SSR-based genetic linkage map of Cucurbita moschata and its synteny with Cucurbita pepo.

The first SSR-based genetic linkage map of Cucurbita moschata was created by integrating the maps of two F2 populations with one common parent developed from the crosses Waltham Butternut (WB) x Nigerian Local (NL) and ZHOU (a hull-less type) x WB. The integrated C. moschata map comprises 205 SSR markers and two morphological traits (Gr and n). The map is composed of 27 linkage groups with a marker density of 7 cM. Comparing the C. moschata map with the published Cucurbita pepo map, we found a high level of macrosynteny. Seventy-two of 76 common SSR markers between C. moschata and C. pepo were located in homologous linkage groups. These markers in general have conserved orders and similar genetic distances; they represent orthologous loci. A reference map based on these SSRs was obtained. No major chromosomal rearrangement between the two species could be detected at present, although four SSR markers were mapped in nonhomologous linkage groups. The comparative alignment of SSR markers did not provide any indication of a possible ancient polyploid origin of the species. The comparative mapping of C. moschata and C. pepo reported here will be useful for further studies on Cucurbit evolution, gene isolation, and breeding work.

Microsatellites for the genus Cucurbita and an SSR-based genetic linkage map of Cucurbita pepo L.

Until recently, only a few microsatellites have been available for Cucurbita, thus their development is highly desirable. The Austrian oil-pumpkin variety Gleisdorfer Olkürbis (C. pepo subsp. pepo) and the C. moschata cultivar Soler (Puerto Rico) were used for SSR development. SSR-enriched partial genomic libraries were established and 2,400 clones were sequenced. Of these 1,058 (44%) contained an SSR at least four repeats long. Primers were designed for 532 SSRs; 500 primer pairs produced fragments of expected size. Of these, 405 (81%) amplified polymorphic fragments in a set of 12 genotypes: three C. moschata, one C. ecuadorensis, and eight C. pepo representing all eight cultivar groups. On an average, C. pepo and C. moschata produced 3.3 alleles per primer pair, showing high inter-species transferability. There were 187 SSR markers detecting polymorphism between the USA oil-pumpkin variety "Lady Godiva" (O5) and the Italian crookneck variety "Bianco Friulano" (CN), which are the parents of our previous F(2) mapping population. It has been used to construct the first published C. pepo map, containing mainly RAPD and AFLP markers. Now the updated map comprises 178 SSRs, 244 AFLPs, 230 RAPDs, five SCARs, and two morphological traits (h and B). It contains 20 linkage groups with a map density of 2.9 cM. The observed genome coverage (Co) is 86.8%.

Genetic and physical mapping of sequence-specific amplified polymorphic (SSAP) markers on the 1RS chromosome arm of rye in a wheat background.

Three rye-specific repeated sequences, pSc10C, pSc20H and R173-1, were used to design sequence-specific anchored primers. These primers and 16 restriction site-specific adaptor primers were used in all possible combinations to establish sequence-specific amplified polymorphic (SSAP) markers for the 1RS chromosome arm of rye in a wheat background. Thirty 1RS-specific SSAP markers were detected in 19 primer combinations. Along with six markers localised previously on 1RS, 26 of the SSAP markers were mapped genetically in wheat genotypes carrying recombinant 1BL.1RS translocations. A clear decrease in recombination frequency from distal to proximal regions was observed. Wheat-rye addition lines for the 1R chromosome with different-sized deletions of the short arm were used to physically localise these markers. Physical mapping suggested an even distribution of the SSAP markers along the total length of the 1RS chromosome arm.

Analysis of relationships between Aegilops tauschii and the D genome of wheat utilizing microsatellites.

Sixty Aegilops tauschii accessions and 60 European hexaploid wheat varieties were analyzed with 14 wheat microsatellite (WMS) primer sets to (i) study the phylogeny of Ae. tauschii, (ii) search for a specific genotype of Ae. tauschii most closely related to the D genome of hexaploid wheat, and (iii) narrow down the presumed birthplace of the latter. An average of 6.5 and 4.0 alleles per locus was detected in Ae. tauschii and in wheat, respectively. The highest genetic diversity of Ae. tauschii was found in Transcaucasia and southeast of the Caspian Sea. Distribution of the 87 alleles (without null alleles) found in Aegilops did not allow differentiation of the species into the two subspecies strangulata and tauschii. Excluding null alleles, 41 alleles occurred parallel in wheat and in Aegilops. Data obtained in this study supports the view of the D genome of hexaploid wheat being a composite of several sources but does not support subsp. strangulata as the possible major source of the D genome. The highest number of region-specific alleles (three) in Ae. tauschii occurring also in the D genome of wheat, and therefore most indicative for its evolution was found in present-day Georgia, where subsp. strangulata is not endemic.

Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat-rye addition lines.

Flow cytometric DNA analysis was used to study changes in nuclear DNA content induced by the addition of complete or telosomic rye chromosomes into the genome of common wheat (Triticum aestivum L.). The DNA content of each addition line was determined by comparison with an internal reference value and was expressed as a difference with respect to the original wheat parental line. A 1.84% difference in the DNA content could be detected. Nuclei were flow sorted and the presence of rye chromatin in the nuclei with the higher DNA content was demonstrated by Southern hybridization. Flow cytometry was proven to be sensitive enough to detect the small DNA content deviations that are expected to occur in aneuploid plants of wheat.

Use of RFLPs to determine the chromosome composition of tetraploid triticale (A/B)(A/B)RR.

Tetraploid triticale, (A/B)(A/B)RR (2n = 28), is a botanical novelty, an amphiploid composed of a diploid rye and a 14 chromosome wheat genome made up of chromosomes of the A and B genomes of tetraploid wheat. Restriction fragment length polymorphism (RFLP) markers were used to elucidate the chromosome composition of the mixed wheat genome of 35 different tetraploid triticale lines. Of 128 possible A/B chromosome pair combinations, only 6 were found among these lines, with a prevalence of the 1A, 2A, 3B, 4B, 5B, 6B, and 7B karyotype. In most triticale lines stable wheat genomes made up of only homologous A or B genome chromosome pairs were identified, however, in some lines homoeologous chromosome pairs were found. In this paper we demonstrate that RFLPs can be used successfully as an alternative to C-banding for the identification of the chromosome composition of tetraploid triticale and discuss the possible selective advantage of specific chromosome composition.

Hybrid necrosis in triticale and the expression of necrosis genes in allopolyploids.

The occurrence in triticale of four different genes causing hybrid necrosis is described: Ne1 and Ne2 in the B genome of wheat and Ner1 and Ner2 in the rye genome. Hybrid necrosis develops due to dominant complementary interaction of two genes. This interaction in triticale, however, may take place not only between genes belonging to the same genome but also between genes of different genomes. In triticale, these genes can cause hybrid necrosis in four different combinations. The inheritance of the phenomenon in triticale is, therefore, more complicated than it is in wheat or rye. To avoid hybrid necrosis in triticale, attention should be paid that no necrosis genes are introduced into the primary triticale stocks from the wheat and rye parents. The expression of necrosis genes is influenced by the level of ploidy. Any additional genome - A, B, D, or R - may exert a suppressing effect on the expression of necrosis genes. Therefore, when identifying genotypes of triticale with regard to their necrosis genes, the level of ploidy has to be accounted for. Moreover, the present results illustrate that gene expression in polyploids is not only determined by interactions with other single genes but that it may also be modified by the total genotype of the respective individual.

Effects of constitutive heterochromatin and genotype on frequency and distribution of chiasmata in the seven individual rye bivalents.

Number and distribution of chiasmata were studied in the single pair of homologous rye chromosomes in 29 chromosomal F1 hybrids between the seven disomic wheat rye addition lines of 'Chinese Spring'/ 'Imperial' and five selected inbred genotypes of cultivated rye by using the differential Giemsa staining technique. The results indicate that the number and position of chiasmata is independent from the amount and position of C-heterochromatin. Genotype had an effect on chiasma number, whereas chiasma distribution within bivalents appeared to be determined by morphological features of chromosomes. Late replicating DNA in constitutive heterochromatin may delay the separation of half bivalents if chiasmata are formed between them and the centromere.

Genetic interactions between wheat and rye genomes in triticale : 1. Cytological results.

Six primary triticale lines were produced from two advanced breeding lines of Triticum durum and three inbred genotypes of Secale cereale. The wheat and rye parents and the triticale derivatives were crossed in all possible combinations within each species group. Chiasma and univalent frequency of parents and hybrids were determined. The primary triticale lines had more univalents and less chiasmata per pollen mother cell than the corresponding wheat and rye parents together. The parental wheat F1 exhibited negative heterosis for chiasma frequency whereas all rye hybrids had much higher chiasma frequencies than their inbred parents. Triticale F1s generally showed lower chiasma frequencies and more univalents than their parents, but the degree of pairing failure was dependent upon which of the parental species within the triticale, wheat or rye, was in the heterozygous state. F1s with heterozygous wheat genome only showed the least reduction in chiasma number (presumably caused by gene actions within the wheat genome), while F1s with heterozygous rye genome showed high reduction in chiasma frequency and an increase in pairing failure (induced by negative interactions between the heterozygous rye and the wheat genome in triticale). A high correlation was found between the frequency of undisturbed pollen mother cells and the frequency of aneuploids in the subsequent generation. A higher number of aneuploids occurred in those populations which were heterozygous for the rye genome.

Genetic interactions between wheat and rye genomes in triticale : 2. Morphological and yield characters.

Six primary triticale lines were produced from two advanced breeding lines of Triticum durum and three inbred genotypes of Secale cereale. The wheat and rye parents as well as the triticale derivatives were then crossed each within the same group of species in all possible combinations. Parents, F1s, and F2 populations were used to study the inheritance of a set of morphological and yield characters. The results suggest that, in general, in triticale allelic interactions within the wheat and the rye components are suppressed in favour of intergenomic interactions. Heterozygosity in the rye genome appeared to be detrimental for triticale, whereas heterozygosity in the wheat genome only resulted in positive interactions between certain characters. The retention of a high level of heterosis for kernels per spike in the F2 generation of such hybrids indicates interactions between genes which are in the homozygous state in the rye but remain in the heterozygous state in the wheat genome. If heterozygosity in the wheat component occurs between genes in the A and B genomes (homoeoalleles), it can be fixed in true breeding lines. This condition may ultimately lead to the fixation of their benefical effect on the interaction between wheat and rye genome. Aneuploidy caused a significant decline in performance for most of the investigated characters.

Chiasma distribution in rye chromosomes of diploid rye and in wheat/rye addition lines in relation to C-heterochromatin.

In five genetically different inbred lines of rye and in the seven 'Chinese Spring'/'Imperial' wheatrye addition lines, chiasma distribution in rye chromosomes was studied with respect to the amount and position of constitutive heterochromatin (Giemsa C-bands). In all inbred lines, rye chromosomes with one primary terminal band were more frequently found as univalents than those with primary bands on both telomeres. These chromosomes were most probably 5R and/or 6R. In the addition lines a highly significant reduction in the number of arms bound by chiasmata was found for rye chromosomes 5R and 6R. Because of the similar chiasma distribution in the inbred lines and in the rye chromosomes of the addition lines, no effect of the wheat genome on the number of chiasmata in the rye chromosomes can be ascertained. However, a relationship between chiasma frequency and chromosome arm length seems to exist, since under reduced chiasma conditions the two shortest arms of the rye complement, those of chromosomes 5R and 6R, frequently fail to form a chiasma. No effect of the large blocks of constitutive heterochromatin in the telomeres of the rye chromosomes on the position of chiasmata within a bivalent could be established.

Origin of nuclear aberrations and seed shrivelling in triticale: a re-evaluation of the role of C-heterochromatin.

The effect of C-heterochromatin on the origin of nuclear aberrations and seed shrivelling was investigated in four triticale lines, each consisting of a pair of genotypes designated A (producing plump, well-filled seeds) and B (with shrivelled seeds). The relative DNA content in the polyploid nuclei of endosperms, 42 h after pollination, was estimated by Feulgen cytophotometry. The observed frequency of polyploid nuclei, 0.85% and 5.69%, respectively, in the two genotypes 1A and 1B caused a reduction in nuclear number of 3.27% and 18.54% at this stage of development. In the B genotype, producing shrivelled grains, polyploidisation started earlier than in the A genotype. An examination of the Giemsa karyotype of the mitotic chromosomes of the rye genome in the four triticale pairs revealed no considerable differences in the banding pattern between the A and B genotypes. Giemsa staining of endosperms, 2-3 days after pollination, clearly showed that bridges without bands, most probably involving wheat chromosomes, were also present. An experiment designed to simulate spindle disturbances in developing endosperms by colchicine treatment revealed that polyploid nuclei can be formed by spindle malfunctions as well.

Localization of Structural and Regulatory Genes for Phosphodiesterase in Wheat (TRITICUM AESTIVUM).

Genes (Pde-A3; Pde-B3; Pde-D3) for phosphodiesterase (PDE; E.C. 3.1.4.1.) isoenzymes in hexaploid wheat were located on the three homoeologous chromosomes of group 3 by testing the electrophoretic banding pattern of monosomic, nullisomic and nullisomic/tetrasomic compensation lines of "Chinese Spring" variety. In plants nullisomic for chromosome 5B, the 3D structural gene is not expressed and this lack of expression can be overcome by four doses of either homoeologous chromosome 5A or 5D. Our data conclusively indicate that there are genes on group 5 chromosomes which positively control the expression of the 3D structural gene. In addition, the expression of the "regulatory genes" is dosage dependent. Thus, our study reveals a complex interaction of the three genomes of wheat for regulation of PDE gene expression.