Márta Molnár-Láng

Utilisation of Aegilops (goatgrass) species to widen the genetic diversity of cultivated wheat

Wild Aegilops species related to cultivated wheat (Triticum spp.) possess numerous genes of agronomic interest and can be valuable sources of resistance to diseases, pests and extreme environmental factors. These genes can be incorporated into the wheat genome via intergeneric crossing, following, where necessary, the development of chromosome addition and substitution lines from the resulting hybrids. The transfer of a single segment from an alien chromosome can be achieved by translocations. The Aegilops (goatgrass) species, which are the most closely related to wheat, exhibit great genetic diversity, the exploitation of which has been the subject of experimentation for more than a century. The present paper gives a survey of the results achieved to date in the field of wheat–Aegilops hybridisation and gene transfer. The Aegilops genus consists of 11 diploid, 10 tetraploid and 2 hexaploid species. Of these 23 Aegilops species, most of the diploids (Ae. umbellulata Zhuk., Ae. mutica Boiss., Ae. bicornis (Forssk.) Jaub. & Spach, Ae. searsii Feldman & Kislev ex Hammer, Ae. caudata L., Ae. sharonensis Eig, Ae. speltoides Tausch, Ae. longissima Schweinf. & Muschl.) and several polyploids (Ae. ventricosa Tausch, Ae. peregrina (Hack. In J. Fraser) Marie & Weiller, Ae. geniculata Roth, Ae. kotschyi Boiss., Ae. biuncialis L.) have been used to develop wheat–Aegilops addition lines. Wheat–Aegilops substitution lines were developed using several species, including Ae. umbellulata, Ae. caudata, Ae. tauschii, Ae. speltoides, Ae. sharonensis, Ae. longissima and Ae. geniculata. Translocations carrying genes responsible for useful agronomic traits were developed with Ae. umbellulata, Ae. comosa, Ae. ventricosa, Ae. longissima, Ae. speltoides and Ae. geniculata. A large number of genes were transferred from Aegilops species to cultivated wheat, including those for resistance to leaf rust, stem rust, yellow rust and powdery mildew, and various pests (cereal cyst nematode, root knot nematode, Hessian fly, greenbug). Many molecular markers are linked to these resistance genes. The development of new molecular markers is also underway. There are still many untapped genetic resources in Aegilops species that could be used as resistance sources for plant breeding.

Physiological and morphological responses to water stress in Aegilops biuncialis and Triticum aestivum genotypes with differing tolerance to drought

The physiological and morphological responses to water stress induced by polyethylene glycol (PEG) or by withholding water were investigated in Aegilops biuncialis Vis. genotypes differing in the annual rainfall of their habitat (1050, 550 and 225 mm year-1) and in Triticum aestivum L. wheat genotypes differing in drought tolerance. A decrease in the osmotic pressure of the nutrient solution from -0.027 to -1.8 MPa resulted in significant water loss, a low degree of stomatal closure and a decrease in the intercellular CO2 concentration (Ci) in Aegilops genotypes originating from dry habitats, while in wheat genotypes high osmotic stress increased stomatal closure, resulting in a low level of water loss and high Ci. Nevertheless, under saturating light at normal atmospheric CO2 levels, the rate of CO2 assimilation was higher for the Aegilops accessions, under high osmotic stress, than for the wheat genotypes. Moreover, in the wheat genotypes CO2 assimilation exhibited less or no O2 sensitivity. These physiological responses were manifested in changes in the growth rate and biomass production, since Aegilops (Ae550, Ae225) genotypes retained a higher growth rate (especially in the roots), biomass production and yield formation after drought stress than wheat. These results indicate that Aegilops genotypes, originating from a dry habitat have better drought tolerance than wheat, making them good candidates for improving the drought tolerance of wheat through intergeneric crossing.

Characterization of a leaf rust-resistant wheat–Thinopyrum ponticum partial amphiploid BE-1, using sequential multicolor GISH and FISH

In situ hybridization (multicolor GISH and FISH) was used to characterize the genomic composition of the wheat–Thinopyrum ponticum partial amphiploid BE-1. The amphiploid is a high-protein line having resistance to leaf rust (Puccinia recondita f. sp. tritici) and powdery mildew (Blumeria graminis f. sp. tritici) and has in total 56 chromosomes per cell. Multicolor GISH using J, A and D genomic probes showed 16 chromosomes originating from Thinopyrum ponticum and 14 A genome, 14 B genome and 12 D genome chromosomes. Six of the Th. ponticum chromosomes carried segments different from the J genome in their centromeric regions. It was demonstrated that these alien chromosome segments did not originate from the A, B or D genomes of wheat, so the translocation chromosomes were considered to be Js type chromosomes carrying segments similar to the S genome near the centromeres. Rearrangements between the A and D genomes of wheat were detected. FISH using Afa family, pSc119.2 and pTa71 probes allowed the identification of all the wheat chromosomes present and the determination of the chromosomes involved in the translocations. The 4A and 7A chromosomes were identified as being involved in intergenomic translocations. The replaced wheat chromosome was identified as 7D. The localization of these repetitive DNA clones on the Th. ponticum chromosomes of the amphiploid was described in the present study. On the basis of their multicolor FISH patterns, the alien chromosomes could be arranged in eight pairs and could also be differentiated unequivocally from each other.

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.

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.

Association between simple sequence repeat-rich chromosome regions and intergenomic translocation breakpoints in natural populations of allopolyploid wild wheats

BACKGROUND AND AIMS Repetitive DNA sequences are thought to be involved in the formation of chromosomal rearrangements. The aim of this study was to analyse the distribution of microsatellite clusters in Aegilops biuncialis and Aegilops geniculata, and its relationship with the intergenomic translocations in these allotetraploid species, wild genetic resources for wheat improvement. METHODS The chromosomal localization of (ACG)(n) and (GAA)(n) microsatellite sequences in Ae. biuncialis and Ae. geniculata and in their diploid progenitors Aegilops comosa and Aegilops umbellulata was investigated by sequential in situ hybridization with simple sequence repeat (SSR) probes and repeated DNA probes (pSc119·2, Afa family and pTa71) and by dual-colour genomic in situ hybridization (GISH). Thirty-two Ae. biuncialis and 19 Ae. geniculata accessions were screened by GISH for intergenomic translocations, which were further characterized by fluorescence in situ hybridization and GISH. KEY RESULTS Single pericentromeric (ACG)(n) signals were localized on most U and on some M genome chromosomes, whereas strong pericentromeric and several intercalary and telomeric (GAA)(n) sites were observed on the Aegilops chromosomes. Three Ae. biuncialis accessions carried 7U(b)-7M(b) reciprocal translocations and one had a 7U(b)-1M(b) rearrangement, while two Ae. geniculata accessions carried 7U(g)-1M(g) or 5U(g)-5M(g) translocations. Conspicuous (ACG)(n) and/or (GAA)(n) clusters were located near the translocation breakpoints in eight of the ten translocated chromosomes analysed, SSR bands and breakpoints being statistically located at the same chromosomal site in six of them. CONCLUSIONS Intergenomic translocation breakpoints are frequently mapped to SSR-rich chromosomal regions in the allopolyploid species examined, suggesting that microsatellite repeated DNA sequences might facilitate the formation of those chromosomal rearrangements. The (ACG)(n) and (GAA)(n) SSR motifs serve as additional chromosome markers for the karyotypic analysis of UM genome Aegilops species.

Genetic mapping of sequence-specific PCR-based markers on the short arm of the 1BL.1RS wheat-rye translocation

Wheat cultivars carrying the 1BL.1RStranslocation were crossed with newly synthesised octoploid triticale lines involving four rye genotypes having ο-secalin banding patterns different from each other and from that of the 1BL.1RS translocation. Homologous recombination was expected between the short arm of the 1R chromosomes of the rye genotypes and the 1RS arm of the 1BL.1RSwheat/rye translocation. Seven sequence-specific PCR-based markers:Xiag95, RMS13, Bmac0213, GPI, Xpsr960, 5Sand SCM9, and ο-secalinproteins were used to detect recombination events in the BC1F2 generation. Segregation analysis demonstrated that a barley SSR marker (Bmac0213) locus was present on the 1RS chromosome arm. Of 834plants tested in four different BC1F2 populations, 246individuals were found to carry recombined1BL.1RS translocation chromosomes. Genetic linkage analysis was performed on the eight markers in the four different mapping populations. The physical positions of the markers are discussed.

Chromosome isolation by flow sorting in Aegilops umbellulata and Ae. comosa and their allotetraploid hybrids Ae. biuncialis and Ae. geniculata.

This study evaluates the potential of flow cytometry for chromosome sorting in two wild diploid wheats Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata. Flow karyotypes obtained after the analysis of DAPI-stained chromosomes were characterized and content of chromosome peaks was determined. Peaks of chromosome 1U could be discriminated in flow karyotypes of Ae. umbellulata and Ae. biuncialis and the chromosome could be sorted with purities exceeding 95%. The remaining chromosomes formed composite peaks and could be sorted in groups of two to four. Twenty four wheat SSR markers were tested for their position on chromosomes of Ae. umbellulata and Ae. comosa using PCR on DNA amplified from flow-sorted chromosomes and genomic DNA of wheat-Ae. geniculata addition lines, respectively. Six SSR markers were located on particular Aegilops chromosomes using sorted chromosomes, thus confirming the usefulness of this approach for physical mapping. The SSR markers are suitable for marker assisted selection of wheat-Aegilops introgression lines. The results obtained in this work provide new opportunities for dissecting genomes of wild relatives of wheat with the aim to assist in alien gene transfer and discovery of novel genes for wheat improvement.

Mixing of maize and wheat genomic DNA by somatic hybridization in regenerated sterile maize plants.

Abstract.Intergeneric somatic hybridization was performed between albino maize (Zea mays L.) protoplasts and mesophyll protoplasts of wheat (Triticum aestivum L.) by polyethylene glycol (PEG) treatments. None of the parental protoplasts were able to produce green plants without fusion. The maize cells regenerated only rudimentary albino plantlets of limited viability, and the wheat mesophyll protoplasts were unable to divide. PEG-mediated fusion treatments resulted in hybrid cells with mixed cytoplasm. Six months after fusion green embryogenic calli were selected as putative hybrids. The first-regenerates were discovered as aborted embryos. Regeneration of intact, green, maize-like plants needed 6 months of further subcultures on hormone-free medium. These plants were sterile, although had both male and female flowers. The cytological analysis of cells from callus tissues and root tips revealed 56 chromosomes, but intact wheat chromosomes were not observed. Using total DNA from hybrid plants, three RAPD primer combinations produced bands resembling the wheat profile. Genomic in situ hybridization (GISH) using total wheat DNA as a probe revealed the presence of wheat DNA islands in the maize chromosomal background. The increased viability and the restored green color were the most-significant new traits as compared to the original maize parent. Other intermediate morphological traits of plants with hybrid origin were not found.

The effect of temperature on seed set and embryo development in reciprocal crosses of wheat and barley

SummaryThe effect of temperature on seed set and embryo development in reciprocal crosses of barley and wheat was assessed in crosses involving two spring barley varieties (Betzes, Martonvásári 50) and one wheat variety (Chinese Spring). Detached tillers placed in nutrient solution were pollinated in controlled environments at constant day-night temperature regimes (12, 15, 18 and 21° C) with a light intensity of 30,000 lux and a relative humidity of 80%. When barley was used as the female, lower temperatures (12 and 15° C) produced the maximum seed set, whereas for the reciprocal cross, the highest temperature (21° C) produced the best seed set in the Chinese Spring × Betzes combination.Low temperature retarded the embryo development. The highest numbers of hybrid plants were produced at 18° C and 21° C in the barley × wheat cross and in the wheat × barley cross, respectively. Embryos of about 1.5 mm length in the barley × wheat cross, and of about 1.0 mm length in the wheat × barley cross germinated successfully. The smallest embryo giving rise to hybrid plants was 0.57 mm in the barley × wheat cross and 0.51 mm in the wheat × barley cross.