Heterozygosity-fitness correlations in zebra finches: microsatellite markers can be better than their reputation

Numerous studies have reported associations between heterozygosity in microsatellite markers and fitness-related traits (heterozygosity-fitness correlations, HFCs). However, it has often been questioned whether HFCs reflect general inbreeding depression, because a small panel of microsatellite markers does not reflect very well an individual's inbreeding coefficient (F) as calculated from a pedigree. Here, we challenge this prevailing view. Because of chance events during Mendelian segregation, an individual's realized proportion of the genome that is identical by descent (IBD) may substantially deviate from the pedigree-based expectation (i.e. F). This Mendelian noise may result in a weak correlation between F and multi-locus heterozygosity, but this does not imply that multi-locus heterozygosity is a bad estimator of realized IBD. We examined correlations between 11 fitness-related traits measured in up to 1192 captive zebra finches and three measures of inbreeding: (i) heterozygosity across 11 microsatellite markers, (ii) heterozygosity across 1359 single-nucleotide polymorphism (SNP) markers and (iii) F, based on a 5th-generation pedigree. All 11 phenotypic traits showed positive relationships with measures of heterozygosity, especially traits that are most closely related to fitness. Remarkably, the small panel of microsatellite markers produced equally strong HFCs as the large panel of SNP markers. Both marker-based approaches produced stronger correlations with phenotypes than the pedigree-based F, and this did not seem to result from the shortness of our pedigree. We argue that a small panel of microsatellites with high allelic richness may better reflect an individual's realized IBD than previously appreciated, especially in species like the zebra finch, where much of the genome is inherited in large blocks that rarely experience cross-over during meiosis.     

On the use of large marker panels to estimate inbreeding and relatedness: empirical and simulation studies of a pedigreed zebra finch population typed at 771 SNPs

In recent years there has been a dramatic increase in the availability of high density genetic marker data for both model and non‐model organisms. A potential application of these data is to infer relatedness in the absence of a complete pedigree. Using a marker panel of 771 SNPs genotyped in three generations of an extensive zebra finch pedigree, correlations between pedigree relatedness and seven marker‐based estimates of relatedness were examined, as was the relationship between heterozygosity and inbreeding. Although marker‐based and pedigree relatedness were highly correlated, the variance in estimated relatedness was high. Further, the correlation between heterozygosity and inbreeding was weak, even though mean inbreeding coefficient is typical of that seen in wild vertebrate pedigrees; the weak relationship was in part due to the small variance in inbreeding in the pedigree. Our data suggest that using marker information to reconstruct the pedigree, and then calculating relatedness from the pedigree, is likely to give more accurate relatedness estimates than using marker‐based estimators directly.     

Can a simple algebraic analysis predict markers-genome heterozygosity correlations?

A current algebraic analysis on genome-wide heterozygosity estimates suggests that correlations between molecular markers and genome-wide heterozygosity, rho, depend on the ratio between the number of markers used, r, and the number of genome loci, n; that is: rho approximately square root r/n. Hence, it is unfeasible to obtain reliable estimates of genome-wide heterozygosity in species of large genome using a few markers. We cast some doubts about this analysis as it assumed that the probability that an individual was heterozygous at a locus is equal to the average heterozygosity of this locus in the population. However, we believe that individual heterozygosity at a given locus depends on individual pedigree. Because the pedigree is common for all loci of an individual, their probabilities of heterozygosity are not independent within the genome. We first performed simulations generating random genomes for 100 individuals. Among these individuals, markers and genome-wide heterozygosities correlated as expected from the above equation. However, when we simulated random mating among these individuals and in successive generations including their descendents, as occur in real populations, the correlations between markers and genome-wide heterozygosity were much higher than those predicted from algebraic analyses, and estimates of genome-wide heterozygosity improved slightly with the increment of the number of loci in the genome.     

基于微卫星标记的斑点叉尾鮰家系鉴定技术及应用

研究旨在建立准确、高效且经济的斑点叉尾鮰(Ictalures punctatus)家系亲缘鉴定体系, 以期为斑点叉尾鮰的遗传评估及家系育种提供科学依据。选用10对具有较高多态性SSR标记, 建立两个5重PCR反应体系。应用建立的斑点叉尾鮰家系亲缘鉴定体系对来源于13个全同胞家系和8个半同胞家系的333尾个体进行亲权鉴定。结果表明: 10个位点平均等位基因数为9.8、平均观测杂合度0.8591、平均期望杂合度0.8092、平均多态信息含量0.7845; 3种情况下的累积排除概率分别为: 0.99996806、0.99833267和0.99999998; 验证群体的鉴定结果与系谱高度一致, 真实鉴定率达到99.1%, 子代与父母本三者之间配对平均LOD值介于13.30—24.70, 且置信度均达到95%。研究选择的微卫星位点等位基因数目较多, 多态性较高, 可以快速、准确地对斑点叉尾鮰混养群体进行家系鉴定。     

INBREEDING INFLUENCES WITHIN‐BROOD HETEROZYGOSITY‐FITNESS CORRELATIONS (HFCS) IN AN ISOLATED PASSERINE POPULATION

Molecular estimates of inbreeding may be made using genetic markers such as microsatellites, however the interpretation of resulting heterozygosity‐fitness correlations (HFCs) with respect to inbreeding depression is not straightforward. We investigated the relationship between pedigree‐determined inbreeding coefficients (f) and HFCs in a closely monitored, reintroduced population of Stewart Island robins (Petroica australis rakiura) on Ulva Island, New Zealand. Using a full sibling design, we focused on differences in juvenile survival associated specifically with individual sibling variation in standardized multilocus heterozygosity (SH) when expected f was identical. We found that within broods, siblings with higher SH at microsatellite loci experienced a higher probability of juvenile survival. This effect, however, was detected primarily within broods that experienced inbreeding or when inbreeding had occurred in their pedigree histories (i.e., at the parents’ level). Thus we show, for the first time in a wild population, that the strength of an HFC is partially dependent on the presence of inbreeding events in the recent pedigree history. Our results illustrate the importance of realized effects of inbreeding on genetic variation and fitness and the value of full‐sibling designs for the study of HFCs in the context of small, inbred populations.     

Molecular and pedigree measures of relatedness provide similar estimates of inbreeding depression in a bottlenecked population

Individual‐based estimates of the degree of inbreeding or parental relatedness from pedigrees provide a critical starting point for studies of inbreeding depression, but in practice wild pedigrees are difficult to obtain. Because inbreeding increases the proportion of genomewide loci that are identical by descent, inbreeding variation within populations has the potential to generate observable correlations between heterozygosity measured using molecular markers and a variety of fitness related traits. Termed heterozygosity‐fitness correlations (HFCs), these correlations have been observed in a wide variety of taxa. The difficulty of obtaining wild pedigree data, however, means that empirical investigations of how pedigree inbreeding influences HFCs are rare. Here, we assess evidence for inbreeding depression in three life‐history traits (hatching and fledging success and juvenile survival) in an isolated population of Stewart Island robins using both pedigree‐ and molecular‐derived measures of relatedness. We found results from the two measures were highly correlated and supported evidence for significant but weak inbreeding depression. However, standardized effect sizes for inbreeding depression based on the pedigree‐based kin coefficients (k) were greater and had smaller standard errors than those based on molecular genetic measures of relatedness (RI), particularly for hatching and fledging success. Nevertheless, the results presented here support the use of molecular‐based measures of relatedness in bottlenecked populations when information regarding inbreeding depression is desired but pedigree data on relatedness are unavailable.     

Understanding the relationship between the inbreeding coefficient and multilocus heterozygosity: theoretical expectations and empirical data

Geneticists have been interested in inbreeding and inbreeding depression since the time of Darwin. Two alternative approaches that can be used to measure how inbred an individual is involve the use of pedigree records to estimate inbreeding coefficients or molecular markers to measure multilocus heterozygosity. However, the relationship between inbreeding coefficient and heterozygosity has only rarely been investigated. In this paper, a framework to predict the relationship between the two variables is presented. In addition, microsatellite genotypes at 138 loci spanning all 26 autosomes of the sheep genome were used to investigate the relationship between inbreeding coefficient and multilocus heterozygosity. Multilocus heterozygosity was only weakly correlated with inbreeding coefficient, and heterozygosity was not positively correlated between markers more often than expected by chance. Inbreeding coefficient, but not multilocus heterozygosity, detected evidence of inbreeding depression for morphological traits. The relevance of these findings to the causes of heterozygosity--fitness correlations is discussed and predictions for other wild and captive populations are presented.     

Effect of selection on the heterozygosity of inbred lines of maize

Although pedigree selection is the most commonly used method for developing inbred lines of maize, there are no studies on its effect on the heterozygosity of the lines. The objective of this work was to study the effect of pedigree selection on their heterozygosity. Thirteen F₅ or F₆ maize inbred lines developed by the pedigree selection method in four breeding programs and their F₁ and F₂ − F₄ ancestors were genotyped with simple sequence repeat markers distributed along the genome. Simulation was also conducted assuming different models of selection to investigate the selective forces needed to explain the data. In the F₂, F₃ and F₄ 40%, 66% and 86% of the markers segregating in the F₁ were fixed; that is, in the F₂ and F₃ fixation was lower than neutral expectation, but higher in the F₄. Due to such opposite apparent directions of selection, the heterozygosity of the lines in the F₅ or F₆ generations did not differ significantly from neutral expectations. The time to fixation differed from that expected with neutrality for most of the chromosomes, indicating that selection is distributed across the genome; but apparent overdominant effects in chromosome 7 were higher than in other chromosomes. In conclusion, the relationship between heterozygosity and vigour may reduce the effectiveness of pedigree selection in its goal of selecting the more vigorous, homozygous individuals. A more effective procedure is proposed using molecular markers for the identification of the more homozygous individuals, the most vigorous of those individuals being selected.     

Multilocus heterozygosity and inbreeding in the Siberian jay

Because of its common negative association with fitness, inbreeding is a major concern in conservation biology. Traditionally it has been measured as individual inbreeding coefficient calculated from the pedigree, but recently multilocus heterozygosity estimates have become commonly used as proxies. However, theoretical and simulation studies have cast doubt on the validity of these surrogates especially when they are based on only a few molecular markers. Yet, empirical studies reporting the correlation between multilocus heterozygosity and inbreeding coefficient are rare. We studied this relationship in a wild Siberian jay (Perisoreus infaustus) population subject to a long-term field study over 30 years. The correlations between inbreeding coefficient and the employed heterozygosity measures—standardized heterozygosity and internal relatedness—based on 21 microsatellite loci were weak. These results together with results from theoretical and simulation studies caution against use of multilocus heterozygosity estimates to study inbreeding in natural populations.     

How many SNPs are enough?

Since the days of allozyme analysis, we have been enamored with the idea that if we just had enough polymorphic mendelian loci, we could gauge the inbreeding level of individuals by measuring heterozygosity and simultaneously measure the degree of genetic relatedness between pairs of individuals. Given Mendel’s Laws, we have always known that we would need numerous independently segregating loci to achieve any reasonable degree of accuracy. Santure et al. (2010, this issue) use a 771 marker SNP panel to assess heterozygosity levels and to assess pairwise relatedness, and compare both with theoretical expectations obtained from a carefully recorded pedigree of a zebra finch breeding colony, as a function of increasing numbers of SNP markers. They also compare the SNP results with those from a 20‐locus microsatellite panel, showing that adding SNPs to a fairly large microsatellite panel improves accuracy, but given an existing panel of 125 SNPs, little is to be gained by adding microsatellites. They show that the accuracy available for estimating individual levels of inbreeding is somewhat limited. They also show that the average pairwise relatedness measures bracket pedigree relationship very nicely, but the variances for individual pairs remain substantial, even with a very large panel.     

Zur Frage der Zuordnung von Genen zu bestimmten Autosomen des Menschen mit Hilfe von Chromosomenaberrationen

A precise localization of a gene on a definite autosome has not yet been successful. We dispose only of some linkage data obtained from pedigree analyses. The classical methods of experimental crossing cannot be applied to man. The possibilities for a methodical approach towards the establishment of human chromosome maps has been substantially imporved by new methods in human genetics, especially cytogenetics. The authors provide a survey of the methods which can at present be used. These are based on the analyses of chromosome aberations, above all duplications and deficiences. Duplications may lead to (1) gene dosage effects, (2) triallelicaly controlled characters, (3) a deviation from frequency distributions which were theoretically to be expected of definite phenotypes. Deficiencies allow visualization of (1) gene dosage effects and (2) monoallelic characters in expected heterozygosity, as well as (3) the exclusion of a localization in heterozygosity. The methodological possibilities for each of these aspects are discussed, and the difficulties and limitations are alsommentioned. Finally, the experimental findings on each aspect are taken into consideration.     

Molecular and genetic polymorphism of cultivated soybean detected by AP-PCR, SSRP and SSR

Molecular-genetic polymorphism of cultivated soybean from different geographic regions was analysed using three PCR methods (AP-PCR, SSRP and ISSR). The dendrogram of phenogenetic relationship was constructed on the base of obtained data. The allocation of varieties are in a good agreement with pedigree information. Main parameters such as polymorphism level, expected heterozygosity, and marker index were calculated. It was shown that ISSR is the method of choice for the differentiation of soybean varieties.     

Genetic Diversity of Early Maturing Indian Maize (Zea mays L) Inbred Lines Revealed by SSR Markers

A set of 16 popular inbred lines, (8 released and 8 experimental) were analyzed using 24 Simple Sequence Repeats (SSR) markers. In total 71 SSR alleles were identified with a mean of 2.96 alleles per locus. The study revealed 28 rare alleles among the total, out of which 9 were unique to some of the inbred lines. The average Polymorphism Information Content (PIC) and Discrimination Rate (DR) were 0.39 and 0.61, respectively. Genetic similarity expressed as Jaccard’s coefficient varied from 0.23–0.68 with an average of 0.41. Five clusters were obtained by using Unweighted Paired Group Method using Arithmetic Averages (UPGMA). The pattern of grouping did not match well with available pedigree information, which may be attributed to inadequate pedigree information. Inbred lines used in present study revealed heterozygosity ranges from 8.3–33.3% and were clearly distinguished with a minimum set of three markers with high PIC and DR. However, fingerprints obtained using 13 markers with high DR revealed a probability of identical match by chance at 4.06×10^?8. In this study we found SSR as a good tool for characterization of maize genotypes along with morphological markers.     

Inbreeding and genetic structure in the endangered Sorraia horse breed: implications for its conservation and management

The Sorraia horse is a closed breed with reduced effective population size and considered in critical maintained risk status. The breed exists in 2 main breeding populations, one in Portugal and one in Germany, with a smaller population size. A set of 22 microsatellite loci was used to examine genetic diversity and structure of the Sorraia horse breed and to compare individual inbreeding coefficient F, estimated from pedigree data, with individual heterozygosity and mean d(2). The Sorraia horse shows lower levels of microsatellite diversity when compared with other horse breeds. Due to management strategies, there are clear differences in the genetic structure of the 2 main Sorraia horse populations. Individual heterozygosity was shown to be a good estimator, used together with or as an alternative to inbreeding coefficient, in predicting fitness and evaluating the inbreeding level of the Sorraia horse. The information gathered in this study, combined with information available from previous studies, offers an important and wide information base for the future development of an effective breeding management of the Sorraia horse in order to preserve this endangered breed.     

The imprecision of heterozygosity-fitness correlations hinders the detection of inbreeding and inbreeding depression in a threatened species

In nonpedigreed wild populations, inbreeding depression is often quantified through the use of heterozygosity-fitness correlations (HFCs), based on molecular estimates of relatedness. Although such correlations are typically interpreted as evidence of inbreeding depression, by assuming that the marker heterozygosity is a proxy for genome-wide heterozygosity, theory predicts that these relationships should be difficult to detect. Until now, the vast majority of empirical research in this area has been performed on generally outbred, nonbottlenecked populations, but differences in population genetic processes may limit extrapolation of results to threatened populations. Here, we present an analysis of HFCs, and their implications for the interpretation of inbreeding, in a free-ranging pedigreed population of a bottlenecked species: the endangered takahe (Porphyrio hochstetteri). Pedigree-based inbreeding depression has already been detected in this species. Using 23 microsatellite loci, we observed only weak evidence of the expected relationship between multilocus heterozygosity and fitness at individual life-history stages (such as survival to hatching and fledging), and parameter estimates were imprecise (had high error). Furthermore, our molecular data set could not accurately predict the inbreeding status of individuals (as 'inbred' or 'outbred', determined from pedigrees), nor could we show that the observed HFCs were the result of genome-wide identity disequilibrium. These results may be attributed to high variance in heterozygosity within inbreeding classes. This study is an empirical example from a free-ranging endangered species, suggesting that even relatively large numbers (>20) of microsatellites may give poor precision for estimating individual genome-wide heterozygosity. We argue that pedigree methods remain the most effective method of quantifying inbreeding in wild populations, particularly those that have gone through severe bottlenecks.     

Pedigree Analysis of the Sika Deer (Cervus nippon) using Microsatellite Markers

The usefulness of microsatellite markers in pedigree analysis of the sika deer (Cervus nippon) was tested in a herd in which the maternal lineages were recorded. Eighteen sets of microsatellite primers originally designed for bovine, ovine, and cervine loci successfully amplified polymorphic DNA in the deer. The numbers of alleles per locus ranged from two to seven, and the observed heterozygosity ranged from 0.350 to 0.900. The resolution power of the markers in paternity testing was then determined by calculating exclusion probabilities and paternity indices. Parentages of the study population were efficiently discriminated by genotyping 17 microsatellite loci. The microsatellite data were also used to calculate the genetic relatedness between individuals, which significantly correlated with coancestry coefficients for the pairs. Our results demonstrate that the microsatellite markers are efficient tools in studying the social structure and behavior of the sika deer, as well as in monitoring the inbreeding status.     

The role of pedigree information in combined linkage disequilibrium and linkage mapping of quantitative trait loci in a general complex pedigree

Combined linkage disequilibrium and linkage (LDL) mapping can exploit historical as well as recent and observed recombinations in a recorded pedigree. We investigated the role of pedigree information in LDL mapping and the performance of LDL mapping in general complex pedigrees. We compared using complete and incomplete genotypic data, spanning 5 or 10 generations of known pedigree, and we used bi- or multiallelic markers that were positioned at 1- or 5-cM intervals. Analyses carried out with or without pedigree information were compared. Results were compared with linkage mapping in some of the data sets. Linkage mapping or LDL mapping with sparse marker spacing ( approximately 5 cM) gave a poorer mapping resolution without considering pedigree information compared to that with considering pedigree information. The difference was bigger in a pedigree of more generations. However, LDL mapping with closely linked markers ( approximately 1 cM) gave a much higher mapping resolution regardless of using pedigree information. This study shows that when marker spacing is dense and there is considerable linkage disequilibrium generated from historical recombinations between flanking markers and QTL, the loss of power due to ignoring pedigree information is negligible and mapping resolution is very high.     

Microsatellites reveal heterosis in red deer.

The fitness consequences of inbreeding and outbreeding are poorly understood in natural populations. We explore two microsatellite-based variables, individual heterozygosity (likely to correlate with recent inbreeding) and a new individual-specific internal distance measure, mean d2 (focusing on events deeper in the pedigree), in relation to two measures of fitness expressed early in life, birth weight and neonatal survival, in 670 red deer calves (Cervus elaphus) born on the Isle of Rum between 1982 and 1996. For comparison, we also analyse inbreeding coefficients derived from pedigrees in which paternity was inferred by molecular methods. Only 14 out of 231 calves (6.1%) had non-zero inbreeding coefficients, and neither inbreeding coefficient nor individual heterozygosity was consistently related to birth weight or neonatal survival. However, mean d2 was consistently related to both fitness measures. Low mean d2 was associated with low birth weight, especially following cold Aprils, in which foetal growth is reduced. Low mean d2 was also associated with low neonatal survival, but this effect was probably mediated by birth weight because fitting birth weight to the neonatal survival model displaced mean d2 as an explanatory variable. We conclude that in the deer population fitness measures expressed early in life do not show evidence of inbreeding depression, but they do show evidence of heterosis, possibly as a result of population mixing. We also demonstrate the practical problems of estimating inbreeding via pedigrees compared with a direct marker-based estimate of individual heterozygosity. We suggest that, together, individual heterozygosity and mean d2, estimated using microsatellites, are useful tools for exploring inbreeding and outbreeding in natural population.     

Diagnostic efficacy of screening tests for hereditary hemochromatosis.

Hereditary hemochromatosis is transmitted as an autosomal recessive trait. Analyses of pedigrees suggest that the frequency of disease (proportion of homozygous individuals) in the general population is approximately 0.3% and that approximately 11% of the population are heterozygous. The genotype of 194 persons in 38 pedigrees was determined by HLA-A and HLA-B haplotyping. Likelihood analysis was then used to appraise the transferrin saturation test when used alone and in combination with the serum ferritin test to detect homozygosity and heterozygosity in these pedigrees. A single cut-off point of 55% for transferrin saturation and a cut-off point at the 90th percentile for the serum ferritin level were adequate for the detection of hemochromatosis if homozygosity was considered to be present when the results of one or both tests were positive. To further assess the value of the transferrin saturation test the percentages were stratified into five intervals. A percentage transferrin saturation of 75 or greater and a serum ferritin level above the 90th percentile ruled in homozygosity, whereas a percentage transferrin saturation of less than 55 and a serum ferritin level at or below the 90th percentile ruled it out with confidence. The probability of heterozygosity rose to 90% when the percentage transferrin saturation was between 35 and 55 and the serum ferritin level was at or below the 90th percentile. The use of five cut-off points allowed the probability of homozygosity and heterozygosity in a pedigree to be estimated for all values of transferrin saturation. Although these screening tests are not recommended for use in the general population, they may be worth while in selected groups of patients.     

Validation of a microsatellite panel for parentage testing of locally adapted and commercial goats in Brazil

Brazilian goats are generally kept in small herds and extensive rearing systems, mainly in the northeastern region of the country. Despite production improvement in recent years, the lack of pedigree control has affected genetic progress. This study aimed to validate a panel of 16 microsatellites for parentage testing in locally adapted and commercial goats breeds raised in Brazil, as well as to compare its efficiency with the panel recommended by the Brazilian Ministry of Agriculture, Livestock and Supplies (MAPA) in 2004. The number of alleles and expected heterozygosity (He) per marker ranged from four to 18, and from 0.051 to 0.831, respectively. Using all markers, 100% of parentage cases of the validation dataset were resolved with a strict confidence level of 95%. The 16 microsatellites panel showed adequate exclusion power (99.99%) and identity accuracy (99.99%). Suggestions for improvement of the marker panel endorsed by MAPA are provided.