A procedure for detailed cytogenetic localizations and the production of small interstitial duplications

Improved procedures for the genetic detection of dosage series and cytogenetic localizations have previously been developed (J. L. Birchler, MNL 1979, 1980; Genetics 94:687; J. L. Birchler, M. Alleman and M. Freeling, Maydica, in press). These methods for chromosomal manipulation could be advanced further with the ability to induce and selectively recognize particular reciprocal translocations that are broken in predetermined regions of the genome. Here a system is described that will allow the recognition of newly induced translocations which will saturate a pre-selected chromosomal segment and provide the basis for finely detailed cytogenetic definition of practically any region of the maize chromosomes.

The procedure involves inducing the appropriate translocations to produce the desired aneuploids that include the alcohol dehydrogenase-1 gene as a marker and another region of interest. The system is possible because each segmental trisomic includes portions of two chromosome arms. The first requirement is the previous existence of one translocation broken in 1L and in the other chromosome arm at the appropriate point. If such exists, the second translocation, satisfying the necessary conditions to segregate segmental trisomics, can be induced and easily selected. These conditions have been outlined by Birchler (Genetics, 1980).

This is accomplished by using the translocation homozygote as a female for X-irradiated pollen from plants homozygous for Adh-Cm. This allele produces a polypeptide that is enzymatically very inactive and has a rare electrophoretic mobility. In pollen the in situ staining reaction (Freeling, Genetics 1976) for Cm is weak compared to normal, allowing ready classification. If no translocation or one involving another chromosome is induced, the F1 plants will segregate 1:1 for ADH positive and negative in the unaborted pollen grains. If, however, a translocation is induced that allows the production of overlapping segmental duplications including Adh, the ratio of ADH positive to ADH negative unaborted pollen will be 2:1. Skewed ratios might also be produced if a linked translocation is formed that segregates duplications adjacent to but excluding Adh. Whether these would be skewed toward positive or negative grains depends upon the relative position of the translocations used in the selection and the newly induced one. The two types of duplications, i.e., including or excluding Adh, can be discerned by subjecting an extract of the pollen to electrophoresis. If Adh is present in duplicated form, the Cm subunit will form dimers with the other allele present in the same gametophyte. Since pollen ADH is formed after meiosis (D. Schwartz, Genetics 1971), the presence of heterodimers is indicative of duplicated gametes. The remaining situations that produce skewed ratios of + and - grains would not result in heterodimer formation. Thus, the appropriate breakpoints can be recognized. After selection of the F1 plants on the basis of pollen phenotype, the Cm allele will serve as a marker in embryo classification. Although this polypeptide lacks normal levels of activity, it forms a distinct active heterodimer that is sufficient for genetic manipulation. This approach shows promise for genetic dissection in maize because it will allow the genetic (as opposed to cytological) recognition and marking of segmental aneuploids with previously defined cytological limits for any region of the genome. Using this protocol one can induce and recover a host of aberrations in a region of interest that can be subsequently used for very precise cytological study of selected chromosomal segments.

J. A. Birchler


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