Brookhaven National Laboratory

Mapping new mutations using RFLPs

--Eileen C. Matz, Frances A. Burr and Benjamin Burr

The usual means of mapping a new mutation in maize employ either multiple marked testers, B-A translocations, or wx translocations. The latter two methods have been successfully used on a routine basis by a number of investigators. We have used B-A translocations ourselves to locate molecular markers to chromosome arms in initial stages of the construction of a molecular map. These stocks provide an elegant means of mapping a gene to a chromosome arm in one generation, providing the gene resides in the 85% of the genome covered by the available set of B-A translocations. We found that considerable time was spent in maintaining and assaying these stocks as well as in making the multiple crosses to mutant lines.

We thought that it would be interesting to explore the feasibility of mapping new mutations solely with molecular markers. We reasoned that if a modest number of well chosen molecular markers were used to screen a segregating population, the entire genome could be surveyed. Once linkage was detected, a fairly precise map location could be determined. Additionally, this work could be done at a time that would not conflict with field observations, genetic crosses, and data collection.

Linkage between two genes separated by 35% recombination in a small F2 or by 30% in a backcross can be detected at the 5% level of probability. This means that approximately 21 markers distributed over the genome are sufficient to detect linkage. The three examples presented here use backcross populations. In two cases we analyzed members of each phenotypic class and in the third case we examined only the homozygous mutants. A more efficient method, however, would be to examine only the homozygous mutant class in an F2 population.

Candidate RFLP markers were tested to see if they detected polymorphism between the two parents of the segregating population. Initially, parental DNA was digested with BamHI, BglII, or EcoRI; however, we subsequently learned that screening for polymorphism was more efficient if HindIII was included in this group. Because of the extensive levels of polymorphism present in maize, most probes yield useful polymorphism. It must be emphasized that the selection of RFLP markers and restriction enzymes used to show polymorphism is specific to each population. Once polymorphism was detected for a specific RFLP marker, DNA from the segregating population was digested with the appropriate enzyme, prepared for Southern hybridization, and the distribution of alleles was scored from the resulting autoradiograms. Reprobing of filters reduces the number of times this entire procedure has to be repeated. The Chi-square method was used to test deviation of allele distributions from those expected by chance (indicating no linkage). Chi-square values that exceeded the 5% level of probability indicated that other markers in the same region of the genome required testing. These markers confirmed linkage and provided a clearer indication of the map position of the mutant.

Example 1: Ufo1 is a dominant mutation giving rise to orange plant color (E. D. Styles, MNL 61:100, 1987). Derek Styles gave us homozygous mutant plants, a backcross population, and sibs of the normal backcross parent. Because of the heterozygosity present in the parental stocks, this was an unusual situation in that the + allele was undefined. Nevertheless, having the homozygous mutant allele allowed us to detect linkage.

Example 2: rd3 is a plant with reduced stature that was obtained by screening segregating F2s from an EMS mutagenesis experiment designed to detect new mutations affecting plant color in a B-Bolivia, pl, r-g background. The original mutant plants were small and had reduced plant color. These were crossed to an unrelated B-S, pl, R-g stock and backcrossed to the mutant. Of the 22 plants obtained from the backcross, 10 had reduced stature. All of the normal plants were pigmented, but 5 of the small plants were nearly colorless. In this population we assayed only the reduced stature class for RFLP alleles. The colorless phenotype segregating in the reduced plants maps near b1 on 2S. It appears that the recessive rd3 genotype is a precondition for the expression of the colorless phenotype in the double mutant. Because rd3 maps on 3L, we have made the cross to test allelism with na1, but the phenotypes are so dissimilar we do not expect them to be allelic.

Table 1. RFLP mapping of mutant phenotypes.
 

Mutant	Loci scored	Linked loci	Map position	Homozygousa class	Heterozygousa class	% Recomb.
Ufo1	25	pio200626	10S-12	8/11	3/13	25
		bnl3.-04	10S07	9/9	3/13	14
		npi285	10S006	7/10	2/12	23
		npi264	10L038	7/10	3/13	26
rd3	25	bnl6.06	3L069	9/10		10
		vp1	3L076	9/10		10
		bnl5.37	3L084	9/10		10
		bnl10.24A	3L089	9/10		10
		bnl8.01	3L095	9/10		10
		bnl5.14	3L097	9/10		10
sh*-459b	25	bnl6.22	5S024	10/10	0/14	0
		bnl5.71	5L051	9/10	3/14	17

^aFraction that are homozygous for the marker allele present in the recessive parent.
^bAllelic with sh5.

Example 3: sh*-459 is our lab designation for what is actually a brittle kernel phenotype. The mutation is from an EMS treated population generated by Gerry Neuffer. His designation is sh*-op1992. As can be seen in Table 1, we mapped this mutant to 5S. Based on this information we tested for allelism with sh5 and obtained a positive allelism test. The sh*-459 allele has a more severe phenotype than the allele identified by George Sprague.

Table 1 indicates that 25 marker loci were scored for allele distribution in the case of each mutant. This number is probably on the high side because we did not detect linkage until most chromosome arms had been screened in all three cases. Several other marker loci were then scored to narrow the approximate map position. Given the small populations screened in all cases, the present results only indicate approximate map location.

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