The Journal of Heredity 2002:93(1)
© 2002 The American Genetic Association 93:67-70
Brief Communication |
Inheritance of Null Alleles for Microsatellites in the White Pine Weevil (Pissodes strobi [Peck] [Coleoptera: Curculionidae])
From Department of Forest Sciences, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
Address correspondence to Y. A. El-Kassaby at the address above or e-mail: yelkassaby{at}cellfor.com.
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Abstract |
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Four families of the white pine weevil (Pissodes strobi) produced by controlled breeding were used to study the mode of inheritance at four microsatellite loci. The results confirmed the Mendelian segregation of all loci. Three of the four loci showed the presence of null alleles. The observed high polymorphism of P. strobi microsatellites increased their usefulness for paternity determination. The presence of null alleles predicates the need to redesign primers before using them in population genetics studies where pedigree is unknown.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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White pine weevil (Pissodes strobi [Peck] [Coleoptera: Curculionidae]) is a native pest of many spruce species in North America. The weevil attacks terminal leaders of young trees, resulting in their death and thus dramatically decreasing the timber's commercial value (Alfaro 1994). Several biocontrol methods have been attempted to control this insect, but with very limited success (Alfaro 1994). Understanding weevil biology and its genetics are prerequisites to its control. Kennedy (1993) demonstrated that the level of genetic variation within a population could determine the uniformity in the long-term evolutionary response of a pest population to a pest management method. Using allozyme and randomly amplified polymorphic DNA markers Lewis et al. (2000, 2001) studied the genetic variation and structure of P. strobi from several Canadian populations. They reported the presence of high within-population genetic variation, a low level of inbreeding, and a fine-scale genetic structure among white pine weevil population.
Sugg and Chesser (1994) demonstrated that multiple paternity affects the rate of change in genetic diversity and inbreeding in a population. The use of isozyme markers in studying multiple paternity has several limitations due to the fact that the amount of variation among parents is often too low, resulting in an ambiguous assignment of paternity. At present the use of microsatellites markers for studying multiple paternity has increased because of their codominant and highly polymorphic nature. Microsatellites are short, tandemly repeated sequences whose core sequences are between 1 and 10 bp, but they can reach a length of up to 150 bp (Schlötterer and Pemberton 1998). The applications of microsatellite markers for population genetics study have been widely reviewed (Beaumont and Bruford 1998; Schlötterer and Pemberton 1998). However, one of the limitations is the presence of null alleles, which has been considered one of the impediments to their utility. The misidentification of null alleles could lead to distortion in segregation analyses, resulting in false estimates. Thus it is imperative that the mode of inheritance for these markers be studied as a prerequisite for their use in parentage and population studies.
In this study we report on the mode of inheritance of four microsatellite markers in white pine weevil. The mode of inheritance is determined from offspring collected from controlled breeding experiments. The four microsatellite markers are then used to determine the mode of mating system for this insect.
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Materials and Methods |
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Weevil samples were obtained from caged controlled cross-matings between one virgin female and one virgin male (1 F:1 M) on Sitka spruce (Picea sitchensis [Bong.] Carr) trees. Four replications were used for this study. Total genomic DNA was isolated from individual weevils by a modification of the CTAB method (Boyce et al. 1989). Four of the six microsatellite primers developed from P. strobi were used to test the mode of inheritance. The polymerase chain reaction (PCR) conditions and loci nomenclatures are described in Liewlaksaneeyanawin et al. (2001). The four primers chosen for this study (we27.2, we3-19, we3-16, and we3-18) demonstrated simple band patterns. All genotypes were scored using tailed primer labeling (Oetting et al. 1995) on a LiCor 4200 automated sequencer (LiCor Inc., Lincoln, NE). For each replication the female and male parents, as well as all offspring, were analyzed (see Table 1). All observed progeny ratios of each primer set were tested against the expected Mendelian segregation ratio using chi-square analysis (Sokal and Rohlf 1995).
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Results |
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The various genotypic combinations of mated males and females produced three different segregation ratios: 1:1, 1:1:1:1, and 3:1 (Table 1). Of the 16 genotypic ratios examined, 8 genotypic ratios exhibited expected Mendelian segregation. For example, replication 4 produced the expected segregation of 1:1:1:1 ratio at locus we2-7.2 due to the fact that both male and female parents were heterozygous for 230/234 and 226/230 genotypes, respectively (Figure 1a). Offspring produced four genotypes (226/230, 226/234, 230/230, and 230/234) that segregated according to expectation (
2 = 3.605, df = 3, P = .307) (Table 1).
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The remaining eight genotypic ratios confirmed Mendelian expectations when unexpected offspring genotypes were considered as homozygotes and heterozygotes for null alleles (Table 1). For example, for locus we3-16, both parents carry a null allele in the heterozygote state in replication 4 (Table 1). If male and female parental genotypes are 289/289 and 295/295, respectively, then only one genotype should be observed in all offspring (289/295, n = 43). However, two unexpected genotypes, 289/289 and 295/295, were detected in 21 of 43 offspring, as well as 12 individuals with no bands (see example in Figure 1b). Therefore it should be assumed that both male and female parents were heterozygous for the null alleles with genotypes of 289/null and 295/null, respectively. The expected offspring genotypes are 289/295, 289/null, 295/null, and null/null with a 1:1:1:1 ratio. Chi-square analysis showed that there was no significant difference from the expected genotype 1:1:1:1 ratio with the hypothesis of a null allele heterozygous in both parents (
2 = 1.372, df = 3, P = .712) (Table 1).
In addition, locus we2-19 (Figure 1c) had both male and female parents with a single homozygous genotype with the same allele size (188). In this situation it is expected that all offspring genotypes will be identical to both parents. Segregation analysis of this parental combination produced two genotypes. One of them is indicative of a null/null genotype, indicating that both parents are heterozygous for a null allele. This was shown by the presence of eight offspring homozygous for nonamplifying alleles, confirming the expectation of offspring genotype null/null (Table 1). Under this model it is expected that offspring would segregate following the 1:2:1 ratio for 188/188, 188/null, and null/null genotypes, respectively. Because the genotypes 188/188 and 188/null cannot be distinguished from each other, the chi-square analysis was performed to test the deviation from the expected genotype 3:1 ratio. There was no significant difference from the expected genotype 3:1 ratio (2 = 0.938, df = 1, P = .333) (Table 1).
The availability of family structure with the known genotype of both parents allowed the detection of the presence of null alleles for the remaining replications at three microsatellite loci (we2-19, we3-18, and we3-16) (Table 1). Locus we2-19 showed the presence of a null allele in both homozygote and heterozygote genotypes (Table 1). Homozygote (null/null) and heterozygote (212/null and 159/null) genotypes were observed in male parents of replication 1 and replications 2 and 3, respectively (Table 1). Locus we3-16 produced similar results with homozygote and heterozygote genotypes for null alleles (Table 1). A homozygote (null/null) genotype was detected for the female parent of replication 1, while the heterozygote (289/null) genotype was observed for male parents of replications 1 (Table 1). Finally, for locus we3-18, homozygote (null/null) and heterozygote (101/null) genotypes for null alleles were detected in male parents of replications 2 and 3, respectively (Table 1). After considering the presence of null alleles, all segregation ratios for the four tested loci showed no significant difference (P 
.05) between observed and expected progeny ratios (Table 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of inheritance analyses indicated that all four microsatellite loci studied segregated as expected in a Mendelian fashion. Although microsatellites developed for P. strobi have null allele problems, they have been shown to be more polymorphic than isozyme markers. Lewis et al. (2000) reported 66% of the isozyme loci were polymorphic and the mean number of allele per locus was 2.1 in one population (Benson River), while the percentage of polymorphic loci and the mean number of allele per locus for six microsatellite loci (with the four included in the present study) examined in the same population were 100% and 10, respectively (Liewlaksaneeyanawin et al. 2001). With the high polymorphism observed in P. strobi microsatellites, the combinations of these four microsatellite loci have proven to be useful for parentage assignment when controlled crosses are performed (Liewlaksaneeyanawin 2000).
The presence of null alleles represents the major limitation of microsatellite markers for population genetics studies in white pine weevil. These limitations are similar to those reported in previous studies conducted on various other organisms. Null alleles result from mutations such as substitutions, insertions, or deletions in one or both priming sites preventing the binding of the DNA strand and oligoprimers (Callen et al. 1993). Similar to the present study, inheritance analysis from controlled crosses and studies on parentage analysis from mother-offspring known genotypes revealed the presence of null alleles at microsatellite loci in various organisms. Null alleles have been reported in humans (Callen et al. 1993), deer (Pemberton et al. 1995), bears (Paetkau and Strobeck 1995), plants (Falque et al. 1998; Fisher et al. 1998), fish (Arden et al. 1999; Banks et al. 1999), and insects (Cooper et al. 1996; Oldroyd et al. 1996).
In the present study, null alleles were observed at three of the four loci examined from controlled crosses (i.e., 75%). The estimates of null allele frequency (based on the heterozygote deficiency according to Summers and Amos (1997) from 75 parents used in multiple paternity studies were 0.235, 0.433, and 0.330 for we3-18, we2-19, and we3-16, respectively. Similarly the null allele homo- and heterozygote genotypes of those parents were inferred by the known parental-offspring genotypes in each cross and used to calculate allele frequency. The null allele frequencies for these three loci were 0.188 ± 0.71, 0.387 ± 0.081, and 0.225 ± 0.110, respectively. The percentage of null alleles found in this study was higher than that reported for two insect species in which the mother-offspring genotypes were known. Oldroyd et al. (1996) and Copper et al. (1996) reported 33% of three studied loci and 50% of two studied loci displaying null alleles in honeybees and damselflies, respectively.
Although the high level of polymorphism of P. strobi microsatellite loci makes them useful for parentage assignment when controlled crosses are performed, it could be a drawback when using them in population genetics studies with individuals from unknown pedigree. The caveat of null alleles for using microsatellite markers in population genetics studies or parentage assignment has been reported for the past several years (Pemberton et al. 1995; Schlötterer and Pemberton 1998). Thus we recommend that determination of the inheritance of the microsatellite markers for any species be a prerequisite for their use.
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Acknowledgments |
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We thank K. Lewis for conducting the breeding experiments and D. Andrucko, D. Brescia, G. Brown, P. Blake, D. Levesque, A. Shand, and L. Van Akker for assistance throughout the experiments. This work was partially funded through a Forest Renewal BC grant (to Y.A.E.).
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Footnotes |
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Corresponding Editor: Philip Hedrick
Received April 11, 2001
Accepted September 5, 2001
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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