New hybrids between Agropyron and wheat

Summary Intergeneric hybrids of Triticum aestivum (2n=42,AABBDD) with Agropyron ciliare (2n= 28,SSYY), A. trachycaulum (2n=28,SSHH), A. yezoense (2n=28,SSYY) and A. scirpeum (2n=28) are reported for the first time. F₁ hybrids of T. aestivum were also produced with A. intermedium (2n=42,E₁E₁E₂E₂Z₁Z₁) and A. junceum (2n=14,JuJu). All wheat-Agropyron hybrids were obtained by embryo rescue technique. Cultivars and reciprocal crosses differed for seed set, seed development and F₁ plant production. The F₁ hybrids were sterile. Attempts to obtain amphiploids were unsuccessful. However, backcross derivatives were obtained with wheat as the recurrent parent.The level of chromosome pairing in A. trachycaulum x wheat, A. yezoense x wheat and wheat x A. junceum hybrids provided no evidence of homologous or homoeologous pairing. Mean pairing frequencies in A. ciliare x wheat, wheat x A. scirpeum and wheat x A. intermedium hybrids indicated homoeologous or autosyndetic pairing. Ph gene was more effective in regulating homoeologous pairing in A. yezoense x wheat hybrids than in A. ciliare x wheat hybrid. Chromosome pairing data of BC₁ derivatives indicated that either some of the wheat chromosomes were eliminated or Agropyron chromosomes caused reduced pairing of wheat homologues.Contribution No. 82-653-J, Department of Plant Pathology, Kansas State Agricultural Experiment Station, Manhattan, Kan, USA     

Homoeologous relationships of Aegilops speltoides chromosomes to bread wheat

 Homoeologous pairing at metaphase I was analysed in the standard-type, ph2b and ph1b hybrids of Triticum aestivum (AABBDD) and Aegilops speltoides (SS). Data from relative pairing affinities were used to predict homoeologous relationships of Ae. speltoides chromosomes to wheat. Chromosomes of both species, and their arms, were identified by C-banding. The Ae. speltoides genotype carried genes that induced a high level of homoeologous pairing in the three types of hybrids analyzed. All arms of the seven chromosomes of the S genome showed normal homoeologous pairing, which implies that no apparent chromosome rearrangements occurred in the evolution of Ae. speltoides relative to wheat. A pattern of preferential pairing of two types, A-D and B-S, confirmed that the S genome is very closely related to the B genome of wheat. Although this pairing pattern was also reported in hybrids of wheat with Ae. longissima and Ae. sharonensis, a different behaviour was found in group 5 chromosomes. In the hybrids of Ae. speltoides, chromosome 5B-5S pairing was much more frequent than 5D-5S, while these chromosome associations reached similar frequencies in the hybrids of Ae. longissima and Ae. sharonensis. These results are in agreement with the hypothesis that the B genome of wheat is derived from Ae. speltoides. Received: 8 January 1998 / Accepted: 4 February 1998     

Hypothesis: Transgene establishment in wild relatives of wheat can be prevented by utilizing the Ph1 gene as a senso stricto chaperon to prevent homoeologous recombination

Durum and bread wheat need transgenic traits such as herbicide and disease resistance due to recent evolution of herbicide resistant grass weeds and an intractable new strain of stem rust. Transgenic wheat varieties have not been commercialized partly due to potential transgene movement to wild/weedy relatives, which occurs naturally to closely related Aegilops and other spp. Recombination does not occur in the F₁ hybrid between wheat and its relatives due to the presence of the Ph1 gene on wheat chromosome arm 5BL, which acts as a chaperone, preventing promiscuous homoeologous pairing to similar, but not homologous chromosomes of the wild/weedy species. Thus recombination must occur during backcrossing after the wheat Ph1 gene has been eliminated. Based on these findings, we speculate that Ph1 could be used to prevent gene introgression into weedy relatives. We propose two methods to prevent such transgene establishment: (1) link the transgene in proximity to the wheat Ph1 gene and (2) insert the transgene in tandem with the lethal barnase on any chromosome arm other than 5BL, and insert barstar, which suppresses barnase on chromosome arm 5BL in proximity to Ph1. The presence of Ph1 in backcross plants containing 5BL will prevent the homoeologous establishment of barnase coupled to the desired transgene in the wild population. 5BL itself will be eliminated during repeated backcrossing to the wild parent, and progeny bearing the desired transgene in tandem with barnase but without the Ph1-barstar complex will die.     

Production and cytogenetic analysis of BC1, BC2, and BC3 progenies of an intergeneric hybrid between Triticum aestivum (L.) Thell. and tetraploid Agropyron cristatum (L.) Gaertn.

Summary Intergeneric hybrids between Triticum aestivum cv Chinese Spring and Agropyron cristatum 4x (2n= 5x=35, ABDPP genomes) with a high level of homoeologous meiotic pairing between the wheat chromosomes were backcrossed 3 times to wheat. Pollination of the F₁ hybrid with Chinese Spring resulted in 22 BC₁ seeds with an average seed set of 1.52%. Five BC₁ plants with 39–41 chromosomes were raised using embryo rescue techniques. Chromosome pairing in the BC₁ was characterized by a high frequency of multivalent associations, but in spite of this there was no evidence of homoeologous pairing between chromosomes of wheat and those of Agropyron. All of the plants were self sterile. The embryo rescue technique was again essential to produce 39 BC₂ plants with chromosome numbers ranging from 37 to 67. The phenomenon of meiotic non-reduction was also observed in the BC₃ progenies. In this generation male and female fertility greatly increased, and meiotic pairing was fairly regular. Some monosomic (2n=43) and double monosomic (2n=44) lines were produced. Analysis of these progenies should permit the extraction of the seven possible wheat-Agropyron disomic addition lines including those with the added chromosomes carrying the genes involved in meiotic non-reduction and in suppression of Ph activity.     

Physical distribution of translocation breakpoints in homoeologous recombinants induced by the absence of the Ph1 gene in wheat and triticale

The physical distribution of translocation breakpoints was analyzed in homoeologous recombinants involving chromosomes 1A, 1B, 1D of wheat and 1R of rye, and the long arms of chromosome 7S of Aegilops speltoides and 7A of wheat. Recombination between homoeologues was induced by removal of the Ph1 gene. In all instances, translocation breakpoints were concentrated in the distal ends of the chromosome arms and were absent in the proximal halves of the arms. The relationship between the relative distance from the centromere and the relative homoeologous recombination frequency was best explained by the function f(x)=0.0091e^0.0592x. The pattern of recombination in homoeologous chromosomes was essentially the same as in homologues except that there were practically no double exchanges. Among 313 recombinant chromosomes, only one resulted from a double crossing-over. The distribution of translocation breakpoints in translocated arms indicated that positive chiasma interference operated in homoeologous recombination. This implies that the reduction of the length of alien chromosome segments present in translocations with wheat chromosomes may be more difficult than the production of the original recombinants.     

RFLP-based maps of the homoeologous group-6 chromosomes of wheat and their application in the tagging of Pm12, a powdery mildew resistance gene transferred from Aegilops speltoides to wheat

Genetic maps of the homoeologous group-6 chromosomes of bread wheat, Triticum aestivum, have been constructed spanning 103 cM on 6A, 90 cM on 6B and 124 cM on 6D. These maps were transferred to a Chinese Spring (CS) x line #31 cross to locate a dominant powdery mildew resistance gene, Pm12, introgressed into line #31 from Aegilops speltoides. Pm12 was shown to lie on the short arm of translocation chromosome 6BS-6SS.6SL in line #31, but could not be mapped more precisely due to the lack of recombination between the 6S Ae. speltoides segment and chromosome 6B. Possible strategies to reduce the size of the alien segment, which probably encompasses the complete long arm and more than 82% of the short arm of chromosome 6B, are discussed.     

The effect of B chromosomes on meiotic and pre-meiotic spindles and chromosome pairing in Triticum/Aegilops hybrids

Diploid populations of Aegilops mutica and Aegilops speltoides containing B chromosomes have been used as male parents in crosses with aneuploid genotypes of Triticum aestivum to investigate the effect of B chromosomes on meiotic homologous and homoeologous chromosome pairing. F₁ hybrids of T. aestivum/Ae. mutica and T. aestivum/Ae. speltoides segregated into four classes with regard to the degree of meiotic chromosome pairing, irrespective of the presence of B chromosomes. The B chromosomes do not introduce factors altering the level of pairing other than that due to the natural allelic and gene variation occurring in the diploids. Similarly no reduction in pairing of homologous chromosomes was observed in genotypes in which pairs of homologues co-existed with B chromosomes. However, a significant drop in chiasma frequency was observed in F₁ hybrids of T. aestivum × Ae. mutica with B chromosomes and T. aestivum × Ae. mutica nullisomic for wheat chromosome 5D with B chromosomes, in temperature regimes of 12° C. No asynapsis occurred in similar hybrids in the absence of Mutica B chromosomes at low temperatures. The low-temperature sensitive phase lies early in the pre-meiotic interphase. In this instance the Mutica B chromosomes are interacting with specific gene loci of the A chromosomes. Synaptic pairing has been observed between A and B chromosomes in Ae. mutica. A high frequency of pollen mother cells with twice the number of chromosomes was observed in hybrids in the presence of Mutica B chromosomes due to failure of spindle formation at the last pre-meiotic mitosis. Meiotic spindle irregularities occurred in hybrids containing Speltoides B chromosomes. Hybrids of Ae. speltoides + B's X Ae. mutica + B's displayed the mitotic and meiotic spindle abnormalities introduced by the presence of the B chromosomes of each parent.     

The effect of Ph gene alleles on synaptonemal complex formation in Triticum aestivum × T. kotschyi hybrids

Summary Chromosome pairing at zygotene-pachytene was studied in Triticum aestivum × T. kotschyi hybrids (2n=5x=35, genomic constitution ABDC^US^v) by electron microscopy of synaptonemal complexes in spread microsporocyte nuclei. Hybrids carrying either the Ph allele or the ph allele, which differ markedly in metaphase I pairing, are both capable of greater than 90% pachytene pairing, although pairing in the Ph hybrids appeared slower or less synchronised. In both genotypes branched synaptonemal complexes were formed by intra-and interchromosomal pairing. The Ph gene control on homoeologous pairing does not act on the ability to pair into synaptonemal complexes. It may act on the rate of pairing or the time of crossing over.     

Effectiveness of ph gene in inducing homoeologous chromosome pairing in agrotricum

Summary We observed pairing, when the ph gene was present, between wheat (Triticum aestivum L. em. Thell.) chromosome 4B, and an Agropyron intermedium (Host) Beauv. chromosome (Ai) carrying a gene resistant to wheat streak mosaic (WSM). In a monosomic addition polyhaploid [2n = 22 = 19' + 5B' (ph) + 4B' + Ai'], we recorded an average of 4.1 bivalents and 0.3 trivalents per cell. Induced homoeologous pairing was most effective when both 5B chromosomes carrying ph gene were present. Our data suggest that chromosome 4B of wheat and the Agropyron chromosome (Ai) carrying a gene for resistance to WSM are homoeologous and that it is possible to use either ph mutant or nullisomic 5B stock to induce genetic recombination between the two chromosomes.Contribution No. 1657-j, Kansas State Agric. Expt. Sta., Manhattan, KS. The research is partially supported by a grant from Kansas Wheat Commission     

C-banding in 6x-Triticale xSecale cereale L. hybrid cytogenetics

Summary The meiotic behaviour of F₁ hybrids of hexaploid Triticale that differed in their genotypic or chromosomic constitution, and diploid rye, was investigated. Meiotic analysis were done by Feulgen and C-banding staining methods. A differential desynaptic effect in the hybrids was detected and explained in terms of genetic differences in pairing regulators. The high homoeologous pairing (A-B wheat chromosomes and wheat-rye chromosomes) observed in the hybrids can be explained in terms of an inhibition of the effect of a single dose of thePh allele of the 5B chromosome produced by two doses of the 5R chromosome. The higher homoeologous pairing detected in the hybrid 188 x Canaleja could be the overall result of the balance between thePh diploidizing system (1 dose), the pairing promoter of the 5R chromosome (2 doses) and that of the 3D chromosome (1 dose coming from the parental line Triticale with the substitution 3R by 3D).     

Synthesis and evaluation of Triticum durum — T. monococcum amphiploids

Summary Nine Triticum durum — T. monococcum amphiploids (AABBA^mA^m) were synthesized by chromosome doubling of sterile triploid F₁ hybrids involving nine T. durum (AABB) cultivars and a T. monococcum (A^mA^m) line. The triploid F₁ hybrids had a range of 4–7 bivalents and 7–13 univalents per PMC. The synthetic amphiploids, however, showed a high degree of preferential pairing of chromosomes of the A genomes of diploid and tetraploid wheats. The amphiploids were meiotically stable and fully fertile. Superiority of four amphiploids for tiller number per plant, 100-grain weight, protein content and resistance to Karnal bunt demonstrated that these could either be commercially exploited as such after overcoming certain inherent defects or used to introgress desirable genes into durum and bread wheat cultivars. Methods for improvement of these amphiploids are discussed.     

Somatic hybrids between Brassica juncea (L). Czern. and Diplotaxis harra (Forsk.) Boiss and the generation of backcross progenies

An attempt to transfer genes from droughttolerant Diplotaxis harra, a wild relative of Brassica species, to an elite oil-yielding cultivar, B-85, of mustard (Brassica juncea) was made through protoplast fusion, as the two plant systems are sexually incompatible. By following the standard protocol for PEG-mediated protoplast fusion followed by high pH, high Ca⁺⁺, DMSO treatment and appropriate cell-culture technique, 16 presumptive somatic hybrid plants could be regenerated. Chromosomal analysis of four such somatic hybrids revealed that three of them were asymmetric. Analysis of morphological characters, meiotic chromosomes, and esterase isoenzyme pattern revealed that all the somatic hybrids were different from each other. Furthermore four chromosomes of each genome could undergo homoeologous pairing at meiosis indicating the possibilities for genetic recombination and chromosomal rearrangements. Irregular distribution of chromosomes at anaphase-II at meiosis has been a consistent feature of these plants. Eventually, pollen of all the somatic hybrids showed complete infertility preventing the recovery of any selfed seed. Nevertheless, ovule fertility of one somatic hybrid was not totally impaired as it had set some seeds upon backcrossing with the B. juncea parent. The esterase isoenzyme banding pattern of 24 individual progeny plants of this backcross provided evidence for their recombinant nature. It was thus confirmed that a transfer of genetic traits from Diplotaxis harra to B. juncea had indeed taken place. Furthermore, it was conceptualised that a transfer of alien genes through the protoplast-fusion technique is primarily possible in situations where meiotic pairing of the chromosomes of the two participating genomes generates recombinant gametocytes which can pass through subsequent filial generations.     

Diploidization and chromosomal pairing affinities in the tetraploid wheats and their putative amphiploid progenitor

Summary The genomes of the diploid wheats Triticum boeoticum and T. urartu are closely related, giving 7II in the f₁ hybrid (T^bT^u) and 8.4 (0–14) II + 2.5 (0–7) IV in the derived amphiploid (T^bT^bT^uT^u). The genomes of the tetraploid wheats are also closely related, giving up to 7II at the polyhaploid level (AB) in the absence of the gene Ph but 14II at the tetraploid level (AABB) in the normal presence of Ph. If the amphiploid is the progenitor of the tetraploids, one or the other homoeologue (T^b or T^u) in each of the 7 homoeologous groups (the 7 potential IV) must have differentiated with respect to pairing affinity in order to account for 14II in the tetraploid. Consequently, in tetraploid X amphiploid hybrids (T^bT^uAB) carrying the Ph gene from the tetraploid, the seven differentiated chromosomes (B) would be expected to give 7I while, on the basis of their observed chiasma frequency, T^b, T^u and the less differentiated A would be expected to give 4.17I + 3.57II + 3.23III), assuming homoeologous pairing. The expected chromosomal configuration freqencies at MI (11.17I + 3.57II + 3.23III) closely fit the observed values (11.22I + 3.45II + 3.19III + 0.071IV) for such hybrids (X² = 0.0046; P>0.99). Thus diploidization of the boeoticum-urartu amphiploid clearly could account for the origin of the tetraploid wheats. Furthermore, T. aestivum X amphiploid hybrids (T^bT^uABD) with and without Ph indicated that B as well as A chomosomes tended to pair with their presumed T^bT^u homologues in the absence of Ph. Other tests showed that the tetraploid wheats could not plausibly have originated from any postulated Triticum-Sitopsis (TTSS) parental combinations with or without such chromosomal differentiation.     

Synthesis and cytological characterization of trigeneric hybrids involving durum wheat,Thinopyrum bessarabicum,and Lophopyrum elongatum

Summary In an attempt to transfer genes for salt tolerance and other desirable traits from the diploid wheatgrasses, Thinopyrum bessarabicum (2n=2x=14; JJ genome) and Lophopyrum elongatum (2n=2x=14; EE genome), into durum wheat cv Langdon (2n=4x=28; AABB genomes), trigeneric hybrids with the genomic constitution ABJE were synthesized and cytologically characterized. C-banding analysis of somatic chromosomes of the A, B, J, and E genomes in the same cellular environment revealed distinct banding patterns; each of the 28 chromosomes could be identified. They differed in the total amount of constitutive heterochromatin. Total surface area and C-banded area of each chromosome were calculated. The B genome was the largest in size, followed by the J, A, and E genomes, and its chromosomes were also the most heavily banded. Only 25.8% of the total chromosome complement in 10 ABJE hybrids showed association, with mean arm-pairing frequency (c) values from 0.123 to 0.180 and chiasma frequencies from 3.36 to 5.02 per cell. The overall mean pairing was 0.004 ring IV + 0.046 chain IV + 0.236 III + 0.21 ring II + 2.95 rod II + 20.771. This is total pairing between chromosomes of different genomes, possibly between A and B, A and J, A and E, B and J, B and E, and J and E, in the presence of apparently functional pairing regulator Ph1. Because chromosome pairing in the presence of Ph1 seldom occurs between A and B, or between J and E, it was inferred that pairing between the wheat chromosomes and alien chromosomes occurred. The trigeneric hybrids with two genomes of wheat and one each of Thinopyrum and Lophopyrum should be useful in the production of cytogenetic stocks to facilitate the transfer of alien genes into wheat.     

The effect of ph mutations on homoeolgous pairing in hybrids of wheat with Triticum longissimum

Homoeologous pairing at metaphase-I was analyzed in wild-type, ph2b, and ph1b hybrids of wheat and a low-pairing type of T. longissimum in order to study the effect of ph mutations on the pairing of T. longissimum chromosomes with wheat chromosomes. Chromosomes of both species, and their arms, were identified by C-banding. The three types of hybrids, with low-, intermediate-, and high-pairing levels, respectively, exhibited a very similar pairing pattern which was characterized by the existence of two types, A-D and B-S¹, of preferential pairing. These results confirm that the S¹ genome of T. longissimum is closely related to the B genome of wheat. The possible use of ph1b and ph2b mutations in the transfer to wheat of genes from related species is discussed.     

Standard karyotype of Triticum searsii and its relationship with other S-genome species and common wheat

C-banding polymorphism was analyzed in 14 accessions of Triticum searsii from Israel, and a generalized idiogram of the species was established. One accession was homozygous for whole arm translocations T1S^sS·4S^sS and T1S^sL·4S^sL. C-banding analysis was also used to identify 7 T. aestivum cv Chinese Spring-T. searsii disomic chromosome addition lines, 14 ditelosomic chromosome addition lines, 21 disomic whole chromosome, and 31 ditelosomic chromosome substitution lines. The identity of these lines was further confirmed by meiotic pairing analysis. Sporophytic and gametophytic compensation tests were used to determine the homoeologous relationships of the T. searsii chromosomes. The results show that the T. searsii chromosomes do not compensate well for their wheat homoeologues. The C-banding patterns of T. searsii chromosomes are distinct from those of other S-genome species and from the B-genome chromosomes of wheat, indicating that T. searsii is not a direct B-genome donor species of T. turgidum and T. aestivum.Contribution No. 95-72-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas, USA     

Absence of qualitative genes controlling interspecific pairing in rye B chromosomes

Summary The meiotic behaviour of hybrids between Secale cereale carrying B chromosomes and S. vavilovii has been studied in order to estimate the effects of B chromosomes on hybrid meiotic pairing. The possible effect of Bs on the meiotic pairing of the offspring from backcrosses with S. vavilovii has been studied also. The results obtained clearly indicate that no detectable differences existed in chromosome pairing of hybrids with or without B chromosomes. The hypothetical existence of epistatic genes on cereale genome masking the effect of Bs has been rejected after the results obtained in backcrosses. Therefore, lack of qualitative genes controlling interspecific pairing on rye B chromosomes has been concluded. A quantitative effect of B chromosomes was detected only when they were in alien cytoplasm.     

Development of monosomic alien addition lines and introgression of genes from Oryza australiensis Domin. to cultivated rice O. sativa L.

Oryza australiensis, a diploid wild relative of cultivated rice, is an important source of resistance to brown planthopper (BPH) and bacterial blight (BB). Interspecific hybrids between three breeding lines of O. sativa (2n=24, AA) and four accessions of O. australiensis (2n=24, EE) were obtained through embryo rescue. The crossability ranged from 0.25% to 0.90%. The mean frequency of bivalents at diakinesis/metaphase I in F₁ hybrids (AE) was 2.29 to 4.85 with a range of 0–8 bivalents. F₁ hybrids were completely male sterile. We did not obtain any BC₁ progenies even after pollinating 20,234 spikelets of AE hybrids with O. sativa pollen. We crossed the artificially induced autotetraploid of an elite breeding line (IR31917-45-3-2) with O. australiensis (Acc. 100882) and, following embryo rescue, produced six F₁ hybrid plants (AAE). These triploid hybrids were backcrossed to O. sativa. The chromosome number of 16 BC₁ plants varied from 28 to 31, and all were male sterile. BC₂ plants had 24–28 chromosomes. Eight monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. australiensis were selected from the BC₂ F₂ progenies. The MAALs resembled the primary trisomies of O. sativa in morphology, and on the basis of this morphological similarity the MAALs were designated as MAAL-1, -4, -5, -7, -9, -10, -11, and -12. The identity of the alien chromosome was verified at the pachytene stage of meiosis. The alien chromosomes paired with the homoeologous pairs to form trivalents at a frequency of 13.2% to 24.0% at diakinesis and 7.5% to 18.5% at metaphase I. The female transmission rates of alien chromosomes varied from 4.2% to 37.2%, whereas three of the eight MAALs transmitted the alien chromosome through the male gametes. BC₂ progenies consisting of disomic and aneuploid plants were examined for the presence of O. australiensis traits. Alien introgression was detected for morphological traits, such as long awns, earliness, and Amp-3 and Est-2 allozymes. Of the 600 BC₂ F₄ progenies 4 were resistant to BPH and 1 to race 6 of BB. F₃ segregation data suggest that earliness is a recessive trait and that BPH resistance is monogenic recessive in two of the four lines but controlled by a dominant gene in the other two lines.     

Cytogenetic relationship ofAegilops longissima chromosomes with common wheat chromosomes

The relationships of three wheat-Aegilops longissima chromosome addition lines A, C, and D with homoeologous wheat chromosomes were studied in PMC meiosis. Substitutions of alien chromosome A for wheat chromosome 6 B, chromosome C for 1 B and chromosome D for 4 B were obtained. The production of 4 BS/C and 7 BS/D chromosome translocations indicated cytogenetic relationships of C partially to homoeologous wheat chromosomes of group 1 and 4, and D partially to homoeologous wheat chromosomes of group 4 and 7.     

Morphological, yield, cytological and molecular characterization of a bread wheat X tritordeum F1 hybrid

The morphological, yield, cytological and molecular characteristics of bread wheat X tritordeum F₁ hybrids (2n =6x = 42; AABBDH^ch) and their parents were analysed. Morphologically, these hybrids resembled the wheat parent. They were slightly bigger than both parents, had more spikelets per spike, and tillered more profusely. The hybrids are self-fertile but a reduction of average values of yield parameters was observed. For the cytological approach we used a double-target fluorescencein situ hybridization performed with total genomic DNA fromHordeum chilense L. and the ribosomal sequence pTa71. This technique allowed us to confirm the hybrid nature and to analyse chromosome pairing in this material. Our results showed that the expected complete homologous pairing (14 bivalents plus 14 univalents) was only observed in 9.59% of the pollen mother cells (PMCs) analysed. Some PMCs presented autosyndetic pairing of Hch and A, B or D chromosomes. The average number of univalents was higher in the wheat genome (6.8) than in the Hch genome (5.4). The maximum number of univalents per PMC was 20. We only observed wheat multivalents (one per PMC) but the frequency of trivalents (0.08) was higher than that of quadrivalents (0.058). We amplified 50 RAPD bands polymorphic between the F₁ hybrid and one of its parents, and 31 ISSR polymorphic bands. Both sets of markers proved to be reliable for DNA fingerprinting. The complementary use of morphological and yield analysis, molecular cytogenetic techniques and molecular markers allowed a more accurate evaluation and characterization of the hybrids analysed here.