High Allelic Polymorphism Research Articles

New genetic markers for <i>Burkholderia pseudomallei</i> strains typing

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease in humans and animals that may be asymptomatic long-term and quickly develop to pneumonia and septicemia in immunocompromised state and due to predisposing factors. Melioidosis is endemic in countries with tropical and subtropical climates, where B. pseudomallei is a part of the soil and water microbiota of stagnant water bodies, as well as the plant rhizosphere. The recording of imported melioidosis cases in countries with temperate climate zone, along with the continued threat of using this pathogen as a bioterrorism agent, indicate the relevance of research aimed at developing modern methods for its diagnosing and typing. Current research in the intraspecific differentiation of pathogenic strains of infectious diseases tends to use two or more types of molecular markers. A promising direction for the B. pseudomallei strains genotyping is based on using a combination of VNTR loci, which provides a high discriminating ability of the method of multi-locus variable tandem repeat number analysis (MLVA), and slowly evolving single nucleotide polymorphisms (SNPs). The aim of this work was to identify new VNTR and SNP loci suitable for use as genetic markers in molecular typing of the melioidosis causative agent. 20 strains of B. pseudomallei from the collection of the Volgograd Plague Control Research Institute and whole genome sequences of 85 B. pseudomallei strains from the GenBank NCBI database were analyzed. While typing melioidosis causative agent strains from the GenBank NCBI database for 4 VNTR loci, 74 genotypes were identified, of which 64 were unique (HunterGaston index 0.997). It was found that the high allelic polymorphism of VNTR loci limited a potential to determine the geographical and phylogenetic relationships of B. pseudomallei isolates by using the MLVA-4 scheme, and the identified null alleles increased the risk of homoplasia. In this regard, the MLVA-4 scheme was supplemented with a VNTR marker in the BPSS1974 locus encoding a collagen-like protein as well as with SNP markers in the genes of lipid A biosynthesis lauroyl acyltransferase, RNA polymerase sigma factor RpoH, and glutamine amidotransferase. For amplification of the select loci, primers and probes were designed as part of the developed scheme, which were tested in the typing of B. pseudomallei collection strains. According to the results of a cluster analysis for 105 strains of the melioidosis causative agent, an integrated approach turned out to be optimal, allowing differentiation of strains within SNP groups based on VNTR profiles. The methodological approach developed for B. pseudomallei genetic typing, based on a comprehensive analysis of 4 SNP- and 5 MLVA-markers in our modification, allowed to differentiate strains according to the geographical regions of their origin and establish the clonal origin of isolates upon revealing cases of melioidosis.

Mining and characterization of genic-microsatellites associated with sucrose synthesis and their utility in population structure analysis in sugarcane

Sucrose content is a highly valued trait in sugarcane breeding that is regulated by several genes. Tracking of multiple genes involved in the expression of a complex trait in a highly heterozygous crop like sugarcane during the breeding programme is a huge challenge. Trait tagging through expressed sequence tags (ESTs) derived molecular markers has been considered as powerful tool because they are encoded by functionally relevant elements of the genome and directly linked with the target traits. Functional markers related to sucrose content are very limited in sugarcane, which hindered the genetic improvement of this trait. Seventy-three sucrose rich and fifteen low sucrose clones chosen from twelve full-sib families were used for the study on the development and characterization of genic microsatellites associated with sucrose synthesis. A total of 186 alleles were obtained, with an average of 6.20 alleles per microsatellite marker. Polymorphism Information Content (PIC) values were ranged from 0.31 to 0.83, with a mean value of 0.61. The average values of gene diversity (GD) and resolving power (RP) were 0.60 and 5.14, respectively. The present study identified genic SSR markers for four major enzymes viz., CA076844187 (FK), CA155160295 (SREBF), CA163325191,213 (SREBF), TA38405-4547232 (PPFK), and CA135596178, 225 (DFPP) which could be useful to identify sucrose rich clones in the initial testing stages of the selection programme in sugarcane. Analysis of molecular variance (AMOVA) showed 74 per cent of the total variation within population and 26 per cent among population. The EST-SSR primers showed a high allelic polymorphism, as demonstrated by high genetic diversity estimates (I =1.17, Ho = 0.78). The low mean values of fixation index (FST = 0.078) and the high estimates of gene flow (Nm = 4.166) indicates the low level of population differentiation and maximum genetic exchange between two populations. Based on SSR marker data, eighty-eight clonal accessions were classified broadly into high and low sugar population which corresponded to the molecular diversity of the sucrose specific alleles through structure analysis and principal coordinate analysis. Key wordsSugarcane, sucrose content, genic-microsatellites, AMOVA, population structure

POLYMORPHISM OF APPLE SCAB VENTURIA INAEQUALIS IN BELARUS

Study of Venturia inaequalis (Cooke) Wint. polymorphism causing apple scab is due to the need to elaborate and implement breeding programs allowing to develop highly resistant apple varieties and systems of phytosanitary protection against the pathogen. The objects of research were 125 V.inaequalis monoisolates isolated in pure culture from the affected apple leaves of 31 varieties of various genetic and geographical origins, growing in the collection orchard of the Institute for Fruit Growing. The study aimed to define the morphological and genetic structure of apple scab pathogen populations. Differences between scab isolates were identified by the growth pattern and a complex of morphological characters that made it possible to divide them into three morphotypes. The study of the morphocultural characters of scab isolates made it possible to determine the diversity of isolated strains that will be used to
 create artificial infectious backgrounds. The method of DNA fragment analysis of V.inaequalis monospore isolates using
 SSR markers allowed to identify high allelic polymorphism in the population of the apple scab causative agent — 218
 alleles were identified due to the presence of a sexual process in the pathogen’s life cycle. A high degree of genetic
 diversity of the apple scab causative agent was identified both within the population and a group of strains isolated
 from the same variety.

Leukoencephalopathy with calcifications and cysts: Genetic and phenotypic spectrum.

Biallelic mutations in SNORD118, encoding the small nucleolar RNA U8, cause leukoencephalopathy with calcifications and cysts (LCC). Given the difficulty in interpreting the functional consequences of variants in nonprotein encoding genes, and the high allelic polymorphism across SNORD118 in controls, we set out to provide a description of the molecular pathology and clinical spectrum observed in a cohort of patients with LCC. We identified 64 affected individuals from 56 families. Age at presentation varied from 3 weeks to 67 years, with disease onset after age 40 years in eight patients. Ten patients had died. We recorded 44 distinct, likely pathogenic, variants in SNORD118. Fifty two of 56 probands were compound heterozygotes, with parental consanguinity reported in only three families. Forty nine of 56 probands were either heterozygous (46) or homozygous (three) for a mutation involving one of seven nucleotides that facilitate a novel intramolecular interaction between the 5' end and 3' extension of precursor-U8. There was no obvious genotype-phenotype correlation to explain the marked variability in age at onset. Complementing recently published functional analyses in a zebrafish model, these data suggest that LCC most often occurs due to combinatorial severe and milder mutations, with the latter mostly affecting 3' end processing of precursor-U8.

English

Development of improved varieties of sweet potato to meet specific end users in the sub-Saharan African region has the potential of transforming the crop’s market, consumption and health benefits. Molecular biology techniques such as fingerprinting are key to crop improvement, and critical for numerous genetic studies. This study, carried out in Ghana, seeks to determine genetic relatedness among two populations of sweet potato (released and elite), as well as to identify polymorphic and informative markers for diversity studies. Twenty expressed sequence tags-simple sequence repeat (EST-SSR) markers were used for analysis of DNA assembled. A total of 169 alleles were obtained, with an average of 8.5 alleles per locus. Pairwise genetic distance among genotypes ranged from 12 – 89. GenAlex v6.4 analysis showed that a higher genetic diversity existed within elite genotypes than the released lines. Results analyzed using Popgene version 1.31 software indicated that on the basis of both populations studied, EST-SSR markers utilized were very polymorphic (95%) and showed genetic diversity (I =0.888, He= 0.548) suggesting high heterozygosity in the collected varieties. The overall Fit (0.372%) and Fis (0.088%) suggest high genetic diversity within populations, and gene flow (1.95) indicate an exchange of genes between released sweet potatoes and elite ones. Ewens-Watterson test was conducted to test the neutrality. From the results obtained, markers CB329940, CB330477 and CB330936 showed high allelic polymorphism among varieties used and were recommended to be included in diversity studies. Key words: Genetic diversity, genetic relatedness, expressed sequence tags-simple sequence repeat (EST-SSR), populations, released sweet potato, elite sweet potato.

Genetic diversity analysis in drought stress tolerant cottons

Cotton is a major fiber product and an important source for edible oil in the world. Cotton productivity is limited by different abiotic and biotic factors such as drought stress. Moreover, long time cultivation practice and artificial selection has led to narrow genetic variability in cotton. Cotton cultivars tolerating drought stress are desirable for arid region cultivation. Different approaches have been used to study drought stress tolerance including conventional plant breeding and molecular finger printing of tolerant plants to estimate genetic variability. In this study eleven SSR and nine EST-SSR primers were used for studying genetic diversity in 26 drought stress tolerant cotton cultivars. EST-SSR and SSR markers used in the present study revealed a moderate degree of allelic polymorphism (19.82 and 13.22%, respectively) in the cotton plants studied. The cultivars Varamin and Sayar-314 showed high allelic polymorphism based on SSR data while, Tabladila showed high allelic polymorphism in EST-SSR markers. Based on clustering and STRUCTURE plot of molecular data and agronomic characteristics, two cultivars of Tabladila and Nazilyare suggested for further hybridization.

Genetic diversity and population structure of indigenous chicken breeds in South China

A total of 587 individuals from 12 indigenous chicken breeds from South China and two commercial breeds were genotyped for 26 microsatellites to investigate the genetic diversity and population structure. All micro- satellites were found to be polymorphic. The number of alleles per locus ranged from 5 to 36, with an average of 12.10 7.00 (SE). All breeds, except White Recessive Rock, had high allelic polymorphism (>0.5). Higher genetic diversity was revealed in the indigenous chicken breeds rather than in the commercial breeds. Potential introgression from the commercial breeds into the indigenous chickens was also detected. The population structure of these indigenous chicken breeds could be explained by their geographical distribution, which suggested the presence of independent history of breed formation. Data generated in this study will provide valuable information to the conservation for indigenous chicken breeds in future.

Surface expression, peptide repertoire, and thermostability of chicken class I molecules correlate with peptide transporter specificity

The chicken major histocompatibility complex (MHC) has strong genetic associations with resistance and susceptibility to certain infectious pathogens. The cell surface expression level of MHC class I molecules varies as much as 10-fold between chicken haplotypes and is inversely correlated with diversity of peptide repertoire and with resistance to Marek's disease caused by an oncogenic herpesvirus. Here we show that the average thermostability of class I molecules isolated from cells also varies, being higher for high-expressing MHC haplotypes. However, we find roughly the same amount of class I protein synthesized by high- and low-expressing MHC haplotypes, with movement to the cell surface responsible for the difference in expression. Previous data show that chicken TAP genes have high allelic polymorphism, with peptide translocation specific for each MHC haplotype. Here we use assembly assays with peptide libraries to show that high-expressing B15 class I molecules can bind a much wider variety of peptides than are found on the cell surface, with the B15 TAPs restricting the peptides available. In contrast, the translocation specificity of TAPs from the low-expressing B21 haplotype is even more permissive than the promiscuous binding shown by the dominantly expressed class I molecule. B15/B21 heterozygote cells show much greater expression of B15 class I molecules than B15/B15 homozygote cells, presumably as a result of receiving additional peptides from the B21 TAPs. Thus, chicken MHC haplotypes vary in several correlated attributes, with the most obvious candidate linking all these properties being molecular interactions within the peptide-loading complex (PLC).

The dominantly expressed class I molecule of the chicken MHC is explained by coevolution with the polymorphic peptide transporter (TAP) genes.

In most mammals, the MHC class I molecules are polymorphic and determine the specificity of peptide presentation, whereas the transporter associated with antigen presentation (TAP) heterodimers are functionally monomorphic. In chickens, there are two classical class I genes but only one is expressed at a high level, which can result in strong MHC associations with resistance to particular infectious pathogens. However, the basis for having a single dominantly expressed class I molecule has been unclear. Here we report TAP1 and TAP2 sequences from 16 chicken lines, and show that both genes have high allelic polymorphism and moderate sequence diversity, with variation in positions expected for peptide binding. We analyze peptide translocation in two MHC haplotypes, showing that chicken TAPs specify translocation at three peptide positions, matching the peptide motif of the single dominantly expressed class I molecule. These results show that coevolution between class I and TAP genes can explain the presence of a single dominantly expressed class I molecule in common chicken MHC haplotypes. Moreover, such coevolution in the primordial MHC may have been responsible for the appearance of the antigen presentation pathways at the birth of the adaptive immune system.

Potential use of major royal jelly proteins (MRJPs) as molecular markers for royal jelly production in Africanized honeybee colonies

The present study determined the genetic variation at mrjps loci (mrjp3, mrjp5 and mrjp8) and evaluated the potential use of MRJPs as molecular markers for higher royal jelly production in Africanized honeybee colonies. The three analyzed loci produced a total of 17 alleles. This high allelic polymorphism indicated these loci could serve as genetic markers. The potential use of MRJPs as molecular markers for royal jelly production was evaluated by analyses of multiple linear regressions with EPD (expected progeny differences) values for royal jelly production. The variance analyses indicated that the mrjp3 repetitive region influenced the genetic value of queen’s offspring for royal jelly production. The determination coefficient (R2) for the significant alleles of the repetitive region of mrjp3 indicated that 36.85% of the EPD variation is explained by the variation of C, D and E alleles. Therefore, these three alleles present a considerable genetic effect on the variation of RJ production.

Development and characterization of 11 microsatellite loci for the Mona Island iguana (Cyclura cornuta stejnegeri)

We isolated and characterized 11 microsatellite loci in the Mona Island iguana (Cyclura cornuta stejnegeri). Eleven loci exhibit moderate to high allelic diversity (two to 12 alleles, mean=4.5) and polymorphism (mean observed heterozygosity,0.56; range, 0.26 to 0.78) in 41 adults. This marker set has low probability of identity and high parentage exclusion power and will be suitable for studies of paternity, social organization and relatedness in this species.

High allelic polymorphism, moderate sequence diversity and diversifying selection for B-NK but not B-lec, the pair of lectin-like receptor genes in the chicken MHC

We previously characterised the C-type lectin-like receptor genes B-NK and B-lec, located next to each other in opposite orientations in the chicken major histocompatibility complex (MHC). We showed that B-NK is an inhibitory receptor expressed on natural killer cells, whereas B-lec is an activation-induced receptor with a broader expression pattern. It is interesting to note that the chicken MHC has been linked with resistance or susceptibility to Marek's disease virus (MDV), an oncogenic herpes virus. Recent reports show that the C-type lectin-like receptors in mouse and rat (Ly49H, NKR-P1 and Clr) are associated with resistance to another herpesvirus, cytomegalovirus (CMV). Therefore, B-NK and B-lec are potential candidate genes for the MHC-mediated resistance to MDV. In this paper, we report that both genes encode glycosylated type II membrane proteins that form disulphide-linked homodimers. The gene sequences from nine lines of domestic chicken representing seven haplotypes show that B-lec is well conserved between the different haplotypes, apparently under purifying selection. In contrast, B-NK has high allelic polymorphism and moderate sequence diversity, with 21 nucleotide changes in the complementary deoxyribonucleic acids (cDNAs) resulting in 20 amino acid substitutions. The allelic variations include substitutions, an indel and loss/gain of three predicted N-linked glycosylation sites. Strikingly, there is as much as 7% divergence between protein sequences of B-NK from different haplotypes, greater than the difference observed between the highly polymorphic human KIR NK receptors. Analysis of ds and dn reveal evidence of strong positive selection for B-NK to be polymorphic at the protein level, and modelling demonstrates significant variation between haplotypes in the predicted ligand binding face of B-NK.

Genetic characterization of six microsatellites in colombian horses Equus caballus (Equidae) and its use for parentage testing

In terms of commercial and cultural implications Co-lombian equines are very valuable; however their genetic potential is unknown. As a result, programs regarding selection and improvement have been limited. In order to contribute to the genetic potential, the variability of 264 horses Criollo Colombiano, Paso Fino Colombiano y Trocha Pura Colombiana breeds was evaluated. The allelic characterization was made with six molecular markers, micro satellite sort (ASB17, AHT4, AHT5, HTG4, HMS3 y HMS6) for further population studies. The analysis of the six microsatellites indicated high allelic polymorphism for the ASB17 with 18 alleles, the less variable was the HTG4 with seven alleles. The allele’s standard was 9.83. Besides, the allelic frequency showed that the populatio-ns are represented by few alleles. The gene diversity He0.7773 and Ho 0.765 was calculated to determine the genetic variability of the population. It indicated a large variability and high gene diversity in equines from the de-partment of Cundinamarca with an endogamy coefficient of FIS 0.0011. In the paternity test, the AHT4 and HMS3 demonstrated that they are the most informative ones; which is due to their high exclusion probability. Neverthe-less, the accumulated probability validity of 0.9543 with the six markers was low. It is recommended to change the AHT4 and HMS6 markers for more informative ones and also to increase the number of markers.

HLA‐G alleles and HLA‐G 14 bp polymorphisms in a Brazilian population

Brazilians represent one of the most heterogeneous populations in the world as the result of five centuries of interethnic crosses. We investigated the polymorphism of the HLA-G locus in a Brazilian urban sample. HLA-G alleles were determined by SBT analysis of exons 2-4 and 8 in 103 unrelated individuals. The genotyping analysis identified 11 different HLA-G alleles, including the recently described G*010110 allele, and four new variants in this population. These data indicate that this Brazilian population presents high HLA-G allelic polymorphism, suggesting increased levels of intrapopulational genetic diversity that may be because of the admixed nature of the sample. This is the first study regarding HLA-G genotyping that was performed at such refined resolution level in this heterogeneous population.

Gene duplication, allelic diversity, selection processes and adaptive value of MHC class II DRB genes of the bank vole, Clethrionomys glareolus

The generation and maintenance of allelic polymorphism in genes of the major histocompatibility complex (MHC) is a central issue in evolutionary genetics. Recently, the focus has changed from ex situ to in situ populations to understand the mechanisms that determine adaptive MHC polymorphism under natural selection. Birth-and-death evolution and gene conversion events are considered to generate sequence diversity in MHC genes, which subsequently is maintained by balancing selection through parasites. The ongoing arms race between the host and parasites leads to an adaptive selection pressure upon the MHC, evident in high rates of non-synonymous vs synonymous substitution rates. We characterised the MHC class II DRB exon 2 of free living bank voles, Clethrionomys glareolus by single-strand conformation polymorphism and direct sequencing. Unlike other arvicolid species, the DRB locus of the bank vole is at least quadruplicated. No evidence for gene conversion events in the Clgl-DRB sequences was observed. We found not only high allelic polymorphism with 26 alleles in 36 individuals but also high rates of silent polymorphism. Exceptional for MHC class II genes is a purifying selection pressure upon the majority of MHC-DRB sequences. Further, we analysed the association between certain DRB alleles and the parasite burden with gastrointestinal trichostrongyle nematodes Heligmosomum mixtum and Heligmosomoides glareoli and found significant quality differences between specific alleles with respect to infection intensity. Our findings suggest a snapshot in an evolutionary process of ongoing birth-and-death evolution. One allele cluster has lost its function and is already silenced, another is loosing its adaptive value in terms of gastrointestinal nematode resistance, while a third group of alleles indicates all signs of classical functional MHC alleles.

Pas de deux: natural killer receptors and MHC class I ligands in primates.

Major histocompatibility complex (MHC) class I and NK cell receptor gene regions are a paradigm of genomic plasticity as they reveal a considerable degree of diversity, exemplified by high allelic polymorphism, genomic duplications and contractions, and formation of gene families. Both genetic components show signs of rapid evolution due to strong selective pressure to combat pathogens. Comparative analyses of these genomic regions in various primates revealed considerable differences, reflecting species-specific adaptations to pathogenic threat or different strategies to combat infections. MHC and NK receptor genomic diversity in populations are important factors that determine susceptibility or resistance to a variety of diseases including autoimmune and infectious diseases as well as reproductive success.

Historical events and allelic polymorphism at the gametophytic self-incompatibility locus in Solanaceae

The historical migration rate of a species is often difficult to estimate with neutral markers, because the relationship between the turnover time of the markers and the age of the species commonly remains unknown. Compared with neutral markers, the plant self-incompatibility locus (S) provides a much better source of data for migration-rate estimation due to its high allelic polymorphism and antiquity. Here, the results from extensive surveys of S alleles in two wild solanaceous species, Solanum carolinense and Physalis longifolia, indicate that historical migration rates have differed significantly between the species; the higher migration rate found in S. carolinense appears to have interacted with the balancing selection at the S locus to result in fewer S alleles being maintained in the species. Historical population growth rates estimated via a modified coalescent approach also suggest a faster growing population for S. carolinense than for P. longifolia, which would have further widened their interspecific difference in S-allelle polymorphism. These historical factors may have reduced the probability of new S alleles to prevailing in S. carolinense, leaving old ones segregating at the S locus with little signature of positive selection being currently detectable.

MHC diversity and the association to nematode parasitism in the yellow‐necked mouse (Apodemus flavicollis)

In vertebrates, the genes of the major histocompatibility complex (MHC) are among the most debated candidates accounting for co-evolutionary processes of host-parasite interaction at the molecular level. The exceptionally high allelic polymorphism found in MHC loci is believed to be maintained by pathogen-driven selection, mediated either through heterozygous advantage or rare allele advantage (= frequency dependent selection). While investigations under natural conditions are still very rare, studies on humans or mice under laboratory conditions revealed support for both hypotheses. We investigated nematode burden and allelic diversity of a functional important MHC class II gene (DRB exon2) in free-ranging yellow-necked mice (Apodemus flavicollis). Twenty-seven distinct Apfl-DRB alleles were detected in 146 individuals with high levels of amino acid sequence divergence, especially at the antigen binding sites (ABS), indicating selection processes acting on this locus. Heterozygosity had no influence on the infection status (being infected or not), the number of different nematode infections (NNI) or the intensity of infection, measured as the individual faecal egg count (FEC). However, significant associations of specific Apfl-DRB alleles to both nematode susceptibility and resistance were found, for all nematodes as well as in separate analyses of the two most common nematodes. Apodemus flavicollis individuals carrying the alleles Apfl-DRB*5 or Apfl-DRB*15 revealed significantly higher FEC than individuals with other alleles. In contrast, the allele Apfl-DRB*23 showed a significant association to low FEC of the most common nematode. Thus, our results provide evidence for pathogen-driven selection acting through rare allele advantage under natural conditions.

Relative roles of mutation and recombination in generating allelic polymorphism at an MHC class II locus in Peromyscus maniculatus.

The MHC class II loci encoding cell surface antigens exhibit extremely high allelic polymorphism. There is considerable uncertainty in the literature over the relative roles of recombination and de novo mutation in generating this diversity. We studied class II sequence diversity and allelic polymorphism in two populations of Peromyscus maniculatus, which are among the most widespread and abundant mammals of North America. We find that intragenic recombination (or gene conversion) has been the predominant mode for the generation of allelic polymorphism in this species, with the amount of population recombination per base pair exceeding mutation by at least an order of magnitude during the history of the sample. Despite this, patchwork motifs of sites with high linkage disequilibrium are observed. This does not appear to be consistent with the much larger amount of recombination versus mutation in the history of the sample, unless the recombination rate is highly non-uniform over the sequence or selection maintains certain sites in linkage disequilibrium. We conclude that selection is most likely to be responsible for preserving sequence motifs in the presence of abundant recombination.

Genetic architecture of sexual and asexual populations of the aphid Rhopalosiphum padi based on allozyme and microsatellite markers.

Cyclical parthenogens, including aphids, are attractive models for comparing the genetic outcomes of sexual and asexual reproduction, which determine their respective evolutionary advantages. In this study, we examined how reproductive mode shapes genetic structure of sexual (cyclically parthenogenetic) and asexual (obligately parthenogenetic) populations of the aphid Rhopalosiphum padi by comparing microsatellite and allozyme data sets. Allozymes showed little polymorphism, confirming earlier studies with these markers. In contrast, microsatellite loci were highly polymorphic and showed patterns very discordant from allozyme loci. In particular, microsatellites revealed strong heterozygote excess in asexual populations, whereas allozymes showed heterozygote deficits. Various hypotheses are explored that could account for the conflicting results of these two types of genetic markers. A strong differentiation between reproductive modes was found with both types of markers. Microsatellites indicated that sexual populations have high allelic polymorphism and heterozygote deficits (possibly because of population subdivision, inbreeding or selection). Little geographical differentiation was found among sexual populations confirming the large dispersal ability of this aphid. In contrast, asexual populations showed less allelic polymorphism but high heterozygosity at most loci. Two alternative hypotheses are proposed to explain this heterozygosity excess: allele sequence divergence during long-term asexuality or hybrid origin of asexual lineages. Clonal diversity of asexual lineages of R. padi was substantial suggesting that they could have frozen genetic diversity from the pool of sexual lineages. Several widespread asexual genotypes were found to persist through time, as already seen in other aphid species, a feature seemingly consistent with the general-purpose genotype hypothesis.