II. 33. Combining chromosomes: the construction of "2-chromosome, double interchanges."
C. E. Fastnaught-McGriff and R. T. Ramage. Department of Plant Sciences, College of Agriculture, University of Arizona, Tucson, Arizona 85721, U.S.A. "R"
Translocations have been used as a tool in the breeding and genetics of barley for close to thirty years. At the suggestion of Dr. C. R. Burnham, we would like to pursue the possibility of using a new type of multiple translocation in a much used method of detecting linkage, the linkage tester set. This new multiple translocation has been designated a "2-chromosome, double interchange" (Burnham, 1968). The new interchanged chromosomes can be illustrated using fictitious chromosomes 5 and 6:
55556666066666555 6666555505555666
These could be written in a shorthand notation as 565 and 656. Because an interchange breakpoint is located on all four arms of the two chromosomes involved, the "2-chromosome, double interchange" would be more effective than single interchanges in detecting linkage over the entire chromosome. For the same reason, it may prove useful in other ways.
The construction of the "2-chromosome, double interchange" was first suggested for use in maize (Burnham, 1968). The method of construction entails crossing two single reciprocal translocations involving the same two chromosomes, but having the interchange breakpoints in the opposite arms. This is referred to as an "opposite arms" type intercross. The presence of the double interchange in the F2 is dependent on crossing over occurring in the differential segments in the F1, which in turn is dependent on the type of pairing at meiosis in the F1, this being dependent on the length of the differential segments of the parental interchanges. Two types of pairing have been observed in maize. When the differential segments were less than .3 of the arms, the ends paired homologously and the differential segments paired non-homologously, resulting in 7II's at metaphase I. When the differential segments were greater than .3 of the arms, the differential segments paired homologously in some of the cells, resulting in associations of four and 7II's at metaphase I. Nine "2-chromosome, double interchanges" have been isolated in maize and tests indicate that they seem to be effective in detecting linkage (Kowles, 1972).
Kasha (1965) found only 7II's at metaphase I in F1's of "opposite arms" intercrosses in barley. This suggests that pairing is initiated only at the ends in barley and that the differential segments could not pair homologously. However, a multiple translocation of the "2-chromosome, double interchange" type was recently identified by N. A. Tuleen fram intercrosses in barley. The morphology of the double interchange chromosomes was distinguishable from the parental interchanges. Tuleen isolated a plant heterozygous for the double interchange by examining root tips of F2 progeny from the cross of T3-7c with T3-7d (personal communication).
One-hundred and twenty-four barley translocations have been used in "opposite arms" intercrosses. The F1's are being grown this winter. F1 microsporocytes will be examined for any indication of homologous pairing. This may be in the form of an association of four or possibly a "pair" in which the differential segments pair and the ends do not. The F1 will be crossed to a standard normal male sterile. Examination of the progeny should confirm our observations of the meiotic configurations in the F1. If we observe only 7III's in a certain F1, then we would expect all the progeny to be heterozygous for one of the parental exchanges and exhibit about 25% sterility. If homologous pairing is evident, we would expect two additional classes of sterility in the progeny, representing the recombinants. One of the recombinants would be normal and fertile. The second recombinant would be the "2-chromosome, double interchange" which is expected to be about 50% sterile when heterozygous.
The availability of one double interchange in barley suggests that in some "opposite arms" intercrosses, regions of the differential segments may be paired homologously. This is possible if a small percentage of pairing is initiated in the proximal regions as has been suggested in maize (Burnham, et al., 1972). The desired multiple interchange is a product of two single crossovers in the same cell, one in each of the differential segments. Thus, in most cases, the "2-chromosome, double interchange" will be isolated only if a large number of progeny are examined. However, this insures that when used in a linkage tester set, very little crossing over will occur in the interstitial region.
The lack of crossing over in the region between the two breakpoints suggests other uses for this double interchange. Genes on both sides of the centromere could be transferred from one line to another. The procedure would be identical to the one used with single interchanges. A gene located in the interstitial region would always be associated with the interchange and 50% sterility. Though such a gene may not have an easily recognized phenotype, it could be "locked" into a population by selecting only the 50% sterile plants to maintain the population. Work of this type has been started by R. F. Eslick in Montana (personal communication).
Our goal is to produce a linkage tester set consisting of five double interchanges which includes all seven chromosomes. In the F2 of the tester set crossed with an unlocated gene, plants will segregate in a 1:1 ratio for fertility vs. partial sterility. Segregation of the unlocated gene within these two classes will indicate linkage. If a gene is not linked to an interchange, it will segregate in a 1:2:1 genotypic ratio within each class. If a gene is absolutely linked to an interchange, recessives will not be found in the partially sterile class. If a gene is not absolutely linked to the interchange, the number of recessives in the partially sterile class will indicate how close the linkage is.
Once a complete set has been isolated, tests will be made to determine the efficiency of the "2-chromosome, double interchange" in assigning a gene to a chromosome. In the future, it may be possible that only five crosses will be necessary to achieve such a task.
References:
Burnham, C. R. 1968. A new method of using interchange as chromosome markers. Crop Science 8:357-360.
Kowles, R. V. 1972. Maize Genetics Cooperation Newsletter. 46:127.
Kasha, K. J. and Burnham, C. R. 1965. The location of interchange breakpoints in barley. II. Chromosme Pairing and the Intercross Method. Canadian Journal of Genetics and Cytology. 7:620-632.
Burnham, C. R., Stout, J. T., Weinheimer, W. H., Kowles, R. V. and Phillips, R. L. 1972. Chromosome pairing in maize. Genetics 71:111-126.