The lg3 locus maps to the short arm of chromosome 3
--Yong Chi, John Fowler and Michael Freeling

Although lg3 is usually represented on the genetic map of chromosome 3 as being on the long arm of the chromosome, no definitive placement on either side of the centromere has previously been possible. Some data (R. S. Poethig, MNL 62:99, 1988) suggested that lg3 is on the short arm of chromosome 3. We have used a method described first by Weber and Helentjaris (Genetics 121:583-90, 1989) to locate lg3 to chromosome 3S.

Essentially, a DNA probe corresponding to the lg3 locus (Fowler and Freeling, unpublished) was used to determine the copy number of the lg3 locus in plants that were either hypoploid or hyperploid for various segments of chromosome 3. Genomic DNA from these plants was digested with various enzymes to give distinct RFLPs for both alleles that were potentially present, Southern blotted, and probed. If the probe corresponded to a locus that was located on a chromosomal segment for which a particular plant was hypoploid, only one band (DNA fragment) appeared; otherwise, two distinct bands were present. In the converse situation, a probe corresponding to a locus on a segment that was hyperploid in a plant yielded two bands, with the RFLP allele present in two copies producing a band twice as intense in signal as the other.

Plants hypoploid and hyperploid for the B-A translocation TB-3Sb were generated by crossing the translocation stock as a male to an h1 v19/ h1 + stock. Hypoploid embryos expressed the h1 (starchy) phenotype, and produced plants hyperploid for the translocated segment of 3S. Plants hypoploid for the same segment in the population were recognized by the expression of the v19 (virescent) phenotype. Testers homozygous for ys3 a1; R-scm were used as females in crosses with a TB3-La translocation stock; colorless endosperm/colored scutellum kernels produced 3L hyperploids and ys3 (yellow stripe) plants were hypoploid for the same segment of 3L.

A chromosome stock provided by K. L. Rose and R. W. Staub (Carleton College) was used to generate plants hypoploid for either the entire 3S or 3L chromosome arm. This stock resulted from the centric fission of chromosome 3, followed by recovery of two stable telocentric chromosomes corresponding to arms 3S and 3L. In the presence of B chromosomes, at a low frequency, one or the other of the telosomes is not transmitted to the zygote through the pollen, perhaps due to non-disjunction at the second microspore division (K.L. Rose and R.W. Staub, personal communication). This results in a plant hypoploid for the entire length of either chromosome arm. A plant carrying both telocentric 3 chromosomes was crossed as a male to several female testers, which were either heterozygous for the v19 marker on 3S or homozygous for the ys3 marker on 3L. One plant out of 90 in a population derived from the v19 tester cross expressed the v19 phenotype and was classified as a 3S hypoploid. One plant out of 37 in a population derived from the ys3 tester expressed the ys3 phenotype and was classified as a 3L hypoploid.

DNA from one of each of these types of plants (hypo- and hyperploid for TB-3Sb, hypo- and hyperploid for TB-3La, hypoploid for the entire 3S arm, and hypoploid for the entire 3L arm) was subjected to Southern analysis. When probed with the lg3 probe, all of the plants except the entire 3S hypoploid exhibited two bands, corresponding to the two alleles of lg3 present in each plant. The 3S hypoploid exhibited only one band, indicating only one copy of lg3 in this plant. These data indicate that lg3 is on the short arm of 3S, proximal to the TB-3Sb translocation breakpoint. The same filters were stripped and reprobed with both the umc92 RFLP probe (located on 3S distal to the TB-3Sb breakpoint) and the a1 gene probe (located on 3L distal to the TB-3La breakpoint) to confirm the chromosomal constitutions of these plants. In all cases, the predicted number and intensity of bands for each DNA sample was observed (i.e., umc92 picked up two bands in each of the plants aneuploid for segments of 3L, one band in the entire 3S and TB-3Sb hypoploids, and two bands, one twice as intense as the other, in the TB-3Sb hyperploid). 

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