microsatellite locus was due to a 4 bp de- letion in one of the original PCR priming sites. Furthermore, the reamplifications re- vealed five distinct size classes of alleles that had been masquerading as the orig- inal null. These null alleles did not overlap in length with the nonnull alleles, and they also differed consistently by a linked nu- cleotide substitution. Results suggest that the original null allele (as well as the non- null class) has diversified considerably since its origin and has not recombined frequently with the nonnull class of alleles. Microsatellite markers have become valu- able tools for studies involving population genetics, kinship, and parentage assess- ment (Bruford and Wayne 1993; Queller et al. 1993), but a potential complication aris- es from the presence of nonamplifying or null alleles (Pemberton et al. 1995). Null alleles can produce serious problems for population-level studies by creating an ap- parent excess of homozygotes, resulting in incorrect allele frequency estimates and overestimates of inbreeding. In parentage studies they can result in false exclusions. Null alleles have been encountered in studies of numerous organisms, including mammals (Hulme et al. 1994; Phillips et al. 1993), birds (Primmer et al. 1995), fish (Jones and Avise 1997), insects (Cooper et al. 1996; Oldroyd et al. 1996), and crusta- ceans (Tam and Kornfield 1996). Despite the pervasiveness of microsatellite null al- leles and the difficulties associated with their assay, little is known about their mo- lecular basis (but see below), and even less is known about the evolutionary his- tories of the null alleles relative to their The Molecular Basis of a nonnull allele counterparts. The state-listed endangered White Microsatellite Null Allele Sands pupfish (Cyprinodon tularosa)is From the White Sands known from only four locations (Salt Pupfish Creek, Lost River, Malpais Spring, and Mound Spring), all of which are in New A. G. Jones, C. A. Stockwell, D. Walker, and J. C. Avise Mexico. During the cloning and character- ization of microsatellites from this species we encountered a null allele at high fre- Microsatellite loci were cloned and char- quency in the Malpais Spring population. acterized from the White Sands pupfish Here we investigate the molecular basis of (Cyprinodon tularosa), a New Mexico this null allele and assess its evolutionary state-listed endangered species. One lo- relationships to the nonnull alleles by re- cus exhibited a high-frequency nonampli- designing PCR primers and sequencing fying allele localized to a single popula- multiple alleles at the region surrounding tion. This null allele was PCR amplified by the microsatellite locus. redesign of one of the original primers and multiple individuals homozygous for null as well as for nonnull alleles were se- Materials and Methods quenced using the new primer. These mo- Microsatellite Cloning lecular dissections revealed that the orig- Total genomic DNA was extracted from a inal failure to amplify some alleles from this single C. tularosa specimen (from the Salt Brief Communications 339 Creek population) by a standard phenol/ CTGCTGCCTCAAAG-3Ј) external to chloroform procedure. The DNA was di- WSP11LO was designed from the original gested with NdeII and size selected by ex- cloned sequence. When used in conjunc- cising the 300–700 bp fragments from a 2% tion with WSP11UP, the new flanking prim- agarose gel after electrophoresis. Frag- er (WSP11LO-A) produced excellent am- ments were purified using the Prep-A-Gene plification of the WSP11 locus from all as- DNA Purification System (BioRad) and li- sayed individuals from all four popula- gated into BamHI-digested, dephosphory- tions. lated pBluescript phagemid (Stratagene). To determine the molecular basis of the Ligations were heat-shock transformed original null allele, several individuals (N into competent XL1-Blue E. coli (Stratage- ϭ 5) that yielded no PCR product from the ne). The transformation was spread on LB original primers, and that were homozy- plates containing ampicillin and X-gal. Ap- gous when assayed with the new primer proximately 1000 white colonies were set (WSP11UP and WSP11LO-A), were se- picked to four patch plates for screening. quenced using the fmol kit (Promega). We Colonies were transferred to Hybond-N ny- also sequenced 10 individuals that were lon membranes (Amersham International) recorded as homozygotes with both the following the manufacturer’s recommen- original and new primer pairs, and thus Figure 1. Autoradiographs of sequencing gels resolv- dations and probed twice at 42ЊC (in 6ϫ did not contain the null allele. ing the amplified products from 15 pupfish from the Malpais Spring population. (A) Amplification using the SSC, 0.1% SDS, 5ϫ Denhardt’s reagent) original WSP11 primers (WSP11UP and WSP11LO). (B) with two different cocktails of end-labeled Amplification of the same templates using the alterna- Results oligonucleotides. Each hybridization was tive primers (WSP11UP and WSP11LO-A). Note in (B) that the alternative primers revealed a host of different followed by two 30 min washes at 42ЊCin The Null Alleles alleles that had appeared as a null in the original as- 6ϫ SSC, 0.1% SDS. First, the oligonucleo- Amplification of a sample of 30 individuals says (A). The allele 177 amplified with the original tides (GT) , (GGAT) , (GACA) , and (TAG) from the Malpais Spring population with primers in (A) corresponds to the 202 bp fragment pro- 10 4 4 6 duced by the new primers in (B). were used, followed by stripping and re- the original WSP11 PCR primers resulted probing with (GA)10, (GATA) 4, (TTAGGG)3, in eight apparent homozygotes for a frag- and (TTC)5. Phagemid DNA was prepared ment 177 bp in length. The other 22 indi- cleotides flanking the microsatellite, in- from the 12 colonies that hybridized to the viduals yielded no product whatsoever. cluding the original lower priming site. A probes (Qiagen QIAprep spin plasmid min- Other populations (Salt Creek, Lost River, total of 15 sequences were analyzed, in- iprep kit). The inserts were sequenced us- and Mound Spring) assayed displayed ap- cluding null alleles of size 179 (N ϭ 1), 181 ing the fmol DNA sequencing system (Pro- parently normal genotypes: all individuals (N ϭ 2), and 187 (N ϭ 2), as well as non- mega) and primers were designed for mi- amplified and the samples did not deviate null alleles of size 194 (N ϭ 4), 200 (N ϭ crosatellite-containing loci. significantly from Hardy-Weinberg expec- 4), and 202 (N ϭ 2). All nonnull alleles The PCR was performed in 10 ␮l reac- tations (Stockwell et al., in preparation). were identical in flanking sequence to one tion volumes containing 1ϫ Promega Taq Thus the null allele appears to occur only another and to the original sequence buffer, 1.5 mM MgCl2, 0.15 ␮M of each in the Malpais Spring population. cloned from the C. tularosa library (Figure primer, 0.1 mM of each dNTP, and 0.5 units The redesigned primer WSP11LO-A, ex- 2). The five sequenced null alleles were of Promega Taq polymerase. The thermal ternal to the original primer, permitted also identical to one another and differed cycling consisted of 30 cycles of denatur- amplification of all individuals from the from the cloned sequence by a 4 bp dele- ation at 94ЊC for 1 min, annealing at 55ЊC Malpais Spring sample (Figure 1). For the tion within one of the original priming for 1 min, and extension at 72ЊC for 1 min, three additional populations, amplification sites. Thus this deletion was the cause of preceded by 2 min of denaturation at 94ЊC by the new primer pair left the microsa- the original null condition. The null alleles and followed by an additional 4 min exten- tellite genotypes unchanged with the fol- also differed consistently from the nonnull sion at 72ЊC. Microsatellite polymorphisms lowing exception: the resulting fragments alleles by a single G to A transition at a were detected by labeling one primer with were larger than the original products nucleotide position 1 bp removed from 1 ␮Ci ␥-32P ATP per 5 pmol of primer and since they were being amplified using a the start of the microsatellite array (Fig- electrophoretically resolving fragments on primer external to one of the original ure 2). standard 6% polyacrylamide denaturing primers (e.g., amplification of the original sequencing gels. 177 bp allele, for example, now yielded a Discussion product of 202 bp). The nonnull alleles Null Allele Analysis corresponded to 194, 196, 198, 200, and Null alleles have been reported in a num- A high-frequency null allele was suspected 202 bp with the new primer pair. The null ber of studies employing microsatellite for locus WSP11 in the Malpais Spring class of alleles, endemic to the Malpais loci. In our case the null allele was readily population of pupfish because many indi- Spring population, included fragments of apparent because it occurred with suffi- viduals failed to amplify from the original size 173, 179, 181, 187, and 189 bp. So an ciently high frequency as to be present in PCR primer pair (WSP11UP, 5Ј-AACAA- entire class of previously undetected al- homozygotes. Null alleles usually are more ATCCAATAATGTATTAGAA-3Ј; and WSP11LO, leles, distinct and nonoverlapping in size difficult to document. For example, had 5Ј-GATGAACGAGGAGAAAGAATAG-3Ј), de- compared to the nonnull alleles, had been the null allele been infrequent in our pop- spite the fact that they amplified success- hidden within the single null allele in the ulation, it would have occurred primarily fully for other loci. To solve this problem original assays (Figure 1). in heterozygotes and likely would have re- a new PCR primer (WSP11LO-A; 5Ј-CCC- Sequence data were generated for 38 nu- mained undetected. Null heterozygotes 340 The Journal of Heredity 1998:89(4) itive DNA.