A goal of this laboratory is to ascertain the molecular mechanisms controlling paramutation and the expression of the B gene, a regulatory locus involved in anthocyanin pigment synthesis in plant tissues. A necessary first step toward analyzing the B locus is to clone the genomic sequences. The B gene product is unknown and is likely to be present in small quantities; therefore, transposon tagging appears to be the best approach for cloning this gene. We have been using Robertson's Mutator as a transposon tag for this purpose. Several years ago Mutator stocks were obtained from D. Robertson and used to isolate 4 variegated mutations at B-Peru (V. Chandler and V. Walbot), an allele that specifies anthocyanin pigment in the aleurone and embryo. The mutations were isolated at frequencies of 1-3 X 10-5, while in control experiments the spontaneous mutation frequency of B-Peru was determined to be <5 x 10-6. All 4 alleles have been mapped to the B locus on chromosome 2S using 3-point testcrosses. In this report we describe the phenotypes and segregation properties of these B-Peru alleles. In a separate report in this issue we describe our molecular analyses of these insertion mutations.
The phenotypes of the progenitor B-Peru allele and the 4 Mutator-induced
variegated alleles are described in the accompanying table. All of the
independently isolated insertions affect both the kernel and plant tissues,
suggesting that each insertion is in a region of the gene required for
expression in all tissues. Three of the mutant alleles have colorless backgrounds,
while 1 allele has pale pigment in both kernel and plant tissues. Each
mutant allele has a characteristic pattern of somatic reversion that is
observed in both kernel and plant tissues.
|Allele||Kernel Phenotype||Plant Phenotype|
|B-Peru||purple aleurone &||spotted irregular|
|embryo||pigment on leaf sheaths|
|and plant stalk; strong|
|red pigment on tassel|
|branches and glumes|
|b-Perumu5||colorless background;||green plant; tassel has|
|low frequency of tiny||low frequency of tiny re-|
|revertant sectors||vertant sectors|
|b-Perumu216||colorless background;||green plant; tassel has|
|medium frequency of||revertant sectors of|
|tiny to medium sized||variable size and|
|b-Perumu218||colorless background;||green plant; tassel and|
|high frequency of tiny||plant with high|
|to large revertant||frequency of revertant|
|sectors||sectors of variable size|
|b-Perumu220||pale background;||pale pigment in tassel|
|medium frequency of||and plant with small re-|
|tiny revertant sectors||vertant sectors of vari-|
Most characterized Mutator-induced mutations have been the result of a Mu1 element insertion into the gene of interest. The typical reversion pattern of these alleles shows small and frequent somatic reversion events and rare germline reversion. The transmission of several Mu1 - induced alleles through several generations has been reported. Typically the allele segregates as a single gene (when heterozygous, 1:1 segregation in outcrossed progeny and 3:1 in selfed progeny), with occasional plants transmitting more colorless kernels than expected to progeny (J.L. Bennetzen, V.L. Chandler, M. Freeling, D.S. Robertson, V. Sundaresan, and V. Walbot). The colorless kernels may still contain the mutant allele, but the element is no longer able to cause somatic reversion. DNA modification of Mu1 elements has been correlated with this loss of somatic reversion for two mutant alleles (bz2-mu1, Chandler and Walbot; a Mu1 insertion at bz1, Sundaresan and Freeling).
To determine the segregation properties of the 4 unstable B-Peru alleles, each has been outcrossed for several generations to a b r-g tester (W23/K55 hybrid) and the ratios of spotted, colorless and purple kernels determined. The genetic properties of each allele are summarized below.
b-Perumu5: This allele segregates as a single gene, and ears that transmit fewer spotted kernels than expected are rarely observed. No loss of somatic instability was observed after 4 generations of self-pollination and 2 generations of outcrossing to the b r-g tester. In the next 5 sequential outcrosses of b-Perumu5/b individuals to the b r-g tester, 9 in 10 ears showed the expected segregation: 50% spotted, 50% colorless (.95>P>.5). One in 10 outcrossed ears had an increased number of colorless kernels, usually 60-70% instead of the expected 50% (P<.01). With more than 105 kernels examined no germline revertants have been recovered from plants transmitting the typical pattern of infrequent, tiny somatic revertant sectors.
The b-Perumu5 allele has a similar phenotype and transmission pattern to several other Mutator-induced alleles, although variants showing different patterns of somatic and germline reversion of b-Perumu5 have been isolated. In progeny from both outcrosses and self-pollinations, kernels with more frequent and larger somatic revertant sectors are observed in approximately 1/300 kernels. Plants resulting from these kernels also produce progeny with larger somatic revertant sectors and produce germline revertants at a frequency of approximately 10-4. In addition, we have identified 2 genetic stocks that contain a factor or factors that cause a dramatic alteration in this allele's phenotype: every kernel receiving the b-Perumu5 allele and the factors contains frequent and large somatic revertant sectors, and germline revertants are isolated at frequencies between 10-1 and 10-3 (G. Patterson and V. Chandler). Bedinger et al. have reported another type of background effect on the b-Perumu5 allele. When b-Perumu5 is crossed by certain maize inbreds fewer spotted kernels are transmitted than expected (MNL, this issue). These results suggest that different backgrounds contain factors that can increase or decrease the activity of the element at b-Perumu5.
b-Perumu216: This allele has never segregated as expected for a single gene. In all selfed and outcrossed progeny fewer spotted kernels are observed than predicted for a single heterozygous gene. Three classes of segregation ratios are observed upon outcrossing a b-Perumu2l6/b plant to a b r-g tester. For example, in one generation a total of 32 ears were examined; 18 of the ears contained 3/4 colorless, 1/4 spotted kernels (.95>P>.2); 6 ears contained 5/8 colorless, 3/8 spotted kernels (.95>P>.3); and 8 ears contained more than 80% colorless kernels (P<.001 that the deviation from 3:1 or 1:1 is due to chance alone). Purple kernels are observed in each class and range from 0-2% of the kernels on each ear.
When the b-Perumu216 allele is transmitted as female, approximately 1 in 25 plants contain large ear sectors of germline revertants, and I in 8 plants contain large ear sectors of colorless kernels. This demonstrates that reversion and inactivation can happen early in ear development. Small sectors of revertant tissue are frequently observed in the tassel, but rarely observed in the plant body. In about 1 in 200 plants larger revertant sectors of 1/8 to 1/32 of the tassel are observed as well as sectors on the plant body.
The b-Perumu216 allele does not have the phenotype and segregation properties of the typical Mutator-induced mutation. The class segregating 3/4 colorless, 1/4 spotted may represent the segregation of an unlinked regulatory locus necessary for the somatic reversion of the element at b-Perumu216, and the class segregating 5/8 colorless, 3/8 spotted may represent the segregation of two unlinked regulatory loci. The class with greater than 80% colorless kernels may represent the inactivation of the element at b-Perumu216 or the regulatory gene. Alternatively, the observed segregation ratios may not represent the segregation of unlinked regulator genes, but may represent a high frequency of inactivation of the element at b-Perumu216. Whatever the mechanism, these segregation patterns are reproducible in that they have been observed every generation for 5 sequential outcrosses.
b-Perumu218: This allele segregates as a single gene, with plants heterozygous for the allele producing 50% spotted and purple kernels upon outcrossing and 75% spotted and purple kernels upon selfing (.95>P>.2). Ears that have an increased number of colorless kernels (P<.01) are rarely observed (I in 15 ears). However, the b-Perumu218 allele is unstable, producing a large number of revertant kernels each generation. The number of revertants on each ear varies between 5 and 2517c of the kernels, and when the b-Perumu218 allele is transmitted as female large sectors of revertant kernels are seen in approximately I in 7 ears. No sectors of colorless kernels have been observed. Somatic revertant sectors are also frequently observed in the tassel and plant body. The large and frequent somatic sectors and the high frequency of germline revertants is not typical of most Mutator-induced mutations, but is similar to that observed for b-Perumu5 in certain backgrounds.
b-Perumu220: The original b-Perumu220/b kernel had a pale pigmented background with tiny dark purple sectors. When planted and outcrossed to b r-g tester 4 kernel phenotypes were observed: pale purple with darker revertant sectors, colorless with revertant sectors, uniformly pigmented with no sectors, and colorless. The same 4 phenotypes were observed upon self-pollination. In the original crosses 2 of 4 outcrossed ears segregated as expected for a single gene, with 50% of the kernels spotted and purple and 50% colorless (.7>P>.5). However, the other 2 outcrossed ears segregated 65% and 70% colorless kernels (P<.05). The uniformly pigmented kernels varied in intensity, ranging from pale to dark purple, and their phenotypes were stable in subsequent generations, suggesting they were partial and full revertants, respectively. The spotted kernels with colorless or pigmented backgrounds have given rise to all 4 kernel classes in 2 subsequent generations of outcrossing. The uniformly pigmented kernels accounted for 1-3% of the ear, and sectors of revertant kernels are observed in 1 in 25 ears. Approximately 1/3 of the ears segregate 50% spotted and purple and 50% colorless kernels. The other ears contain larger numbers of colorless kernels, usually in the range of 55-65% of the total kernels. No ear sectors of colorless kernels have been observed. The coloration of plants arising from the variegated kernels is also highly variable, but the majority of plants have pale pigmentation and small, infrequent revertant sectors. The somatic reversion frequency and kernel phenotype is typical of many Mutator-induced mutations, but the high germline reversion frequency (10-2) is not typical.
In summary, 3 of these B-Peru alleles have phenotypes or reversion frequencies different from most Mutator-induced mutations. Other groups have also reported Mutator-induced mutations with exceptional phenotypes. Robertson has reported a Mutator-induced waxy allele, wx-mum2, that has large somatic revertant sectors and a germline reversion frequency of 10-4 (MNL, this issue), and Freeling's laboratory has reported a high reversion frequency of an Adh1 allele caused by a Mu3 insertion. One possibility is that the differences in phenotypes between these mutants and most Mutator-induced mutations is because the B-Peru and waxy mutations are not caused by Mu1 elements. Support for this idea comes from the molecular characterization of 3 of these B-Peru alleles which demonstrated that no characterized Mu transposable elements are linked to the mutant alleles (Chandler and Turks, this issue).
Vicki L. Chandler, Susan Belcher and Devon Turks
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