Gabriella Linc

Detection of wheat-barley translocations by genomic in situ hybridization in derivatives of hybrids multiplied in vitro

Wheat-barley translocations were identified by genomicin situ hybridization (GISH) in backcross progenies originating from in vitro regenerated wheat (Triticum aestivum L. cv. Chinese Spring) × barley (Hordeum vulgare L. cv. Betzes) hybrids. The regenerated hybrids were pollinated with the wheat line Martonvásári 9 kr1. Five translocated wheat-barley chromosomes were recovered among 51 BC2F2 progeny from the in vitro regenerated wheat × barley hybrids. All were single breakpoint translocations with the relative positions of the breakpoints ranging from the centromere to about 0.8 of the relative arm length. Of the four translocations with intercalary breakpoints, three were transfers of terminal barley segments to wheat chromosomes; one was a transfer of a terminal wheat segment to a barley chromosome. Because of the absence of diagnostic N-bands, the identity of three barley segments could not be determined; in one translocation the barley chromosome involved had a NOR so it must have been 5H or 6H, and the centric translocation was 4HS.2BL. Following selfing, homozygotes of four translocations were selected. The experiment suggests that in vitro culture conditions are conducive for major genome rearrangements in wheat-barley hybrids.

Robertsonian translocations in wheat arise by centric misdivision of univalents at anaphase I and rejoining of broken centromeres during interkinesis of meiosis II

The mechanism of origin of Robertsonian translocations was investigated in plants monosomic for chromosome 1A of wheat and 1Ht of Elymus trachycaulus by GISH. Chromosomes 1A and 1Ht stayed univalent in all metaphase I cells analyzed, suggesting that Robertsonian translocations do not originate from meiotic recombination in centromeric regions with shared DNA sequence homology. At ana-/telophase I, the 1Ht and 1A univalents underwent either chromosome or chromatid segregation and misdivided in 6–7% of the pollen mother cells. None of the ana-/telophases I analyzed had Robertsonian translocations, which were only observed in 2% of the “half tetrads” at ana-/telophase II. The frequency of Robertsonian translocations observed at ana-/telophase II corresponds well with the number of Robertsonian translocations (1–4%) detected in progenies derived from plants monosomic for group-1 chromosomes of wheat (1A, 1B, and 1D) and 1Ht of E. trachycaulus. Our data suggest that Robertsonian translocations arise from centric misdivision of univalents at ana-/telophase I, followed by segregation of the derived telocentric chromosomes to the same nucleus, and fusion of the broken ends during the ensuing interkinesis.

Transfer of the recessive crossability allele kr1 from Chinese Spring into the winter wheat variety Martonvasari 9

SummaryThe recessive of crossability allele kr1 was transferred from the spring wheat variety Chinese Spring (CS) into the winter wheat variety Martonvásári 9 (Mv9) by backcrossing the Mv9 × CS hybrids with Mv9. The Mv9 variety possesses dominant Kr1 alleles and is heterogeneous at the kr2 locus, so that some individual plants carry recessive kr2 alleles. The selection of plants possessing the recessive kr alleles from the (Mv9 × CS)Mv9 BC1 generation was carried out according to the seed set achieved when pollinated with rye (Secale cereale L. cv. Mercator). The partial dominance of the Kr alleles made it possible to differentiate between plants heterozygous at the Kr1 locus and Kr1Kr1 homozygous dominant plants. Two selfed consecutive progenies were tested by pollination with rye to select the homozygous recessive kr1kr1kr2kr2 plants and to check the result of the selection after each backcross.As a result of three backcrosses with Mv9 and two selfings after each backcross the selected progenies had 61.6% seed set with rye tested on sixty individual plants. These data confirm that after the third backcross the selected Mv9 kr1 line carries necessive crossability alleles Kr1 and Kr2, but the genotype is 93.75% Mv9.

Inversions of chromosome arms 4AL and 2BS in wheat invert the patterns of chiasma distribution

In many species, including wheat, crossing over is distal, and the proximal regions of chromosome arms contribute little to genetic maps. This was thought to be a consequence of terminal initiation of synapsis favoring distal crossing over. However, in an inverted rye chromosome arm, the pattern of metaphase I chiasmata was also inverted, suggesting that crossover frequencies were specific to chromosome segments. Here, wheat chromosome arms 2BS and 4AL, with essentially entire arms inverted in reverse tandem duplications (rtd), were studied in the MI of meiosis. Inversion–duplication placed the recombining segments in the middle of the arms. While the overall pairing frequencies of the inverted–duplicated arms were considerably reduced relative to normal arms, chiasmata, if present, were always located in the same regions as in structurally normal arms, and relative chiasma frequencies remained the same. The frequencies of fragment or fragment + bridge configurations in AI and AII indicated that of the two tandemly arranged copies of segments in rtds, the more distal inverted segments were more likely to cross over than the segments in their original orientations. These observations show that also in wheat, relative crossover frequencies along chromosome arms are predetermined and independent of the segment location. The segments normally not licensed to cross over do not do so even when placed in seemingly most favorable positions for it.

A FISH karyotype to study chromosome polymorphisms for the Elytrigia elongata E genome

Elytrigia elongata (Host) Nevski(= Agropyron elongatum, Thinopyrum elongatum, 2n = 2x = 14, EE) has long been used as a source of various types of resistance for wheat improvement, and numerous transfers have been made. However, despite heavy use, no high-resolution karyotype exists. We characterized the E. elongata karyotype of several accessions applying highly repetitive DNA sequences as mcFISH probes for chromosome identification. The complete E. elongata disomic chromosome addition series and 11 ditelosomic addition lines in Chinese Spring wheat were exposed to sequential GISH-mcFISH. Based on the mcFISH results, each complete chromosome and each telocentric studied was unambiguously identified. The validation of the karyotype in 4 E. elongata accessions with different geographical origins showed extensive variations in the probe hybridization patterns, but this did not prevent chromosome identification. The established karyotype will be useful for the rapid identification of potential donor chromosomes in wheat improvement programs, allowing appropriate alien transfer.

Production and Molecular Cytogenetic Identification of Wheat-Alien Hybrids and Introgression Lines

Barley, rye, Aegilops and Thinopyrum (syn. Agropyron) species belonging to the Triticeae tribe have large genetic diversity and serve as a valuable genetic reservoir for wheat improvement. Many of these species have been used for more than a century for the production of wheat × alien hybrids and introgression lines. The most up-to-date molecular cytogenetic techniques make it possible to detect and identify alien chromosomes in the wheat genome. The first methods used to identify rye, barley, Aegilops and Thinopyrum chromosomes in the wheat genome were C- and N-banding. Genomic in situ hybridization (GISH) is the most accurate way of detecting the translocation breakpoint in introgression lines. Alien chromosomes can be identified in the wheat genome using fluorescence in situ hybridization (FISH) with the help of repetitive DNA probes.Multicolor GISH (mcGISH) was developed to demonstrate the various genomes in polyploid plant species and in interspecific and intergeneric hybrids, amphiploids and derivatives. Sequential GISH and FISH are useful methods for identifying alien translocations in the wheat genome.

Recreating stable Brachypodium hybridum allotetraploids by uniting the divergent genomes of B. distachyon and B. stacei

Brachypodium hybridum (2n = 30) is a natural allopolyploid with highly divergent sub-genomes derived from two extant diploid species, B. distachyon (2n = 10) and B. stacei (2n = 20) that differ in chromosome evolution and number. We created synthetic B. hybridum allotetraploids by hybridizing various lines of B. distachyon and B. stacei. The initial amphihaploid F1 interspecific hybrids were obtained at low frequencies when B. distachyon was used as the maternal parent (0.15% or 0.245% depending on the line used) and were sterile. No hybrids were obtained from reciprocal crosses or when autotetraploids of the parental species were crossed. Colchicine treatment was used to double the genome of the F1 amphihaploid lines leading to allotetraploids. The genome-doubled F1 plants produced a few S1 (first selfed generation) seeds after self-pollination. S1 plants from one parental combination (Bd3-1×Bsta5) were fertile and gave rise to further generations whereas those of another parental combination (Bd21×ABR114) were sterile, illustrating the importance of the parental lineages crossed. The synthetic allotetraploids were stable and resembled the natural B. hybridum at the phenotypic, cytogenetic and genomic levels. The successful creation of synthetic B. hybridum offers the possibility to study changes in genome structure and regulation at the earliest stages of allopolyploid formation in comparison with the parental species and natural B. hybridum.

Production and Molecular Cytogenetic Identification of New Winter Wheat/Winter Barley Disomic Addition Lines

Disomic wheat/barley addition lines were developed from hybrids produced with the German two-rowed winter barley cultivar ‘Igri’ and the Ukrainan six-rowed winter barley cultivar ‘Manas’. The 2H, 3H and 4H disomic addition lines of winter wheat ‘Martonvasari 9 kr1’/winter barley ‘Igri’ produced in Martonvasar were identified using GISH, FISH and SSR markers. A disomic addition of the 1HS isochromosome was also identified. The 2H, 3H and 4H addition lines have been multiplied in the nursery and characterized morphologically. Backcross progenies were produced and identified with molecular genetic and cytogenetic methods from the Japanese wheat ‘Asakaze komugi’ × Ukrainan six-rowed winter barley ‘Manas’ hybrids. A deletion and a translocated chromosome facilitated the physical localization of microsatellite markers on chromosome 5H. So far the 4H disomic addition line has been identified from the fertile BC2 plants in this combination, but the development of other disomic additions is in progress

Molecular cytogenetic (FISH) and genome analysis of diploid wheatgrasses and their phylogenetic relationship

This paper reports detailed FISH-based karyotypes for three diploid wheatgrass species Agropyron cristatum (L.) Beauv., Thinopyrum bessarabicum (Savul.&Rayss) A. Löve, Pseudoroegneria spicata (Pursh) A. Löve, the supposed ancestors of hexaploid Thinopyrum intermedium (Host) Barkworth & D.R.Dewey, compiled using DNA repeats and comparative genome analysis based on COS markers. Fluorescence in situ hybridization (FISH) with repetitive DNA probes proved suitable for the identification of individual chromosomes in the diploid JJ, StSt and PP genomes. Of the seven microsatellite markers tested only the (GAA)n trinucleotide sequence was appropriate for use as a single chromosome marker for the P. spicata AS chromosome. Based on COS marker analysis, the phylogenetic relationship between diploid wheatgrasses and the hexaploid bread wheat genomes was established. These findings confirmed that the J and E genomes are in neighbouring clusters.

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

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, Afa-family, 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.