Biodiversity Science ›› 2013, Vol. 21 ›› Issue (1): 117-126.doi: 10.3724/SP.J.1003.2013.10133

• Orginal Article • Previous Article     Next Article

Null alleles in microsatellite markers

Yafeng Wen1, 2, *(), Kentaro Uchiyama2, Wenjun Han1, Saneyoshi Ueno2, Weidong Xie3, Gangbiao Xu1, Yoshihiko Tsumura2   

  1. 1 College of Forestry, Central South University of Forestry and Technology, Changsha 410004, China
    2 Forestry and Forest Products Research Institute, Tsukuba 305-8687, Japan
    3 College of Forestry, Guangxi University, Nanning 530004, China
  • Received:2012-06-20 Accepted:2012-09-04 Online:2013-02-04
  • Wen Yafeng

Microsatellite markers are widely used in genetic analysis, but the common occurrence of null alleles, is an unfortunate drawback, and one which has not been paid enough attention. In this review, we confirm the concept of null alleles and their characteristics, detail methods for estimating the frequency of null alleles and discuss potential causes. We also consider the effect of null alleles on genetic studies, such as measurement of population genetic diversity, parentage analysis and assignment tests. The review has a certain reference value for future studies. One main cause of microsatellite null alleles is poor primer annealing due to nucleotide sequence divergence (point mutations or indels) in one or both flanking primers. Microsatellite null alleles are not a natural characteristic of a specific gene, and are essentially different from isozyme null alleles. Although microsatellite null alleles have the characteristics of universality, complexity and invisibility, we can test and estimate their frequency through multiplex approaches, using Hardy-Weinberg equilibrium tests, parent-offspring genotype analysis, and new primer design. Null alleles have significant effects on the results of genetic analysis, potentially decreasing population genetic diversity and increasing genetic differentiation among populations. Moreover, null alleles may decrease the probability of parentage exclusion, even leading to errors or confusion in parentage analysis. Null alleles should be paid more attention and demand caution in future studies. Through a variety of methods, such as locus selection, null allele data adjustment, and the design of new primers, we can escape the adverse effects of null alleles and achieve greater accuracy in genetic analysis.

Key words: null alleles, microsatellite markers, allele frequency, Hardy-Weinberg equilibrium

62 Selkoe KA, Toonen RJ (2006) Microsatellites for ecologists: a practical guide to using and evaluating microsatellite markers.Ecology Letters, 9, 615-629.
63 Shaw PW, Pierce GJ, Boyle PR (1999) Subtle population structuring within a highly vagile marine invertebrate, the veined squid Loligo forbesi, demonstrated with microsatellite DNA markers.Molecular Ecology, 8, 407-417.
64 Slatkin M (1995) A measure of population subdivision based on microsatellite allele frequencies.Genetics, 139, 457-462.
65 Spritz RA (1981) Duplication/deletion polymorphism 5'- to the human β globin gene.Nucleic Acids Research, 9, 5037-5047.
66 Summers K, Amos W (1997) Behavioral, ecological, and molecular genetic analyses of reproductive strategies in the Amazonian dart-poison frog, Dendrobates ventrimaculatus.Behavioral Ecology, 8, 260-267.
67 Sun T (孙涛), Wang BS (王博石), Liu ZJ (刘志瑾), Que TC (阙腾程), Huang CM (黄乘明), Zhou QH (周岐海), Li M (李明) (2010) Identification and characterization of microsatellite markers via cross species amplification from Francois’ langur (Trachypithecus francoisi).Acta Theriologica Sinica(兽类学报), 30, 351-353. (in Chinese with English abstract)
68 Szabo LJ (2007) Development of simple sequence repeat markers for the plant pathogenic rust fungus, Puccinia triticina.Molecular Ecology Notes, 7, 92-94.
69 Tautz D (1989) Hypervariability of simple sequences as a general source for polymorphic DNA markers.Nucleic Acids Research, 17, 6463-6471.
70 Tóth G, Gáspári Z, Jurka J (2000) Microsatellites in different eukaryotic genomes: survey and analysis.Genome Research, 10, 967-981.
71 Tsui CKM, Feau N, Ritland CE, Massoumi Alamouti S, Diguistini S, Khadempour L, Bohlmann J, Breuil C, Hamelin RC (2009) Characterization of microsatellite loci in the fungus, Grosmannia clavigera, a pine pathogen associated with the mountain pine beetle.Molecular Ecology Resources, 9, 1500-1503.
72 Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data.Molecular Ecology Notes, 4, 535-538.
73 Van Oosterhout C, Weetman D, Hutchinson WF (2006) Estimation and adjustment of microsatellite null alleles in nonequilibrium populations.Molecular Ecology Notes, 6, 255-256.
74 van Oppen MJH, Rico C, Turner GF, Hewitt GM (2000) Extensive homoplasy, nonstepwise mutations, and shared ancestral polymorphism at a complex microsatellite locus in Lake Malawi cichlids.Molecular Biology and Evolution, 17, 489-498.
75 Vogl C, Karhu A, Moran G, Savolainen O (2002) High resolution analysis of mating systems: inbreeding in natural populations of Pinus radiata.Journal of Evolutionary Biology, 15, 433-439.
76 Wagner AP, Creel S, Kalinowski ST (2006) Estimating relatedness and relationships using microsatellite loci with null alleles.Heredity, 97, 336-345.
77 Wang ZR (王中仁) (1994) The genetic bases of allozyme analysis (part 2).Chinese Biodiversity(生物多样性), 2, 213-219. (in Chinese with English abstract)
78 Wattier R, Engel CR, Saumitou-Laprade P, Valero M (1998) Short allele dominance as a source of heterozygote deficiency at microsatellite loci: experimental evidence at the dinucleotide locus Gv1CT in Gracilaria gracilis (Rhodophyta).Molecular Ecology, 7, 1569-1573.
79 Worthington Wilmer J, Allen PJ, Pomeroy PP, Twiss SD, Amos W (1999) Where have all the fathers gone? An extensive microsatellite analysis of paternity in the grey seal (Halichoerus grypus).Molecular Ecology, 8, 1417-1429.
80 Yasuda N (1968) Estimation of the inbreeding coefficient from phenotype frequencies by a method of maximum likelihood scoring.Biometrics, 24, 915-935.
1 Angers B, Bernatchez L (1997) Complex evolution of a salmonid microsatellite locus and its consequences in inferring allelic divergence from size information.Molecular Biology and Evolution, 14, 230-238.
2 Astanei I, Gosling E, Wilson J, Powell E (2005) Genetic variability and phylogeography of the invasive zebra mussel, Dreissena polymorpha (Pallas).Molecular Ecology, 14, 1655-1666.
3 Bacles CFE, Burczyk J, Lowe AJ, Ennos RA (2005) Historical and contemporary mating patterns in remnant populations of the forest tree Fraxinus excelsior L.Evolution, 59, 979-990.
4 Beck NR, Double MC, Cockburn A (2003) Microsatellite evolution at two hypervariable loci revealed by extensive avian pedigrees.Molecular Biology and Evolution, 20, 54-61.
5 Blouin MS, Parsons M, Lacaille V, Lotz S (1996) Use of microsatellite loci to classify individuals by relatedness.Molecular Ecology, 5, 393-401.
6 Brookfield JFY (1996) A simple new method for estimating null allele frequency from heterozygote deficiency.Molecular Ecology, 5, 453-455.
7 Brownlow RJ, Dawson DA, Horsburgh GJ, Bell JJ, Fish JD (2008) A method for genotype validation and primer assessment in heterozygote-deficient species, as demonstrated in the prosobranch mollusc Hydrobia ulvae.BMC Genetics, 9, 55.
8 Callen DF, Thompson AD, Shen Y, Phillips HA, Richards RI, Mulley JC, Sutherland GR (1993) Incidence and origin of null alleles in the (AC)n microsatellite markers.American Journal of Human Genetics, 52, 922-927.
9 Carlsson J (2008) Effects of microsatellite null alleles on assignment testing.Journal of Heredity, 99, 616-623.
10 Cercueil A, Bellemain E, Manel S (2002) PARENTE: computer program for parentage analysis.Journal of Heredity, 93, 458-459.
11 Chakraborty R, De Andrade M, Daiger SP, Budowle B (1992) Apparent heterozygote deficiencies observed in DNA typing data and their implications in forensic applications.Annals of Human Genetics, 56, 45-57.
12 Chapuis MP, Estoup A (2007) Microsatellite null alleles and estimation of population differentiation.Molecular Biology and Evolution, 24, 621-631.
13 Chapuis MP, Loiseau A, Michalakis Y, Lecoq M, Estoup A (2005) Characterization and PCR multiplexing of polymorphic microsatellite loci for the locust Locusta migratoria.Molecular Ecology Notes, 5, 554-557.
14 Chen XY (陈小勇) (1999) Parentage analysis of natural plant populations and its applications in ecology.Chinese Journal of Ecology(生态学杂志), 18(2), 30-35. (in Chinese with English abstract)
15 Chybicki IJ, Burczyk J (2009) Simultaneous estimation of null alleles and inbreeding coefficients.Journal of Heredity, 100, 106-113.
16 Danzmann RG (1997) PROBMAX: a computer program for assigning unknown parentage in pedigree analysis from known genotypic pools of parents and progeny.Journal of Heredity, 88, 333.
17 Dakin EE, Avise JC (2004) Microsatellite null alleles in parentage analysis.Heredity, 93, 504-509.
18 Dempster AP, Laird NM, Rubin DB (1977) Maximum likelihood from incomplete data via the EM algorithm.Journal of the Royal Statistical Society, 39, 1-38.
19 Dubreuil M, Riba M, González-Martínez SC, Vendramin GG, Sebastiani F, Mayol M (2010) Genetic effects of chronic habitat fragmentation revisited: strong genetic structure in a temperate tree, Taxus baccata (Taxaceae), with great dispersal capability.American Journal of Botany, 97, 303-310.
20 Duchesne P, Godbout MH, Bernatchez L (2002) PAPA (package for the analysis of parental allocation): a computer program for simulated and real parental allocation.Molecular Ecology Notes, 2, 191-193.
21 Ellis JR, Burke JM (2007) EST-SSRs as a resource for population genetic analyses.Heredity, 99, 125-132.
22 Foltz DW (1986) Null alleles as a possible cause of heterozygote deficiencies in the oyster Crassostrea virginica and other bivalves.Evolution, 40, 869-870.
23 Gagneux P, Boesch C, Woodruff DS (1997) Microsatellite scoring errors associated with noninvasive genotyping based on nuclear DNA amplified from shed hair.Molecular Ecology, 6, 861-868.
24 Garcia de Leon FJ, Canonne M, Quillet E, Bonhomme F, Chatain B (1998) The application of microsatellite markers to breeding programmes in the sea bass, Dicentrarchus labrax.Aquaculture, 159, 303-316.
25 Gerber S, Mariette S, Streiff R, Bodénès C, Kremer A (2000) Comparison of microsatellites and amplified fragment length polymorphism markers for parentage analysis.Molecular Ecology, 9, 1037-1048.
26 Glenn TC, Schable NA (2005) Isolating microsatellite DNA loci. In: Molecular Evolution: Producing the Biochemical Data, Part B (eds Zimmer EA, Roalson E), pp. 202-222. Academic Press, San Diego, USA.
27 Grimaldi MC, Crouau-Roy B (1997) Microsatellite allelic homoplasy due to variable flanking sequences.Journal of Molecular Evolution, 44, 336-340.
28 Gur-Arie R, Cohen CJ, Eitan Y, Shelef L, Hallerman EM, Kashi Y (2000) Simple sequence repeats in Escherichia coli: abundance, distribution, composition, and polymorphism.Genome Research, 10, 62-71.
29 Harding RM, Boyce AJ, Clegg JB (1992) The evolution of tandemly repetitive DNA: recombination rules.Genetics, 132, 847-859.
30 He TH (何田华), Ge S (葛颂) (2001) Mating system, paternity analysis and gene flow in plant populations.Acta Phytoecologica Sinica(植物生态学报), 25, 144-154. (in Chinese with English abstract)
31 Hoban S, Anderson R, McCleary T, Schlarbaum S, Romero-Severson J (2008) Thirteen nuclear microsatellite loci for butternut (Juglans cinerea L.).Molecular Ecology Resources, 8, 643-646.
32 Jamieson A, Taylor SC (1997) Comparisons of three probability formulae for parentage exclusion.Animal Genetics, 28, 397-400.
33 Jarne P, Lagoda PJL (1996) Microsatellites, from molecules to populations and back.Trends in Ecology and Evolution, 11, 424-429.
34 Jones AG, Ardren WR (2003) Methods of parentage analysis in natural populations.Molecular Ecology, 12, 2511-2523.
35 Kalinowski ST, Taper ML (2006) Maximum likelihood estimation of the frequency of null alleles at microsatellite loci.Conservation Genetics, 7, 991-995.
36 Kalinowski ST, Taper ML, Marshall TC (2007) Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment.Molecular Ecology, 16, 1099-1106.
37 Kimura M, Crow JF (1964) The number of alleles that can be maintained in a finite population.Genetics, 49, 725-738.
38 Kwok S, Kellog DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies.Nucleic Acids Research, 18, 999-1005.
39 Lehmann T, Besanky NJ, Hawley WA, Fahey TG, Kamau L, Collins FH (1997) Microgeographic structure of Anopheles gambiae in western Kenya based on mtDNA and microsatellite loci.Molecular Ecology, 6, 243-253.
40 Lehmann T, Hawley WA, Collins FH (1996) An evaluation of evolutionary constraints on microsatellite loci using null alleles.Genetics, 144, 1155-1163.
41 Lemer S, Rochel E, Planes S (2011) Correction method for null alleles in species with variable microsatellite flanking regions, a case study of the black-lipped pearl oyster Pinctada margaritifera.Journal of Heredity, 102, 243-246.
42 Levinson G, Gutman GA (1987) Slipped-strand mispairing: a major mechanism for DNA sequence evolution.Molecular Biology and Evolution, 4, 203-221.
43 Li G, Hubert S, Bucklin K, Ribes V, Hedgecock D (2003) Characterization of 79 microsatellite DNA markers in the Pacific oyster Crassostrea gigas.Molecular Ecology Notes, 3, 228-232.
44 Li WH, Luo CC, Wu CI (1985) Evolution of DNA sequences. In: Molecular Evolutionary Genetics (ed. Macintryre RJ), pp. 1-94. Plenum Press, New York.
45 Li YC, Korol AB, Fahima T, Beiles A, Nevo E (2002) Microsatellites: genomic distribution, putative functions and mutational mechanisms: a review.Molecular Ecology, 11, 2453-2465.
46 Marshall TC, Slate J, Kruuk LEB, Pemberton JM (1998) Statistical confidence for likelihood-based paternity inference in natural populations.Molecular Ecology, 7, 639-655.
47 Meglecz E, Petenian F, Danchin E, D’Acier AC, Rasplus JY, Faure E (2004) High similarity between flanking regions of different microsatellites detected within each of two species of lepidoptera: Parnassius apollo and Euphydryas aurinia.Molecular Ecology, 13, 1693-1700.
48 Miesfeld R, Krystal M, Arnheim N (1981) A member of a new repeated sequence family which is conserved throughout eucaryotic evolution is found between the human δ and β globin genes.Nucleic Acids Research, 9, 5931-5947.
49 Moriguchi Y, Taira H, Tani N, Tsumura Y (2004) Variation of paternal contribution in a seed orchard of Cryptomeria japonica determined using microsatellite markers.Canadian Journal of Forest Research, 34, 1683-1690.
50 Morand ME, Brachet S, Rossignol P, Dufour J, Frascaria-Lacoste N (2002) A generalized heterozygote deficiency assessed with microsatellites in French common ash populations.Molecular Ecology, 11, 377-385.
51 Oddou-Muratorio S, Vendramin GG, Buiteveld J, Fady B (2009) Population estimators or progeny tests: what is the best method to assess null allele frequencies at SSR loci ?Conservation Genetics, 10, 1343-1347.
52 Ohta T, Kimura M (1973) A model of mutation appropriate to estimate the number of electrophoretically detectable alleles in a finite population.Genetical Research, 22, 201-204.
53 Paetkau D, Strobeck C (1995) The molecular basis and evolutionary history of a microsatellite null allele in bears.Molecular Ecology, 4, 519-520.
54 Paetkau D, Waits LP, Clarkson PL, Craighead L, Strobeck C (1997) An empirical evaluation of genetic distance statistics using microsatellite data from bear (Ursidae) populations.Genetics, 147, 1943-1957.
55 Peakall R, Gilmore S, Keys W, Morgante M, Rafalski A (1998) Cross-species amplification of soybean (Glycine max) simple sequence repeats (SSRs) within the genus and other legume genera: implications for the transferability of SSRs in plants.Molecular Biology and Evolution, 15, 1275-1287.
56 Primmer CR, Møller AP, Ellegren H (1995) Resolving genetic relationships with microsatellite markers: a parentage testing system for the swallow Hirundo rustica.Molecular Ecology, 4, 493-498.
57 Qu NN (曲妮妮), Gong SY (龚世园), Huang GJ (黄桂菊), Tong JG (童金苟), Yu DH (喻达辉) (2010) Isolation and screening of microsatellite markers from the Chinese pearl oyster Pinctada fucata based on FIASCO.Journal of Tropical Oceanography(热带海洋学报), 29, 47-54. (in Chinese with English abstract)
58 Raymond M, Rousset F (1995) GENEPOP (Version 1.2): Population genetic software for exact tests and ecumenicism.Journal of Heredity, 86, 248-249.
59 Rousset F (2008) Genepop’007: a complete re-implementation of the genepop software for Windows and Linux.Molecular Ecology Resources, 8, 103-106.
60 Rungis D, Bérubé Y, Zhang J, Ralph S, Ritland CE, Ellis BE, Douglas C, Bohlmann J, Ritland K (2004) Robust simple sequence repeat markers for spruce (Picea spp.) from expressed sequence tags.Theoretical and Applied Genetics, 109, 1283-1294.
81 Yasuda N, Kimura M (1968) A gene-counting method of maximum likelihood for estimating gene frequencies in ABO and ABO-like systems.Annals of Human Genetics, 31, 409-420.
61 Schlötterer C (2000) Evolutionary dynamics of microsatellite DNA.Chromosoma, 109, 365-371.
[1] Lin Wei, Shenggui Liu, Xianwei Shi. (2008) Genetic diversity of the Xuefeng black bone chicken based on microsatel-lite markers . Biodiv Sci, 16(5): 503-508.
Full text