In situ hibridizáció különböző módszereinek adaptálása és továbbfejlesztése búza genetikai alapanyagok elemzésére = Adaptation and development of different methods of in situ hybridization for the analysis of wheat genetic stocks

Pályázatunk során a legfontosabb célkitűzésünk volt olyan in situ hibridizációs módszerek adaptálása és továbbfejlesztése laboratóriumunkban, amelyek alkalmasak transzgének kimutatására is, valamint a csoportunkban korábban faj- és nemzetségkeresztezéssel létrehozott közeli rokonságban lévő hibridekben és azok származékaiban az idegen fajból származó kromoszómák kimutatása. A fiber-FISH technikát sikerrel adaptáltuk a molekuláris citogenetika csoportban. A kihúzott DNS fonalakat előállítottuk izolált sejtmagokból ill. flow-sorted kromoszómákból. Megkezdtük a különböző technikákkal elkészített preparátumok és a módszer tesztelését, egymástól eltérő, fluorokrómokkal jelölt próbát használtunk, amely a transzgént különböző formában tartalmazta. Sikerrel oldottuk meg a búza-Ae. biuncialis hibridek származékaiban az Ae. biuncialis kromoszómák kimutatását genomikus in situ hibridizációval (GISH). Bevezettük a PRINS in situ hibridizációs technikát (primed in situ labeling). PCR alkalmazásával előállított GAA trinukleotid szekvenciák segítségével végeztük el az Ae. biuncialis kromoszómák azonosítását a búza genomjában. | The main aim of the project was to adapt and improve in situ hybridisation methods that were suitable for the detection of transgenes, and to detect chromosomes from alien species in closely related hybrids previously developed by the team from interspecific and intergeneric crosses, and in their progeny. The fibre-FISH technique was successfully adapted by the molecular cytogenetics team. Extended DNA fibres were produced from isolated cell nuclei or from flow-sorted chromosomes. Work has begun on the testing of the method and of preparations made using various techniques using various probes labelled with fluorochromes and containing the transgene in various forms. The Ae. biuncialis chromosomes in derivatives of wheat?Ae. biuncialis hybrids were successfully detected using genomic in situ hybridisation (GISH). The PRINS in situ hybridisation technique (primed in situ labelling) was also introduced. With the aid of GAA trinucleotide sequences developed using PCR, the Ae. biuncialis chromosomes in the wheat genome have been analyzed

Molecular cytogenetic characterisation of Salix viminalis L. using repetitive DNA sequences

Abstract Salix viminalis L. (2n=38) is a diploid dicot species belonging to the Salix genus of the Salicaceae family. This short-rotation woody crop is one of the most important renewable bioenergy resources worldwide. In breeding for high biomass productivity, limited knowledge is available on the molecular cytogenetics of willow, which could be combined with genetic linkage mapping. The present paper describes the adaptation of a fluorescence in situ hybridisation (FISH) protocol as a new approach to analyse the genomic constitution of Salix viminalis using the heterologous DNA clones pSc119.2, pTa71, pTa794, pAs1, Afafamily, pAl1, HT100.3, ZCF1 and the GAA microsatellite marker. Three of the nine probes showed unambiguous signals on the metaphase chromosomes. FISH analysis with the pTa71 probe detected one major 18S-5.8S-26S rDNA locus on the short arm of one chromosome pair; however, the pTa794 rDNA site was not visible. One chromosome pair showed a distinct signal around the centromeric region after FISH with the telomere-specific DNA clone HT100.3. Two chromosome pairs were found to have pAs1 FISH signals, which represent a D-genome-specific insert from Aegilops tauschii. Based on the FISH study, a set of chromosomes with characteristic patterns is presented, which could be used to establish the karyotype of willow species

Interspecifikus kromoszóma átépülések indukálása és felhasználásuk a kromoszómák fizikai térképezésére a búzában = Induction of interspecific chromosome rearrangements and their use for the physical mapping of chromosomes in wheat

A pályázat célja idegen fajú kromoszómákat, szegmentumokat hordozó búza genotípusok előállítása volt, majd azok azonosítása fluoreszcens in situ hibridizációval (FISH) és molekuláris markerekkel. A pályázati munkában az árpából (Hordeum vulgare), egy kecskebúza (Aegilops buncialis) és egy tarackbúzafajból (Agropyron glael) történő génátvitelre koncentráltunk. Őszi búza/őszi árpa (Mv9 kr1/Igri) 2H, 3H, 4H és 1HS izokromoszómás diszómás addíciós vonalakat állítottunk elő és azonosítottunk FISH-sel illetve molekuláris markerekkel. A 4H(4D) búza(árpa) szubsztitúciós vonalból PhI szuppresszor gént hordozó búzavonallal végzett megporzással búza/árpa transzlokációkat hoztunk létre és mutattunk ki GISH-sel. Az Asakaze komugi × Manasz (búza/őszi árpa) hibrid szövettenyészetben való elszaporítása után az utódokban 3H, 5H deléciós és búza/árpa transzlokációs kromoszómákat mutattunk ki GISH-sel és térképeztünk fizikailag is molekuláris markerekkel. Besugárzással búza × Aegilops biuncialis amfiploidokban intergenomikus kromoszóma átrendeződéseket hoztunk létre és mutattunk ki multikolor GISH-sel, megkülönböztetve a búza és az Ae. biuncialis U és M genomját. A búza × Agropyron glael hibridből szövettenyészetben való elszaporítás után számos utódot állítottunk elő. Az Agropyron kromoszómák kimutatására a GISH módszer adaptálását egy búza-A. elongatum részleges amphidiploidon végeztük, amelyben intergenomikus átrendeződéseket is detektáltunk | The main objective of the present proposal was to produce wheat genotypes with alien chromosomes (segments) and to identify them with fluorescence in situ hybridization (FISH) and molecular markers. Alien gene transfer was carried out from barley (Hordeum vulgare), from a goatgrass (Aegilops biuncialis) and from an Agropyron species (A. glael). 2H, 3H, 4H and 1HS wheat/barley (Mv9 kr1/Igri) disomic addition lines were produced and identified with FISH and using molecular markers. Wheat/barley translocation lines were produced from the 4H(4D) substitution line by pollination with a wheat line carrying the PhI suppressor gene, and they were detected by GISH. 3H, 5H deletion and wheat/barley translocation chromosomes were detected by GISH in the progeny of Asakaze komugi × Manas (winter barley) hybrids after multiplication in tissue culture. The chromosomes were physically mapped using GISH and molecular markers. Intergenomic rearrangements were induced from wheat × Aegilops biuncialis amphiploids using irradiation. The translocations were demonstrated using multicolour GISH, which differentiated the wheat genome and the U and M genomes of Ae. biuncialis. Several progenies were produced from the wheat × Agropyron glael hybrids after multiplication in tissue culture. A modification of the GISH technique, allowing Agropyron chromosomes to be detected, was carried out on a wheat×Agropyron partial amphiploid, in which intergenomic translocations were observed

A termesztett búza diploid őseinek molekuláris citogenetikai elemzése: pachytén- és fiber-FISH = Molecular cytogenetic characterization of diploid donor genomes of cultivated wheat: pachytene- and fibre-FISH

A fluoreszcensz in situ hibridizáció (FISH) lehetővé teszi számunkra, hogy adott DNS szekvenciákat közvetlenül a sejten belül, a kromoszómákon mutassunk ki, illetve azonosítsunk. Nagy felbontású citogenetikai térképek elkészítésére legalkalmasabb technika a pachytén-FISH. Pályázatunk során legfontosabb célunk volt a pachytén-FISH adaptálása a martonvásári Génmegőrzés és Organikus Nemesítés Osztály Molekuláris Citogenetika Laboratóriumában. A termesztett búza diploid őseinek több mint tíz genotípusát folyamatosan szaporítottuk, majd az izolált portokokat használtuk a pachytén-FISH kísérletekhez, sorozatos előkezelést követően. Genomikus in situ hibridizáció (GISH) segítségével az egymástól eltérő genomok DNS-e jól megkülönböztethető. A módszer alkalmazásával pontosan azonosítható és nyomon követhető volt egy Lolium×Festuca hibrid sejtjeiben a meiotikus pachytén fázis. Sikerrel oldottuk meg a pachytén-FISH módszerének adaptálását a termesztett búza diploid ősein. A technika tesztelése során, egyedi DNS szekvenciákat mutattunk ki egyes diploid genomok pachytén kromoszómáin (pl. Dx5 gén). Megkezdtük a termesztett búza modell növénye, a Brachypodium distachyon genomból származó DNS klónok fizikai térképezését a búza diploid donor genomjainak pachytén fázisban lévő kromoszómáin. A pályázat során végzett kísérletek hozzájárulhatnak a hexpaloid búza genom szekvencia adatainak pontos fizikai térképezéséhez és kromoszómális azonosításához. | Fluorescence in situ hybridization technique (FISH) makes it possible to detect and identify certain DNA sequences directly in the cell and along the chromosomes. The most suitable method for producing high resolution cytogenetic maps is pachytene-FISH. The main goal of the project was to implement pachytene-FISH technology in the Department of Plant Genetic Resources and Organic Breeding, Martonvásár. More than 10 accessions of the diploid donor genome of bread wheat were increased and the isolated anthers were used after different pre-treatments for pachytene-FISH experiments. Based on genomic in situ hybridization (GISH) genomes with different origin can be distinguished unequivocally. GISH technique was used to follow and analyze I. meiotic pachytene phase in a Lolium×Festuca hybrid. The pachytene-FISH method was successfully adapted to the diploid donor genome of bread wheat. During testing the pachytene-FISH method, several single copy DNA sequences (Dx5 etc.) were detected in diploid wheat chromosomes. Physical mapping of DNA clones from the model organism of bread wheat, Brachypodium distachyon has been started in the pachytene chromosomes of the diploid donor genome of hexaploid wheat. The adaptation of the pachytene-FISH and our results could redound the fine physical mapping and chromosomal analysis of sequence data of hexaploid wheat

Wheat-barley hybridization – the last forty years

Abstract Several useful alien gene transfers have been reported from related species into wheat (Triticum aestivum), but very few publications have dealt with the development of wheat/barley (Hordeum vulgare) introgression lines. An overview is given here of wheat 9 barley hybridization over the last forty years, including the development of wheat 9 barley hybrids, and of addition and translocation lines with various barley cultivars. A short summary is also given of the wheat 9 barley hybrids produced with other Hordeum species. The meiotic pairing behaviour of wheat 9 barley hybrids is presented, with special regard to the detection of wheat– barley homoeologous pairing using the molecular cytogenetic technique GISH. The effect of in vitro multiplication on the genome composition of intergeneric hybrids is discussed, and the production and characterization of the latest wheat/barley translocation lines are presented. An overview of the agronomical traits (b-glucan content, earliness, salt tolerance, sprouting resistance, etc.) of the newly developed introgression lines is given. The exploitation and possible use of wheat/barley introgression lines for the most up-to-date molecular genetic studies (transcriptome analysis, sequencing of flow-sorted chromosomes) are also discussed

Identification of COS markers specific for Thinopyrum elongatum chromosomes preliminary revealed high level of macrosyntenic relationship between the wheat and Th. elongatum genomes.

Thinopyrum elongatum (Host) D.R. Dewey has served as an important gene source for wheat breeding improvement for many years. The exact characterization of its chromosomes is important for the detailed analysis of prebreeding materials produced with this species. The major aim of this study was to identify and characterize new molecular markers to be used for the rapid analysis of E genome chromatin in wheat background. Sixty of the 169 conserved orthologous set (COS) markers tested on diverse wheat-Th. elongatum disomic/ditelosomic addition lines were assigned to various Th. elongatum chromosomes and will be used for marker-assisted selection. The macrosyntenic relationship between the wheat and Th. elongatum genomes was investigated using EST sequences. Several rearrangements were revealed in homoeologous chromosome groups 2, 5, 6 and 7, while chromosomes 1 and 4 were conserved. Molecular cytogenetic and marker analysis showed the presence of rearranged chromosome involved in 6ES and 2EL arms in the 6E disomic addition line. The selected chromosome arm-specific COS markers will make it possible to identify gene introgressions in breeding programmes and will also be useful in the development of new chromosome-specific markers, evolutionary analysis and gene mapping

Molecular cytogenetic and morphological characterization of two wheat-barley translocation lines.

Barley chromosome 5H, carrying important QTLs for plant adaptation and tolerance to abiotic stresses, is extremely instable in the wheat genetic background and is eliminated in the early generations of wheat-barley crosses. A spontaneous wheat-barley 5HS-7DS.7DL translocation was previously obtained among the progenies of the Mv9kr1 x Igri hybrid. The present work reports on the transfer of the 5HS-7DS.7DL translocation into a modern wheat cultivar, Mv Bodri, in order to use it in the wheat breeding program. The comparison of the hybridization bands of DNA repeats HvT01, pTa71, (GAA)n and the barley centromere-specific (AGGGAG)n in Igri barley and the 5HS-7DS.7DL translocation, together with the visualization of the barley chromatin made it possible to determine the size of the introgressed barley segment, which was approximately 74% of the whole 5HS. Of the 29 newly developed PCR markers, whose source ESTs were selected from the Genome Zipper of barley chromosome 5H, 23 were mapped in the introgressed 1-0.26 FL 5HS bin, three were located in the missing C-0.26 FL region, while three markers were specific for 5HL. The translocation breakpoint was flanked by markers Hv7502 and Hv3949. A comparison of the parental wheat cultivars and the wheat-barley introgression lines indicated that the presence of the translocation improved tillering ability in the Mv9kr1 and Mv Bodri genetic background. The similar or better yield components under high- or low-input cultivation environments, respectively, indicated that the 5HS-7DS.7DL translocation had little or no negative effect on yield components, making it a promising genotype to improve wheat genetic diversity. These results promise to accelerate functional genomic studies on barley chromosome 5H and to support pre-breeding and breeding research on wheat

Out-of-position telomeres in meiotic leptotene appear responsible for chiasmate pairing in an inversion heterozygote in wheat (Triticum aestivum L.)

Chromosome pairing in meiosis usually starts in the vicinity of the telomere attachment to the nuclear membrane and congregation of telomeres in the leptotene bouquet is believed responsible for bringing homologue pairs together. In a heterozygote for an inversion of a rye (Secale cereale L.) chromosome arm in wheat, a distal segment of the normal homologue is capable of chiasmate pairing with its counterpart in the inverted arm, located near the centromere. Using 3D imaging confocal microscopy, we observed that some telomeres failed to be incorporated into the bouquet and occupied various positions throughout the entire volume of the nucleus, including the centromere pole. Rye telomeres appeared ca. 21 times more likely to fail to be included in the telomere bouquet than wheat telomeres. The frequency of the out-of-bouquet rye telomere position in leptotene was virtually identical to the frequency of telomeres deviating from Rabl's orientation in the nuclei of somatic cells, and was similar to the frequency of synapsis of the normal and inverted chromosome arms, but lower than the MI pairing frequency of segments of these two arms normally positioned across the volume of the nucleus. Out-of-position placement of the rye telomeres may be responsible for reduced MI pairing of rye chromosomes in hybrids with wheat and their disproportionate contribution to aneuploidy, but appears responsible for initiating chiasmate pairing of distantly positioned segments of homology in an inversion heterozygote