Materials and methods
Plant material: Seed of 19 accessions belonging to 14 species from Triticum-Aegilops group was procured from PAU, Ludhiana (India) and used for the present study (see Table 1).
SSR probes: Four synthetic SSR oligonucleotide probes including three tri-nucleotide repeats {(CAC)5, (CAG)5, (TCC)5} and one di-nucleotide repeat {(GA)8} were used for in-gel hybridization.
SSR primers: For 13 of the 15 SSR primers, the details on sequences, repeat motifs, locus designations and the expected product sizes are published elsewhere (Prasad et al. 2000; Varshney et al. 2000a). The remaining two primer pairs belonging to WMC404 and WMC415 were developed by Romestant (RAGT, France) and are proprietary in nature (mromestant@ragt.fr).
DNA isolation, digestion, electrophoresis, in-gel hybridization and PCR: The details of methods for DNA isolation, restriction digestion (using AluI and HinfI, HindIII, DraI and EcoRI), electrophoresis, in-gel hybridization, PCR, and PAGE (polyacrylamide gel electrophoresis) are described elsewhere (Varshney et al. 1998; Prasad et al. 2000).
Statistical analysis: Polymorphic information content (PIC) for each SSR locus was calculated using the formula: PIC = 1-Σ(Pi)2, where Pi is the frequency of ith allele at a locus (Botstein et al. 1980).
Results and discussion
In-gel hybridization using synthetic oligonueleotide (SSR) probes: Thirteen (13) probe-enzyme combinations (involving four synthetic SSR probes each in combination with 2-4 enzymes) were used for in-gel hybridization. SSR sequences, homologous to each of the four synthetic probes, were available in all the 19 accessions belonging to 14 different species from Triticum-Aegilops group. This is in agreement with earlier reports suggesting ubiquitous distribution of SSRs in different plant and animal species (for a review, see Gupta et al. 1996). No intraspecific polymorphism was detected in the present study. Even in an earlier study, we found that such intraspecific polymorphism was rather rare (Varshney et al. 1998). This suggested that in-gel hybridization involving synthetic SSRs as probes may not be a suitable technique for detection of intraspecific polymorphism in members of the tribe Triticeae; however, in several other plant species, a high level of intraspecific polymorphism was actually observed (see Weising et al. 1995).
In the present study, 12 of the 13 probe-enzyme combinations hybridized with 1-4 bands against a clear background while the remaining solitary probe-enzyme combination {(GA)8-EcoRI} gave bands on in-lane smear background. In our earlier study on bread wheat also, out of the 142 probe-enzyme combinations, bands were available in 107 cases and a smear with high in-lane background was observed in 35 cases (Varshney et al. 1998). Using 12 different probe-enzyme combinations, a total of 20 bands were visualized (1-4 fragments per probe-enzyme combination) in 14 species. Only hexaploid wheat (T. aestivum, ABD genomes) and no other species gave all the bands, so that there was no unique fragment which wag absent in wheat and present in another species, although as many as four additional genomes occurred in these alien species. In the remaining 13 individual species, a total of 5 to 15 bands were visualized with all the probe-enzyme combinations considered together. In 9 of the above 12 probe-enzyme combinations, a solitary band of >23 kb was observed in 9 of the 14 species examined; in the five remaining species (Ae. kotschyii, Ae. sharonensis, Ae. cylindrica, T. boeoticum and T. urartu), this band was not observed in a few of these 9 probe-enzyme combinations, but other bands were available. In the remaining three of the 12 probe-enzyme combinations {HindIII-(CAC)5 AluI-(CAC)5 AluI-(CAG)5}, in accessions of several species, this characteristic band of>23 kb was observed in association with other bands ranging in size from 7 kb to 0.4 kb; in few species, however, one or more bands of smaller size were present. Similar high molecular weight bands (>21 kb to 30 kb) were also reported in earlier studies on barley, sugar beet and wheat (Beyermarin et al. 1992; Schmidt and Heslop-Harrison 1996; Varshney et al. 1998). The high molecular weight band (>23 kb) that was visualized with different probe- enzyme combinations, may represent same or different repeat sequences of >23 kb harboring SSRs. In a recent study in barley, characterization of clones harboring dinucleotide SSR repeats revealed that a high percentage of such clones are associated with retrotransposon-like and other dispersed repetitive elements (Ramsay et al. 1999). SSRs are, however. now known to be frequent in unique DNA sequences also (Morgante et al. 2002).