Molecular and chromosomal characterization of repeated and single-copy DNA sequences in the genome of Dasypyrum villosum

Abstract

Restriction fragment length polymorphism of ribosomal DNA repeated unit and single‐copy DNA fragments and chromosomal distribution of a highly repeated sequence, have been studied to assess molecular markers and the extent of their heterogeneity in Dasypyrum villosum. Substantial variation has been found for the length of the intergenic spacer of ribosomal genes clustered in different alleles at Nor‐VI locus of heterozygous individuals, but not within the cluster of rDNA of homozygous individuals. After Southern blots and hybridization to an intergenic spacer probe, each cluster of rDNA was detected as a single band with at least four variants differing for the number of 130 bp subrepeats in the intergenic spacer. One recombinant plasmid contained a 2270 bp DNA insert from the D. villosum genome that upon Sph I restriction endonuclease digestion was cleaved in three 380 bp repeat elements and one 1090 bp fragment. When Southern blots of Sph I digested D. villosum DNAs of different genotypes were hybridized to the 32P‐labelled 380 bp repeat, a distinct ladder consisting of multiples of a basic repeat unit of about 380 bp in length was revealed on autoradiograms. The in situ hybridization of the 3H‐labelled 380 bp repeat element showed that one chromosome pair (7V) was not labelled. In the other pairs, silver grains remained clustered at or near the telomeres. Dot‐blot hybridization analysis of DNAs from a range of diploid, tetraploid, and hexaploid Triticeae species indicated that the 380 bp repeated element was a specific feature of the D. villosum genome. Other cloned DNA sequences of D. villosum showed a large restriction length polymorphism and one was located on V chromosomes.

Centromeric distribution of 350-family inDasypyrum villosumand its application to identifyingDasypyrumchromatin in the wheat genome

“…4). The main in situ hybridization sites were similar to that of 3 H‐labeled p380 sequence (De Pace et al 1992; Frediani et al 1994) and that of biotin‐labeled pHvNAU62 sequence (Li et al 1995). It is interested that pDvTU383 has a microsatellite sequence (TG) 7‐9 , which was much different with that in p380 or absent in pHvNAU62.…”

“…This sequence shares 67% homology with the rye 350‐family (Bedbrook et al 1980; Appels et al 1981, 1986). Sequence comparison proved that three clones, pDvTU383, p380 (De Pace et al 1992; Frediani et al 1994) and pHvNAU62 (Li et al 1995) belonged to the same family. These clones were variants of the 350‐family tandemly existed in the genome of D. villosum , because the ladder pattern of the gel blot indicated that the repetitive sequence organized a tandem array of a monomeric 380 bp unit (Fig.…”

“…On the other hand, homologous family had been found in other wheat related species, such as Agropyron cristatum (Appels et al 1989), A. intermedium (Xin and Appels 1988, Appels et al 1989, Ma et al 1994), and Elymus trachycaulus (Tsujimoto and Gill 1991). Also in D. villosum , De Pace et al (1992) identified a repetitive sequence p380, which showed tandem organization of a 380 bp unit. Frediani et al (1994) determined the sequence of p380, which showed 68.5% homology to the rye 350‐family, and Li et al (1995) also isolated a tandem repetitive clone pHvNAU62 (292 bp) from D. villosum , which shares 67% homology to the rye 350‐family.…”

“…However, in the genome of D. villosum , ISH results using radioisotope or biotin labeled 380 bp probes were unsatisfactory in distinguishing individual chromosomes (De Pace et al 1992; Li et al 1995). In this study, we report a new variation of the 350‐family sequences isolated from D. villosum , and its utilization for identifying D. villosum chromatins in the wheat genome by the fluorescence in situ hybridization with the 350‐family sequence.…”

Studies on genome relationship and species-specific PCR marker for Dasypyrum breviaristatum in Triticeae

“…The isolation of molecular markers specific to and ubiquitous to Dasypyrum were reported in several studies. D e P ace et al (1992) firstly isolated a 380 bp single copy sequence in D. villosum . L i et al (1995) cloned a 292 bp species‐specific repeated sequence pHvNAU62 in D. villosum , and localized it on six of seven chromosomes pairs of D. villosum , which gave signals of 24 sites by FISH.…”