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An Example of Using SSR Markers to Map Alleles for Disease Resistance
References:
Demirbas et al. 2001. Simple sequence repeat markers linked to the soybean Rps genes for phytophthora resistance. Crop Sci. 41:1220-1227.
Allard, R.W. 1956. Formulas and tables to facilitate the calculation of recombination values in heredity. Hilgardia 24:235-278.
This manuscript provides an example of using SSR (simple sequence repeat) markers to screen soybean F2 populations to locate major genes for resistance to a disease of soybean. Formulas are provided for estimating the ‘p' value between the marker and within one classification of the disease resistance gene. The authors state that the recombination between the SSR marker and the resistance gene should be no greater than
p = 0.05 for efficient marker assisted selection (MAS).
Resistance to phytophthora root rot is determined by multiple alleles at several loci. The disease resistance is not a quantitative trait and cannot be considered a QTL. Specific resistance genes confer complete resistance to one or more races of phytophthora. For this reason, the segregation ratios of the F2 plants are discreet such as a 1:2:1 or 3:1 ratio. It would be advantageous to identify resistant plants using molecular markers because this approach would require less resources than inoculating seedling to screen directly for the disease. The procedure to identify linkage relationships between the SSR markers and the phytophthora resistance genes consisted of three steps.
The first step was to determine whether polymorphisms existed between the two parents used to form the F2 population for the 228 SSR markers. The second step was to use bulk segregant analysis for those SSR markers that were segregating in the population. One bulk consisted of DNA collected from 15 F2:3 homozygous resistant (RpsRps) lines and the second bulk consisted of DNA collected from the same number of homozygous recessive (rps rps) susceptible lines. If the DNA of the homozygous resistant bulk had a different SSR marker allele than the DNA of the homozygous susceptible bulk, this was evidence of linkage between the SSR locus and the locus for disease resistance. For those SSR loci for which there was evidence of linkage, the third step was to determine the SSR allele for individual F2:3 plants within each of the bulk segregant classes.
One SSR allele was given the allelic symbol A and the other SSR allele was given the symbol B. A Chi-square test was conducted to determine whether there was a 1:1 ratio of the AA:BB markers for the RpsRps and rps rps bulks. A 1:1 ratio would be evidence of independence between the markers and the disease resistance alleles. The degree of linkage or ‘p' was determined within the homozygous recessive (rps rps) class by evaluating the segregation of the AA:AB:BB SSR markers, based on a formula provided by Allard (1956). The formula for determining p within the rps rps class is
s X [2/(p-1)] + r X [(1 - 2p)/p(1 - p)] + t X [2/p] = 0
where s, r, and t are the respective observed numbers of the AA, AB, and BB genotypes within the rps rps class and p is the estimated recombination value.
Example:
On pg. 1223, Table 3 from Demirbas et al. (2001) there are 0 AA:1 AB: 17 BB Satt159 (SSR) genotypes within the rps1 rps1 (homozygous recessive) class. The estimated p value between the Satt159 and rps loci is p = 0.03. We will use the formula provided below to estimate p for this numerical example. In this case s = 0, r = 1, and t = 17.
s X [2/(p-1)] + r X [(1 - 2p)/p(1 - p)] + t X [2/p] = 0
0 X [2/(p - 1)] + 1 X [(1 - 2p)/p( 1 - p)] + 17 X [2/p] = 0
(1 - 2p)/p(1 -p) + 34/p = 0
The first derivative of this
equation is 1/p -34 = 0.
We can re-write this equation
as p = 1/34 and p= 0.029. The first derivative provides
the best estimate of p. The standard error of p is [1/(v
X ip]1/2 where v is the total number of F2 individuals
examined and ip = 1/[2p(1 - p)]. In this example
v = 18
and ip = 1/0.0582 = 17.182. The S.E. of
p = [1/(v X ip]1/2
= 1/[18 X 17.182] = 0.06.
The authors conclude that "linkage
of p = 0.05 or less are generally required for efficient
marker assisted selection , although this restriction
can be relaxed if two markers flanking the locus of interest
are available." They also state that "the SSR genotyping
of just those F2 individuals homozygous recessive for
a gene of interest proved to be a convenient and efficient
means of confirming (or refuting) the tightest putative
linkages, which were the ones of interest."
Copyright
2000©, Ted Helms
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