In barley large sets of reciprocal translocations have been developed and widely used for studies of various problems related to basic and applied purposes in genetic and cytogenetic research. Reliable information on the chromosomal positions of the breakpoints essentially contributes to a more efficient utilization of translocations in general.

Identification of the chromosomes involved in a translocation is a routine practice since translocation tester sets, standardized on the basis of participating linkage groups, are available. Localization of breakpoints as to the chromosome arms or even more specified chromosome regions involved, requires additional efforts. The following methods have, singly or in combination, provided information to the current knowledge: (1) linkage tests relating breakpoints to known marker genes, (2) karyotype analyses of meiotic chromosomes, (3) analyses of hybrids from intercrosses between translocation lines that involve the same two chromosomes but with different break locations. With all these methods remains a certain risk for incorrect conclusions. This was demonstrated by, e.g., Linde-Laursen (1948) who found in a re-examination of 26 translocations by Giemsa C-banding of root-tip chromosomes several of the proposed breakpoint locations to be incorrect. Another limitation is evident from the fact that, as compared to chromosomes 5, 6 and 7, information about breakpoint locations on chromosomes 1, 2, 3, and 4 is relatively scarce since these chromosomes are not identifiable by conventional mitotic karyotype analysis.

The "Intercross Method" for locating breakpoints, as established for barley by Kasha and Burnham (1965) and further refined by Hagberg et al. (1978), makes use of the knowledge that meiotic pairing initiates at the ends of barley chromosomes. Therefore, hybrids from intercrosses between different translocation lines that involve the same two chromosomes form either seven bivalents (7 11) or one quadrivalent plus five bivalents (1 IV 5 11) in meiosis, depending on the positions of the breakpoints. The critical point is whether the two translocations have the same arrangement of chromosome ends (7 11) or differ with respect to the arrangement of chromosome ends (1 IV + 5 11). Via this route the parental translocations can be placed in two groups: those with breaks either in the short arms or in the long arms of both translocated chromosomes (s-s or 1-1) and those which have either one break in the short arm of the first chromosome and the other break in the long arm of the second chromosome or vice versa (s-1 or 1-s). Crosses within a group result in 7 IT whereas crosses between groups result in 1 IV + 5 11.

Crosses within the two groups, resulting in hybrids with 7 II, again fall into two subgroups: (1) Combinations with breakpoints in the same arms of both chromosomes (s-s × s-s and 1-1 × 1-1 or s-1 × s-1 and 1-s × 1-s). Hence, the translocated chromosomes from bivalents which are non- homologous as to the distances between the breaks on the respective chromosome arms; the central (centromere) regions are homologous in this case. (2) Combinations with breakpoints in different arms of both chromosomes (s-s × 1-1 and 1-1 × s-s or s-1 × 1-s and 1-s × s-1). Hence, the bivalents involving the translocated chromosomes comprise non-homologous centromere regions.

A safe subdivision into these subgroups would considerably improve the efficiency as to the localization of breakpoints by the "Intercross Method" for the following reason: If both breakpoint positions with respect to the chromosome arms are known for at least one of the translocations within one of the subgroups, intercrosses with any other translocation of the same subgroup can be used to assign the previously unknown breakpoints.

As indicated by, e.g., Kasha and Burnham (1965) and Hagberg et al. (1978), at least in cases of short breakpoint distances, sometimes a low degree of pollen sterility (less than 50%) in the hybrids is indicative of a combination with breakpoints in the same arms of both chromosomes (Subgroups 1). However, this criterion only in rare cases helps to differentiate between the two subgroups. Our experience with a large number of translocations gave no definitive conclusions for the majority of the combinations studied. This handicap may be overcome, at least for translocations with breakpoints outside the centromeric heterochromatin, by Giemsa banding if differential staining of the centromeric heterochromatin in the bivalents permits to discriminate between combinations producing seven homomorphically banded bivalents (subgroups 1) and combinations with five homomorphically plus two heteromorphically banded bivalents (subgroups 2).

Preliminary studies with a slightly modified Giemsa N- banding technique (Singh and Tsuchiya 1982) revealed more or less distinctly stained regions around the centromeres of the bivalents: intercalary bands were not detectable. A certain degree of variability in band size between the bivalents was observed. For a more detailed study a set of seven hybrids was produced, each of them heterozygous for three translocations involving six of the seven chromosome pairs, and thus containing only one definite bivalent each. Analysis of the seven individual bivalents revealed, as to the band sizes, an order of rank roughly corresponding to the band sizes near the centromeres in mitotic chromosomes. The order of bivalents with decreasing band sizes was found to be 4, 7, 3, 6, 1, 5, 2. The results indicate sufficient differences in band size to recognize heteromorphically banded bivalents for 12 out of the possible 21 different chromosome combinations, namely for translocations involving chromosomes 1-3, 1-4, 1-7, 2-3, 2-41 2-6, 2-7, 3-5, 4-5, 4-6, 5-7, and 6-7. For these translocations, hybrids between all of the lines involving the same two chromosomes and having breakpoints outside the centromeric heterochromatin should be classified as to one of the subgroups mentioned above. This should yield safe results as to chromosome arm involvement of breakpoints for any of these translocations, provided that one translocation per subgroups with breakpoint locations known from other sources of information is available.

The described method is expected to give reliable results only for translocations with breakpoints outside the centromeric heterochromatin. Therefore, in analyzing large sets of translocations a priority sequence of studies is recommended: (1) Identification of the chromosomes by translocation tester sets, (2) analyses of conventionally stained and/or Giemsa banded mitotic chromosomes, (3) use of the "Intercross Method" in connection with the proposed Giemsa banding of meiotic chromosomes.

Giemsa banding of mitotic chromosomes most reliably reveals the break positions involved in translocations if there is a transfer of diagnostically relevant bands. This applies to those translocations with one or both breakpoints inside the heterochromatin near the centromeres. Mitotic analyses, however, may fail to give results for translocations with both breakpoints located in the outermost one-third of the chromosome arms that lack prominent bands according to the presently available banding technique. This important category of translocations, most suitable, e.g., as testers in linkage studies, may become analyzable as to the chromosome arm involvement in translocations via the "Intercross Method" improved by Giemsa banding in meiosis as described above.

References:

Hagberg, A., L. Lehmann, and P. Hagberg. 1978. Segmental interchanges in barley. II. Translocations involving chromosomes 6 and 7. Z. Pflanzenzuchtg. 81:89-110.

Kasha, K. J., and C. R. Burnham. 1965. The location of interchange breakpoints in barley. II. Chromosome pairing and the intercross method. Can. J. Genet. Cytol. 7:620-632.

Linde-Laursen, 1. 1984. Breakpoints localized to chromosome arm or region in 26 translocation lines of barley using Giemsa C-banding. BGN 14:12-13.

Singh, R. J. and T. Tsuchiya. 1982. An improved Giemsa N- banding technique for the identification of barley chromosomes. J. Hered. 73:227-229.