Widespread null alleles and poor cross-species amplification of microsatellite DNA loci cloned from the Pacific oyster, Crassostrea gigas

Journal of Shellfisheries Research, August, 2004 by Dennis Hedgecock, Gang Li, Sophie Hubert, Katherine Bucklin, Vanessa Ribes

ABSTRACT Non-amplifying, PCR-null alleles are detected at 49 (51%) of 96 microsatellite DNA markers tested for Mendelian segregation in three families of the Pacific oyster Crassostrea gigas Thunberg. The average frequency of null alleles among [F.sub.1] hybrid grandparents is 0.093. The frequency of null alleles suggests a high level of sequence polymorphism in PCR primer binding sites and yields a conservative estimate of one single nucleotide polymorphism (SNP) every 82 base pairs. Among 86 markers tested on congeneric species, 83 (96.5%) are likely to be useful markers for the Portuguese oyster Crassostrea angulata, 71 (82.6%) for the Kumamoto oyster C. sikamea, 31 (36.0%) for the Suminoe oyster C. ariakensis, and only 11 (12.8%) for the Eastern oyster C. virginica. PCR product-yield and mean numbers of alleles per locus also decline significantly across this series of congeneric species, which separated from the Pacific oyster <1, ~2, ~4, and >5 million years ago, respectively. Decline in cross-specific PCR yield does not depend on microsatellite repeat-motif but is correlated with the frequency of null alleles across loci. The high nucleotide diversity suggested by these observations for the oyster may be a by-product of high fecundity, consistent with G. C. Williams' (1975) Elm-Oyster evolutionary model and experimental evidence for a high mutational load. Microsatellite loci should be identified de novo for each species of cupped oyster, and their inheritance should be validated before use in population analyses. Homology of microsatellite loci among related species should be confirmed by sequencing of flanking regions.

KEY WORDS: Pacific oyster, microsatellite DNA. null alleles, cross-specific amplification, nucleotide polymorphism, Crassostrea gigas

INTRODUCTION

With the completion or impending completion of genome sequences lot the human, fruit fly, nematode, mouse, and other eukaryotic genetic models, much attention is being paid to DNA sequence polymorphism and its potential use in understanding the genetic basis of complex phenotypes, such as disease susceptibility (Zwick et al. 2000). Whereas nucleotide diversity is becoming very well described for model organisms, which are all low-fecundity species (<[10.sub.3] - [10.sub.4] eggs per female), little is known about DNA polymorphism in high-fecundity species (>106 eggs per female). We might expect highly fecund species to have high nucleotide diversity, owing to large population sizes, and perhaps higher mutation rates. Indeed, G. C. Williams (1975) argued with his Elm Oyster Model that highly fecund species with high early mortality (Type-III survivorship) should reproduce sexually, show tremendous variation in individual fitness, and carry a large load of recessive deleterious mutations.

A large load of recessive deleterious mutations has recently been confirmed for the European flat oyster Ostrea edulis (Bierne el al. 1998) and the Pacific oyster Crassostrea gigas Thunberg (Launey & Hedgecock 200l, Bucklin 2002). Oysters naturally carry dozens of highly deleterious recessive mutations, which explain widespread observations of heterosis for fitness-related traits in bivalve mollusc species, at the whole organism and genetic-marker levels, and distortions of Mendelian segregation ratios at marker loci (Launey & Hedgecock 2001). On a practical level, discovery of genetic load in bivalves suggests that marker inheritance and linkage should be confirmed early in larval development, before selection can substantially distort genotypic proportions. Typing 11-day-old larvae and using double-hybrid crosses to reduce homozygosity by descent and inbreeding depression in mapping families, Hubert & Hedgecock (2004) have produced the first low-density microsatellite DNA marker maps for the Pacific oyster.

Microsatellite DNA markers are short tandem repeats of nucleotide motifs, 2 to 6 base pairs (bp) in length, which are distributed throughout the genome in prokaryotes and eukaryotes (Chambers & MacAvoy, 2000). Because they are highly polymorphic, microsatellites have been widely used as genetic markers for studies of linkage, kinship, and population structure (Goldstein & Schlotterer 1999, Chambers & MacAvoy 2000). Presently, the DNA sequences of 369 microsatellite containing clones from C. gigas are deposited in GenBank. Of these, 123 have been developed into PCR-amplifiable markers with confirmed inheritance (Magoulas et al. 1998, Huvet et al. 2000, McGoldrick et al. 2000, Li et al, 2003, Sekino et al. 2003) and 100 have been placed on linkage maps for the Pacific oyster (Hubert & Hedgecock 2004). Here, we present data on polymorphism of 96 of these microsatellite DNA markers and show that there seems to be little or no dependence of polymorphism on repeat-motif or motif complexity.

In developing microsatellite markers for constructing a linkage map of the Pacific oyster, we also uncovered two lines of evidence that nucleotide diversity and rate of sequence evolution in cupped oysters may be extremely high. Because high nucleotide diversity has important implications for future genetic and genomic studies with oysters, we present these findings here. First, we show that there is a high frequency of nonamplifying PCR null alleles, which likely result front polymorphism in the nonrepetitive flanking sequences to which PCR primers are designed to anneal. The inheritance of these null alleles is confirmed in multigenerational families, which were derived from the same population from which the microsatellite markers were cloned. Second, we show a dramatic decay in ability to amplify these markers across a series of four congeneric species that diverged from the Pacific oyster from 5 million years ago. This decay in cross-species amplification is independent of microsatellite repeat-motif but is correlated with the frequeucy of null alleles across loci. These observations contrast sharply with reports of success in amplifying microsatellites from very divergent vertebrate taxa (Garza et al. 1995, Pepin et al. 1995, Schlotterer et al. 1991, FitzSimmons et al. 1995, Rico et al. 1996) and species groups of Drosophila flies (Colson et al. 1999, Huttunen & Schlotterer 2002).

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