Haploid Components Research Articles

New application for haplotype-specific extraction: Separation of mitochondrial DNA mixtures

A particularly challenging aspect of forensic casework is the accurate interpretation of samples that contain DNA from more than one contributor, since DNA analysis of specific identification markers often results in ambiguous mixed profiles. Physically separating the various haploid components of a diploid or polyploid DNA sample or of a specimen containing DNA from multiple individuals can resolve this problem. We adapted and combined previously developed methods of haplotype-specific extraction (HSE) to separate and analyze mitochondrial DNA (mtDNA) in samples containing DNA from two individuals. After optimizing conditions and probes, we performed HSE to separate mitochondrial DNA mixtures, which manifest as mixed nucleotide calls at single base positions. We were able to subsequently identify the DNA of the two individuals by sequencing. Based on our findings from this proof-of-concept experiment, this novel assay will be useful for distinguishing among the mtDNA of individuals in mixed DNA samples.

Karyotype Analysis of Ten Species of <i>Ipomoea</i> Jacq.

The genus Ipomoea with about 500 species is distributed in the tropical and subtropical regions of the world. The species are not only among the most beautiful ornamental plants, they are also important because of their medicinal value. The present study deals with the karyotype analysis of 10 species of Ipomoea growing in Maharashtra state of India. All the species analysed are diploid, showing 2n=30. I. aquatica has the smallest chromosomes, 1.25 to 2.67 μm, with a mean length of 1.99(±0.38), and the total length of the haploid component as 29.88 μm, while I. carnea has the longest chromosomes, at 2.13 to 4.79 μm, with mean length as 3.39(±0.74) and total length of the haploid component at 50.83 μm. The karyomorphological investigations revealed that none of the species had a symmetrical karyotype. The degree of asymmetry was, however, low. Amongst the several parameters studied, that of Stebbins (1971) and the AI values were found to be the most reliable for assessing asymmetry. The species falling under group 2B of Stebbins had higher AI values as compared to those of group 1B, proving that they are more evolved. The heterogeneity within the 10 species could be traced by AI values.

Individual specific extraction of DNA from male mixtures—First evaluation studies

In forensic work, the interpretation of DNA profiles becomes complicated when samples contain more than one contributor because the simultaneous amplification of individual identification markers results in mixed profiles. To overcome this problem, we present haplotype-specific extraction (HSE) as a more straightforward method to analyze a DNA mixture. HSE has been developed to clarify ambiguous HLA alleles by separating diploid samples into their haploid components to facilitate HLA typing. We have started to establish new protocols and strategies to adapt HSE for the separation of male DNA mixtures in forensic analysis. First results have shown an improved enrichment of male DNA from a single contributor. We have also evaluated a new, optimized buffer composition by testing different concentrations of its components. Improved separation of a male DNA mixture is detected using AmpFℓSTR® Yfiler short tandem repeat analysis.

Molecular genetic analysis of a new B112 allele of ABO blood group

To analyze the molecular genetic basis for a new B112 allele of ABO blood group and the pedigree of the proband. The ABO group antigens on red cells of the proband were identified by monoclonal antibodies. The ABO antibody in serum was detected by using standard A, B and O cells. The exons 5-7 of ABO gene for the proband was amplified by polymerase chain reaction and the amplified products were digested with double enzymes and sequenced for exons 6 and 7. A magnetic bead-based, haplotype specific extraction was used to separate the diploid sample of the proband into its haploid components. The exons 6 and 7 of the two single ABO haplotypes were then amplified and sequenced separately. The samples of the parents of the proband were collected, and the blood group serological test and sequence analysis for exons 6 and 7 of ABO gene were performed. The serum characteristic of the proband was consisted with the normal B phenotype. The DNA sequencing of exons 6 and 7 showed 261G/del, 297A/G, 526C/G, 559C/T, 657C/T, 703G/A, 796C/A, 803G/C and 930G/A heterozygotes and was assigned for B/O genotype. After separation of the two single strands of the proband with haplotype specific extraction, a B112 and an O01 allele were identified after sequencing. The B112 allele had one nucleotide change (C to T) at position 559 compared with B101, which resulted in an amino acid change at position 187 (Arg to Cys). The B112 in the proband was identified to inherit from his mother after pedigree analysis, the ABO blood group serological characteristics and sequences of exons 6 and 7 of the mother were identical to that of the proband. A novel B112 allele of ABO group system with 559C>T was identified. It had normal B antigen expression, suggesting that Arg118Cys of alpha-1, 3 galactosyltransferase did not affect its enzyme activity.

Haplotype inference from diploid sequence data: evaluating performance using non-neutral MHC sequences

The direct sequencing of PCR products from diploid organisms is problematic because of ambiguities associated with phase inference in multi-site heterozygotes. Several molecular methods such as cloning, SSCP, and DGGE have been developed to empirically reduce diploid sequences to their constitutive haploid components, but in theory these empirical approaches can be supplanted by analytical treatment of diploid sequences. Analytical approaches are more desirable than molecular methods because of the added time and expense required to generate molecular data. A variety of analytical methods have been developed to address this issue, but few have been rigorously evaluated with empirical data. Furthermore, they all assume that the sequences under consideration are evolving in a neutral fashion and assume a moderate number of heterozygous sites. Here, we use non-neutral major histocompatibility complex (MHC) sequences comprised of large numbers of heterozygous sites that are under strong balancing selection to evaluate the performance of the popular Bayesian algorithm implemented by the program PHASE. Our results suggest that PHASE performs admirably with non-neutral sequences of moderate length with numerous heterozygous sites typical of MHC class II sequences. We conclude that analytical approaches to haplotype inference have great potential in large-scale population genetic assays, but recommend groundtruthing analytical results using empirical (molecular) approaches at the outset of population-level analyses.

Identification of a New HLA-B Allele (B*1576) by Haplotype Specific Extraction

Routine typing of a potential bone marrow donor by sequence-specific oligonucleotide probes (SSOP) and sequence based typing (SBT) produced inconclusive subtyping results, suggesting a new allele. A magnetic bead-based method, haplotype specific extraction (HSE), was used to separate the diploid sample into its haploid components. The sample was then re-typed using standard SBT, revealing a new human leukocyte antigen (HLA) allele, since named B*1576. HSE used in conjunction with standard SBT is a convenient and simple tool for resolving ambiguous and novel allele combinations without the need for amplification or subcloning.

Haplotype‐specific extraction: a universal method to resolve ambiguous genotypes and detect new alleles – demonstrated on HLA‐B

Haplotype-specific extraction (HSE) allows the collection of individual alleles by separating diploid samples into their haploid components. The separation step is performed using magnetic beads in conjunction with allele-group-specific probes. The haplo-separated DNA samples can be directly typed with downstream applications such as sequence-specific oligonucleotide (SSO) typing, sequence-specific primer (SSP) typing, and sequence-based (SB) typing. Here we show that HSE permits the direct sequencing of an allele in its individual, separated state, including previously unknown alleles. Allele pair combinations that cannot be resolved by SSP, SSO, or generic SB typings can be unambiguously typed after the alleles are separated by HSE, which allows for new alleles to be easily detected without cloning. We show how HSE was performed to separate samples with locus-specific ambiguities in human leukocyte antigen (HLA)-B, which could not be resolved by means of generic SB typing for either sample. After haplotype-specific separation of the respective allele pairs, novel polymorphisms in the HLA-B*56 and HLA-B*44 alleles were clearly detected by SB typing.

Dikaryons of the basidiomycete fungus Schizophyllum commune: evolution in long-term culture.

The impact of ploidy on adaptation is a central issue in evolutionary biology. While many eukaryotic organisms exist as diploids, with two sets of gametic genomes residing in the same nucleus, most basidiomycete fungi exist as dikaryons in which the two genomes exist in separate nuclei that are physically paired and that divide in a coordinated manner during hyphal extension. To determine if haploid monokaryotic and dikaryotic mycelia adapt to novel environments under natural selection, we serially transferred replicate populations of each ploidy state on minimal medium for 18 months (approximately 13,000 generations). Dikaryotic mycelia responded to selection with increases in growth rate, while haploid monokaryotic mycelia did not. To determine if the haploid components of the dikaryon adapt reciprocally to one another's presence over time, we recovered the intact haploid components of dikaryotic mycelia at different time points (without meiosis) and mated them with nuclei of different evolutionary histories. We found evidence for coadaptation between nuclei in one dikaryotic line, in which a dominant deleterious mutation in one nucleus was followed by a compensatory mutation in the other nucleus; the mutant nuclei that evolved together had the best overall fitness. In other lines, nuclei had equal or higher fitness when paired with nuclei of other histories, indicating a heterozygote advantage. To determine if genetic exchange occurs between the two nuclei of a dikaryon, we developed a 24-locus genotyping system based on single nucleotide polymorphisms to monitor somatic exchange. We observed genetic exchange and recombination between the nuclei of several different dikaryons, resulting in genotypic variation in these mitotic cell lineages.

Identification of quantitative trait loci contributing to Fusarium wilt resistance on an AFLP linkage map of flax (Linum usitatissimum)

An AFLP genetic linkage map of flax (Linum usitatissimum) was used to identify two quantitative trait loci (QTLs) on independent linkage groups with a major effect on resistance to Fusarium wilt, a serious disease caused by the soil pathogen Fusarium oxysporum (lini). The linkage map was constructed using a mapping population from doubled-haploid (DH) lines. The DH lines were derived from the haploid component of F2 haploid-diploid twin seed originating from a cross between a polyembryonic, low-linolenic-acid genotype (CRZY8/RA91) and the Australian cultivar ‘Glenelg’. The AFLP technique was employed to generate 213 marker loci covering approximately 1400 cM of the flax genome (n=15) with an average spacing of 10 cM and comprising 18 linkage groups. Sixty AFLP markers (28%) deviated significantly (P<0.05) from the expected segregation ratio. The map incorporated RFLP markers tightly linked to flax rust (Melamspora lini) resistance genes and markers detected by disease resistance gene-like sequences. The study illustrates the potential of the AFLP technique as a robust and rapid method to generate moderately saturated linkage maps, thereby allowing the molecular analysis of traits, such as resistance to Fusarium wilt, that show oligogenic patterns of inheritance.

Inheritance of resistance to flax wilt (Fusarium oxysporum f.sp. lini Schlecht) in a doubled haploid population of Linum usitatissimum L.

The inheritance of resistance to fusarium wilt (Fusarium oxysporum f.sp. lini) was investigated in Linum usitatissimum as a first step towards gaining an understanding of the molecular genetics of the disease and developing a procedure for marker-assisted selection. A recombinant doubled haploid (DH) population was derived from the haploid component of polyembryonic F2 seeds originating from a cross between a wilt resistant, twinning Linola™ Linola is a registered trademark of CSIRO line CRZY8/RA91 and the wilt susceptible Australian flax cultivar Glenelg. The segregation of resistance was studied in 143 DH lines under glasshouse and field conditions. Most of the phenotypic variation was attributable to the segregation of two independent genes with additive effects. Minor resistance genes may have also contributed by modifying the resistance response. A glasshouse screening method of DH lines proved a reliable indicator of field resistance to fusarium wilt.

Genome Size of Bisexual and Unisexual Poeciliopsis

We used flow cytometry to examine the genome size of bisexual and unisexual fishes in the genus Poeciliopsis. Two bisexual species, P. monacha and P. lucida, had genome sizes of 1.45–1.47 pg DNA and 1.37 pg DNA, respectively. These are small genomes for a vertebrate. Hemiclones of the unisexual hybrid P. mona-cha-lucida had genome sizes intermediate to those of P. monacha and P. lucida, its sexual progenitors. The triploid unisexuals P. 2 monacha-lucida and P. monacha-2 lucida had genome sizes equal to the sum of their three haploid components. No ploidy mosaics were observed among the unisexuals. Their genome size has not changed over time from that of the parental species that hybridized and produced them.

Contribution de l'étude du polymorphisme enzymatique à la systématique des Pleurotes des Ombellifères

This study presents a contribution to the systematics of Pleurotus species growing on Umbellifers, which are classically divided into three types. Studies of enzyme polymorphism by electrophoresis performed on synthetized dikaryotic mycelia and their haploid components (homokaryons) allowed a genotypic determination of "wild" dikaryons, and a statistical treatment of homokaryotic genotypes. These results show a distinction between P. nebrodensis on one hand, and P. eryngii and P. ferulae on the other hand, and support the hypothesis of a division of P. nebrodensis into two types at least as distinct from each other as P. eryngii from P. ferulae. Different hypotheses related to internal dynamics and evolution of this group of Basidiomycetes are discussed.

The Effect of Ploidy on the Stability of Plasmodial Heterokaryons in Physarum polycephalum

The stability of actively growing heterokaryons made by fusing haploid and diploid plasmodia of Physarum polycephalum has been investigated with the aid of genetic markers affecting plasmodial colour and amino acid requirements. All heterokaryons initially expressed the dominant alleles present in both component plasmodia, but after a few subcultures every heterokaryon synthesized from a haploid plus a diploid plasmodium changed to express the recessive alleles carried by the haploid component. In contrast, heterokaryons synthesized from combinations of haploid plasmodia remained stable throughout many subcultures. No evidence was found for the segregation of incompatibility alleles responsible for heterokaryon instability, although the possibility could not be excluded that a gene closely linked to matA was involved. A direct test of the effect of ploidy on heterokaryon stability was carried out, in which the use of isogenic haploid and diploid plasmodia allowed the effect of incompatibility genes to be eliminated. Again the phenotype of every haploid plus diploid heterokaryon changed to that of the haploid component, whereas haploid plus haploid heterokaryons remained stable. Variations in the relative sizes of the haploid and diploid plasmodia altered the speed, but not the direction, of the changes observed. Analysis of progeny indicated that the changes in phenotype were due to loss of diploid nuclei from the heterokaryons.

Inheritance of polyembryony in flax (Linum usitatissimum)

The inheritance of polyembryony (haploid–diploid twin seedlings) in the cross between Linum usitatissimum L. cv. Avantgarde (zero twinning) and cv. Rocket 4 (6.6% twins) was examined. It was concluded that the production of polyembryonic seeds is controlled mainly by additive gene action, with a realized narrow-sense heritability of 0.8. The frequency of twinning in the F2 generation displayed substantial transgressive segregation, ranging from zero to 32.0%. It was considered that such transgression could be due to either the presence of residual heterogeneity for twinning alleles in cv. Rocket 4, or the existence of a genetic system suppressing the expression of twinning alleles in cv. Avantgarde. The occurrence of a genotype producing haploid plants from more than 30% of its seeds should enable the efficiency of doubled-haploid line production to be greatly improved, and also lead to a better understanding of the embryological origins of the haploid component of polyembryonic seeds.

Étude du polymorphisme enzymatique d'une population de pleurotes des ombellifères : Pleurotus eryngii (Tournus, France)

Electrophoretic separations followed by revelations of esterase, aminopeptidase, and phosphatase activities were performed on a population of Pleurotus eryngii (Tournus: Saône valley, France). Electrophoresis on controlled dicaryotic mycelia and their haploid components was used as a convenient means to study the genetic determination of some esterase and phosphatase isoenzymes. The electrophoretic patterns obtained from original isolates (wild dicaryons) show little variability, but internal variations are revealed. These variations allowed for a characterization of four genotypically different individual groups. This analytical method seems to give some interesting data, useful for taxonomic studies.

The Effects of Griseofulvin on Diploid Strains of Coprinus lagopus

Summary: Diploid strains of Coprinus lagopus grown on medium supplemented with griseofulvin (20 μg ml−1) produced fast-growing sectors of two types after 7 to 28 days incubation: these were diploid somatic recombinants and haploids. A dikaryon grown on the same medium broke down to give one haploid component only; resolution of this dikaryon and a near-isogenic diploid resulted in selection for sectors carrying a gene for resistance to griseofulvin. The use of this antibiotic is suggested as a tool for routine haploidization in C. lagopus.

Intergenic complementation of glucoamylase and citric acid production in two species of Aspergillus.

All auxotrophs of Aspergillus foetidus and all but two auxotrophs of A. niger which we isolated yield glucoamylase and citric acid, respectively, at levels below that of the prototrophic strain from which they were derived. Results of representative heterokaryon tests suggest that the nucleus was principally responsible for the inheritance of citric acid or glucoamylase production. Most somatic diploid strains of A. foetidus gave rise to higher yields of glucoamylase when compared to their haploid component strains. Both heterokaryons and somatic diploid strains of A. niger synthesized between auxotrophs which were simultaneously reduced in citric acid yields also gave rise to enhanced yields when compared with their haploid components. The yields of a heterokaryon and somatic diploid synthesized between two high producers of citric acid were not higher than those of respective haploid components. We concluded from these results that gene dosage (or ploidy) does not increase the yield of citric acid. The apparent enhancement in yields observed in diploids or heterokaryons synthesized between auxotrophs with reduced yields in both species can be interpreted as resulting from intergenic complementation.

Intergenic Complementation of Glucoamylase and Citric Acid Production in Two Species of Aspergillus

All auxotrophs of Aspergillus foetidus and all but two auxotrophs of A. niger which we isolated yield glucoamylase and citric acid, respectively, at levels below that of the prototrophic strain from which they were derived. Results of representative heterokaryon tests suggest that the nucleus was principally responsible for the inheritance of citric acid or glucoamylase production. Most somatic diploid strains of A. foetidus gave rise to higher yields of glucoamylase when compared to their haploid component strains. Both heterokaryons and somatic diploid strains of A. niger synthesized between auxotrophs which were simultaneously reduced in citric acid yields also gave rise to enhanced yields when compared with their haploid components. The yields of a heterokaryon and somatic diploid synthesized between two high producers of citric acid were not higher than those of respective haploid components. We concluded from these results that gene dosage (or ploidy) does not increase the yield of citric acid. The apparent enhancement in yields observed in diploids or heterokaryons synthesized between auxotrophs with reduced yields in both species can be interpreted as resulting from intergenic complementation.

Recessive lethals induced by nitrours acid in Aspergillus nidulans

The frequency of spontaneous and nitrous acid-induced irreparable recessive lethal mutations has been estimated in a diploid strain of Aspergillus nidulans. Markers on at least one member of each chromosome pair permitted the assay of lethals in all 16 chromosomes of the diploid strain. 50 treated isolates produced 4 lethals giving a frequency of 8% induced lethals for the whole genome at 5% survival. One isolate was heterozygous for 2 non-allelic lethals in trans. After treatment with UV it produced a stable heterokaryon which was balanced by the non-allelic lethals, one on each haploid component. Meiotic and mitotic analysis showed that UV treatment has caused a translocation, to chromosome V, of a segment of a chromosome III carrying one recessive lethal and also the wild-type allele of the second lethal.

Lethal mutations and balanced lethal systems in Aspergillus nidulans.

SUMMARY The frequency of spontaneous and ultraviolet-induced, recessive, lethal mutations was estimated in two diploid strains of Aspergillus nidulans. The parent diploids were heterozygous for recessive and semi-dominant markers; each recessive lethal resulted in failure to recover haploid strains carrying the allele linked in coupling to that lethal. Diploids 1 and 2 carried markers permitting direct assay of lethals on 4 and 8 chromosomes, respectively, out of the total diploid complement of 16. No lethals were detected in 100 isolates from each untreated diploid. One hundred isolates from each treated parent yielded 6 and 16 lethals, respectively, suggesting a frequency for the whole genome, at 5 % survival, of about 28 % lethals. The lethals were nutritionally irreparable and were not temperature-sensitive. Several of them have been located roughly by mitotic crossing-over. One isolate was heterozygous for two unlinked lethals. It segregated to give a stable heterokaryon bearing two classes of non-viable haploid conidia. Meiotic analysis, via a three-component heterokaryon, showed that the stable heterokaryon was balanced by the non-allelic lethals, one in each haploid component. Another isolate had a balanced lethal system resulting from linked, non-allelic lethals in trans. Six isolates from treatment were stable diploids which produced no haploid sectors.