield and biomass was observed for the synthetic population Syn5, obtained by crossing the lines D3, D5 and G1/1, as demonstrated using comparative analysis. A total of 338 polymorphic products were generated using 20 arbitrary primers. Cluster analysis using the Unweighted-Pair Group Method with Arithmetic Mean (UPGMA) in the Molecular image Gel Doc™ XR (Bio-Rad) software based on the Dice’s coefficient of genetic similarity showed a division of the studied forms into two groups based on genetic similarity. The ecotype E16 formed one of the groups whereas all of the other ecotypes observed in this study clustered into the second group. A high level of polymorphism among the studied lucerne forms was detected indicating an interesting gene pool awaiting future exploration. Analysis of variance also supported a high diversity among the studied forms. This study provides insightful information into the heterosis effect of synthetic populations or hybrids of F1 lucerine by providing correlations between the genetic background of the inbred lines and their ability to produce a specific yield.

Possible ancient origin of heterochromatic JNK sequences in chromosomes 2R of Secale vavilovii Grossh.

Employing FISH analysis as well as BLAST and CUSTAL W (1.82) programs, we investigated types of DNA nucleotide sequences building an additional heterochromatic band in 2R chromosomes of 3 lines ofSecale vavilovii Grossh. The probes used in FISH analysis were designed based on the reverse transcriptase sequence of Ty1-copia and Ty3-gypsy retrotransposons and the 5S rRNA gene sequence. No hybridization signals from the reverse transcriptase probes were observed in the chromosome region where the additional band occurs. On the other hand, signals were observed after hybridization with the 5S rDNA probe, clearly suggesting the presence of that type of sequences in the analyzed heterochromatin band. Using BLAST and CUSTAL W programs, we revealed high similarity of the JNK1 sequence to the 5S rRNA gene fromHordeum chilense (HCH1016, HCH1018, 88%) and to a fragment of the 5S rRNA sequence ofH. marinum (HMAR003, 97%). In addition, the same fragment of JNK1 was shown to be very similar to the part of theAngela retrotransposon (92%) as well as to theSNAC 426K20-1 transposon (89%) belonging to CACTA family, both fromTriticum monococcum, and toZingeria biebersteiniana pericentromeric sequences (78%). The similarity of JNK1 to those sequences may be accidental or the JNK1 may represent an ancient mobile genetic element that caught the 5S rRNA sequence. During the evolution those sequences might have been accumulated in the particular region on the 2R chromosome. Our results suggest that the additional heterochromatin band in chromosomes 2R ofS. vavilovii is a collection of defective genes and/or mobile genetic elements.

Genetic relatedness in different Medicago sativa lines based on polymorphism of mitochondrial DNA specific sequences

Two synthetic populations (Syn-5 and Syn-7), four inbred lines and four landraces of alfalfa (Medicago sativa L.) were studied for genetic relatedness. These forms were characterized by different amount of seeds set and green mass yield. Two primer pairs of specific nucleotide sequences of mitochondrial DNA isolated from soybean were used. The mtDNA revealed the existence of significant polymorphism among the investigated forms of alfalfa. The genetic similarity (Dice coefficient) among studied forms of alfalfa ranged from 20.1 to 96.1. The greatest resemblances were noticed between D5 inbred line and the population of Syn-5. The lowest resemblances were noticed between Syn-7 and E1/2. The UPGMA dendrogram split investigated forms of alfalfa into two groups: first group include three landraces, the second consist of the rest analyzed forms. There are two landraces distinct with the highest seed set and yield of green mass: one in the first group, another one in the second group.

Application of ISSR-PCR, IRAP-PCR, REMAP-PCR, and ITAP-PCR in the assessment of genomic changes in the early generation of triticale

Analysis of structural changes of octoploid triticale genomes was conducted in F2 and F3 generations. The plants were derived from crosses of five cultivars and breeding lines of hexaploid wheat (Triticum aestivum L.) with one cultivar of rye (Secale cereale L). The study used four marker systems: inter-simple sequence repeat (ISSR), inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP), and a technique named inter-transposon amplified polymorphism (ITAP) developed by the authors. Most frequently, elimination of specific bands was observed, especially of rye bands. Depending on the cross combination, the percentage of eliminated rye bands ranged from 73.6 to 80.6 %. A lower percentage of wheat bands was eliminated, i.e., from 57.6 to 76.48 %, depending on the combination of crosses. The emergence of new types of bands in hybrids absent in the parental forms was the rarest phenomenon (14.5–17.9 %). The results indicate the ongoing process of genome rearrangements at the molecular level in the early generations of plant crosses that also involve repeated nucleotide sequences of DNA.