Complete Homozygosity Research Articles

Fast development of genetically uniform strains by gynogenesis in Japanese flounder (Paralichthys olivaceus)

The Japanese flounder is a major species in both aquaculture and research. Inbred strains of Japanese flounder were developed efficiently in our laboratory by meiotic and mitotic gynogenetic reproduction techniques. To determine the induction efficiency of gynogenesis and the rate at which full homozygosity is produced, six meiotic gynogenetic females (G1–G6) that had experienced meiotic gynogenesis once and three common females (C1–C3) were selected for production of mitotic gynogenesis in our experiments. Of the nine adult females, all six gynogenetic fish successfully produced viable offspring. However, only one of the three common fish did. Using microsatellite markers, we estimated the homozygosity of gynogenetic fish induced by mitotic gynogenesis. We found that the homozygosity of seven lines (C1, G1–G6) increased quickly, individually from 43%, 72%, 69%, 71%, 78%, 69% and 58% to 100%, 100%, 100%, 90%, 100%, 100% and 90% offspring complete homozygosity. Under mitotic gynogenesis, individuals with higher homozygosity had a higher induction rate. The G4 line showed the highest induction rate, achieving 44.59% convert hatching rate and 29.28% convert normality rate. We conclude that meiotic gynogenesis may be a feasible method to produce DH genetic material in Japanese flounder.

Early embryo achievement through isolated microspore culture in Citrus clementina Hort. ex Tan., cvs. ‘Monreal Rosso’ and ‘Nules’

Microspore embryogenesis is a method of achieving complete homozygosity from plants. It is particularly useful for woody species, like Citrus, characterized by long juvenility, a high degree of heterozygosity and often self-incompatibility. Anther culture is currently the method of choice for microspore embryogenesis in many crops. However, isolated microspore culture is a better way to investigate the processes at the cellular, physiological, biochemical, and molecular levels as it avoids the influence of somatic anther tissue. To exploit the potential of this technique, it is important to separate the key factors affecting the process and, among them, culture medium composition and particularly the plant growth regulators and their concentration, as they can greatly enhance regeneration efficiency. To our knowledge, the ability of meta-Topolin, a naturally occurring aromatic cytokinin, to induce gametic embryogenesis in isolated microspores of Citrus has never been investigated. In this study, the effect of two concentrations of meta-Topolin instead of benzyladenine or zeatin in the culture medium was investigated in isolated microspore culture of two genotypes of Citrus. After 11 months of isolated microspore culture, for both genotypes and for all the four tested media, the microspore reprogramming and their sporophytic development was observed by the presence of multinucleated calli and microspore-derived embryos at different stages. Microsatellite analysis of parental and embryo samples was performed to determine the embryo alleles constitution of early embryos produced in all tested media, confirming their origin from microspores. To our knowledge, this is the first successful report of Citrus microspore embryogenesis with isolated microspore culture in Citrus, and in particular in Citrus clementina Hort. ex Tan, cvs. ‘Monreal Rosso’ and ‘Nules.’

Polyploidy versus endosymbionts in obligately thelytokous thrips.

BackgroundThelytoky, the parthenogenetic development of females, has independently evolved in several insect orders yet the study of its mechanisms has so far mostly focussed on haplodiploid Hymenoptera, while alternative mechanisms of thelytoky such as polyploidy are far less understood. In haplodiploid insects, thelytoky can be encoded in their genomes, or induced by maternally inherited bacteria such as Wolbachia or Cardinium. Microbially facilitated thelytoky usually results in complete homozygosity due to gamete duplication and can be reverted into arrhenotoky, the parthenogenetic development of males, through treatment with antibiotics. In contrast, genetically encoded thelytoky cannot be removed and may result in conservation of heterozygosity due to gamete fusion. We have probed the obligate thelytoky of the greenhouse thrips, Heliothrips haemorrhoidalis (Bouché), a significant cosmopolitan pest and a model species of thelytoky in the haplodiploid insect order Thysanoptera. Earlier studies suggested terminal fusion as a mechanism for thelytoky in this species, while another study reported presence of Wolbachia; later it was speculated that Wolbachia plays a role in this thrips’ thelytokous reproduction.ResultsBy using PCR and sequence analysis, we demonstrated that global population samples of H. haemorrhoidalis were not infected with Wolbachia, Cardinium or any other known bacterial reproductive manipulators. Antibiotic treatment of this thrips did also not result in male production. Some individuals carried two different alleles in two nuclear loci, histone 3 and elongation factor 1 alpha, suggesting heterozygosity. However, the majority of individuals had three different alleles suggesting that they were polyploid. Genetic diversity across both nuclear loci was low in all populations, and absent from mitochondrial cytochrome oxidase I, indicating that this species had experienced genetic bottlenecks, perhaps due to its invasion biology or a switch to thelytoky.ConclusionsGeographically broad sampling and experimental manipulation revealed low genetic diversity, absence of Wolbachia but presence of three different alleles of nuclear loci in most analysed individuals of obligately thelytokous H. haemorrhoidalis. This suggests that polyploidy may be involved in the thelytokous reproduction of this thrips species, and polyploidy may be a contributing factor in the reproduction of Thysanoptera and other haplodiploid insect orders.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0304-6) contains supplementary material, which is available to authorized users.

Selection Strategies for the Development of Maize Introgression Populations

Introgression libraries are valuable resources for QTL detection and breeding, but their development is costly and time-consuming. Selection strategies for the development of introgression populations with a limited number of individuals and high-throughput (HT) marker assays are required. The objectives of our simulation study were to design and compare selection strategies for the development of maize introgression populations of 100 lines with population sizes of 360–720 individuals per generation for different DH and crossing schemes. Pre-selection for complete donor chromosomes or donor chromosome halves reduced the number of simultaneous backcross programs. The investigated crossing and selection schemes differed considerably with respect to their suitability to create introgression populations with clearly separated, evenly distributed target donor chromosome segments. DH crossing schemes were superior to crossing schemes, mainly due to complete homozygosity, which greatly reduced the total number of disjunct genome segments in the introgression populations. The crossing schemes were more flexible with respect to selection and provided economic alternatives to DH crossing schemes. For the DH crossing schemes, increasing population sizes gradually over backcross generations was advantageous as it reduced the total number of required HT assays compared to constant population sizes. For the crossing schemes, large population sizes in the final backcross generation facilitated selection for the target segments in the final backcross generation and reduced fixation of large donor chromosome segments. The suggested crossing and selection schemes can help to make the genetic diversity of exotic germplasm available for enhancing the genetic variation of narrow-based breeding populations of crops.

Single-nucleotide variations defining previously unreportedADAMTS13haplotypes are associated with differential expression and activity of the VWF-cleaving protease in a Salvadoran congenital thrombotic thrombocytopenic purpura family

Background Although autosomal recessive hematologic disorders are individually rare and difficult to ascertain, studies involving one or more homozygous affected children and their unaffected heterozygous parents have led to expanded understanding of known and discovery of previously unknown processes. The son and daughter of two Salvadoran parents were diagnosed with congenital thrombotic thrombocytopenic purpura (cTTP) at 6 and 2 years of age, respectively, after presenting with fever, respiratory symptoms, hemolytic anemia, and thrombocytopenia and being found to have ADAMTS13 activities Methods Prior to a plasma infusion, blood samples were collected from the children and parents. Genomic DNA was isolated for polymerase chain reaction (PCR), and direct Sanger sequencing of all ADAMTS13 exons and flanking intronic segments was performed; all variants identified were confirmed by bidirectional sequencing of a second, independently generated amplicon. Total RNA was isolated and the steady-state level of ADAMTS13 mRNA was measured using a quantitative real-time PCR (q-RT-PCR)-based assay. ADAMTS13 was characterized enzymatically using the fluorogenic FRETS-VWF73 substrate and antigenically by ELISA. Results Both children were found to be homozygous and parents to be heterozygous for the previously described, cTTP-causing ADAMTS13 single-base-substitution mutation 20506C>T, a missense mutation that encodes cDNA-nucleotide 2518 (c.2518C>T) and ADAMTS13 residue 692 (692Arg>Cys [692R>C]) (Fig. A). As expected, the children9s ADAMTS13 antigen and activity levels were undetectable, although notably, steady-state levels of the ADAMTS13 mRNA were >2.5-fold higher in the daughter than in the son. The re-sequenced regions of the ADAMTS13 loci segregating within this family contained 26 additional SNVs, seven of which were nonsynonymous (ns) including two previously unreported ns-SNVs: 27852C>T (c.3362C>T; 972Arg>Trp) and 33325G>A (c.3733G>A; 1096Arg>His) (Fig. A, left panel). The parents9 genotypes differed at nine positions, including three ns-SNVs, creating two distinct, non-mutant haplotypes (designated I and III) at the gene, mRNA and protein levels. The q-RT-PCR assay revealed >4-fold higher steady-state mRNA levels in the father compared to the mother (p Discussion We capitalized on the fortuitous finding of children with complete homozygosity across ancestrally-related ADAMTS13 alleles harboring a null-type, loss-of-function mutation, as this enabled the substantially different levels of gene expression and function observed in the parents to be attributed to their two previously unreported, SNV-based, ADAMTS13 haplotypes. Additional investigation at the molecular, biochemical, cellular, and organismal levels will be necessary to determine which of the myriad potential individual SNV- and/or haplotype-based mechanisms are responsible for the observed parental differences in circulating ADAMTS13 antigen and activity. Disclosures: Kim: Haplomics, Inc.: Membership on an entity’s Board of Directors or advisory committees; Baxter: Honoraria. Marder: Baxter: Research Funding. Howard: Haplomics, Inc.: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Baxter: Research Funding.

Single-Nucleotide Variations Defining Previously Unreported ADAMTS13 Haplotypes Are Associated With Differential Expression and Activity Of The VWF-Cleaving Protease In a Salvadoran Congenital Thrombotic Thrombocytopenic Purpura Family

BackgroundAlthough autosomal recessive hematologic disorders are individually rare and difficult to ascertain, studies involving one or more homozygous affected children and their unaffected heterozygous parents have led to expanded understanding of known and discovery of previously unknown processes. The son and daughter of two Salvadoran parents were diagnosed with congenital thrombotic thrombocytopenic purpura (cTTP) at 6 and 2 years of age, respectively, after presenting with fever, respiratory symptoms, hemolytic anemia, and thrombocytopenia and being found to have ADAMTS13 activities <1% without neutralizing IgG antibodies. They remain without long-term neurologic or renal sequelae following prophylactic infusions of fresh plasma (10 mL/kg every 2.5-weeks). The purpose of this study was to characterize and correlate single-nucleotide variations (SNVs) in each parent's, non-mutant ADAMTS13 allele with its mRNA and protein expression, activity, and enzyme kinetics. MethodsPrior to a plasma infusion, blood samples were collected from the children and parents. Genomic DNA was isolated for polymerase chain reaction (PCR), and direct Sanger sequencing of all ADAMTS13 exons and flanking intronic segments was performed; all variants identified were confirmed by bidirectional sequencing of a second, independently generated amplicon. Total RNA was isolated and the steady-state level of ADAMTS13 mRNA was measured using a quantitative real-time PCR (q-RT-PCR)-based assay. ADAMTS13 was characterized enzymatically using the fluorogenic FRETS-VWF73 substrate and antigenically by ELISA. ResultsBoth children were found to be homozygous and parents to be heterozygous for the previously described, cTTP-causing ADAMTS13 single-base-substitution mutation 20506C>T, a missense mutation that encodes cDNA-nucleotide 2518 (c.2518C>T) and ADAMTS13 residue 692 (692Arg>Cys [692R>C]) (Fig. A). As expected, the children's ADAMTS13 antigen and activity levels were undetectable, although notably, steady-state levels of the ADAMTS13 mRNA were >2.5-fold higher in the daughter than in the son. The re-sequenced regions of the ADAMTS13 loci segregating within this family contained 26 additional SNVs, seven of which were nonsynonymous (ns) including two previously unreported ns-SNVs: 27852C>T (c.3362C>T; 972Arg>Trp) and 33325G>A (c.3733G>A; 1096Arg>His) (Fig. A, left panel). The parents' genotypes differed at nine positions, including three ns-SNVs, creating two distinct, non-mutant haplotypes (designated I and III) at the gene, mRNA and protein levels. The q-RT-PCR assay revealed >4-fold higher steady-state mRNA levels in the father compared to the mother (p<0.001; Fig. B). Plasma ADAMTS13 activity and antigen levels were ∼2-fold greater in the father than in the mother (p=0.00164 and p=0.0633, respectively), but the specific activities of these structurally distinct ADAMTS13 proteins were notably almost identical (253.5 vs. 256.2 U/μg). Moreover, initial velocity kinetic analysis using the Michelis-Menten equation demonstrated that the Vmax of the father's ADAMTS13 was twice that of the mother's (1.4 vs. 0.7; p < 0.0001) while its affinity for substrate was one-third that of her ADAMTS13 (Km = 0.3 vs. 0.1; p = 0.0585). DiscussionWe capitalized on the fortuitous finding of children with complete homozygosity across ancestrally-related ADAMTS13 alleles harboring a null-type, loss-of-function mutation, as this enabled the substantially different levels of gene expression and function observed in the parents to be attributed to their two previously unreported, SNV-based, ADAMTS13 haplotypes. Additional investigation at the molecular, biochemical, cellular, and organismal levels will be necessary to determine which of the myriad potential individual SNV- and/or haplotype-based mechanisms are responsible for the observed parental differences in circulating ADAMTS13 antigen and activity. [Display omitted] Disclosures:Kim:Haplomics, Inc.: Membership on an entity's Board of Directors or advisory committees; Baxter: Honoraria. Marder:Baxter: Research Funding. Howard:Haplomics, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Baxter: Research Funding.

Genetic Mosaicism and Hemoglobinopathies; Uniparental Isodisomy

BackgroundRecently we discovered three independent cases of “severe late onset beta-thalassemia”, all presenting with the mild phenotype of beta thalassemia minor up to adult age and developing a severe transfusion dependent phenotype in the third and fourth decade of life when a presumed homozygosity for the beta-thalassemia mutation is observed. We demonstrate that uniparental isodisomy (UPD) of part of chromosome 11p, a mechanism also seen in 20% of Beckwith-Wiedemann syndrome patients, accounts for the observed mosaicism in all three independent cases. Clonal selection for hematopoietic stem cells containing the UPD for the mutant beta-globin gene during life may account for the progressive development of the disease. Recently we discovered another case showing a similar mosaic UPD of 11p, presenting as a regular beta-thalassemia carrier. MethodDirect sequencing of the beta-globin genes. Multiplex Ligation dependent Probe Amplification (MLPA) analysis of the HBB (beta-globin) gene cluster. Affymetrix GeneChip Human Mapping 262K NspI array (Santa Clara, CA, USA). Illumina OmniExpress 730K SNP Beads array (San Diego, CA, USA). ResultsIn all cases molecular analysis shows sequences in which a near to complete homozygosity for the beta-gene mutation in DNA extracted from leucocytes is seen. Loss of heterozygosity due to allele drop-out or a deletion of one allele was excluded by direct sequencing and MLPA analysis. Affymetrix and/or Illumina SNP-array analysis revealed incomplete homozygosity for SNP's along almost the entire short arm of chromosome 11 containing the beta-globin gene, indicating mosaicism for a partial uniparental isodisomy of chromosome 11p. Three patients were born asymptomatic as beta-thal carriers and developed a severe blood-transfusion dependent beta-thalassemia major at different ages and with different percentages of mosaicism. The fourth patient however did not develop the clinical severity despite of an almost 50% mosaicism determined from the DNA isolated from leucocytes. ConclusionWe demonstrate that uniparental isodisomy of part of chromosome 11 accounts for the observed mosaicism in all four independent cases. The most probable mechanism seems clonal selection for hematopoietic stem cells containing the uniparental isodisomy for the mutant beta-globin gene during life, this may account for the progressive development of the disease. However, there seems to be no correlation between the percentage of mosaicism measured in the DNA isolated from the white cells and the severity of the clinical phenotype related to the expression in red cells, which strongly suggests hematopoietic tissue heterogeneity for the observed UPD containing cell lineages. This may have serious consequences for disease prediction and counseling, as this is largely dependent upon DNA isolated from leucocytes. Disclosures:No relevant conflicts of interest to declare.

English

&nbsp; Traditional basmati rice varieties are very low yielding due to their tendency to lodging and increasing susceptibility to diseases. To improve the characters of basmati rice variety and study the inheritance of various physio-morphological and quality characters,&nbsp;F5&nbsp;population comprising of 204 lines from the cross between NPT II (non-aromatic,&nbsp;japonica) and Taraori Basmati or HBC19 (aromatic,&nbsp;indica), were evaluated. Ample amount of genetic variability was observed for the characters plant height, tillers per plant, kernel length, kernel breath and L/B ratio. The grain yield/plant showed positive correlation with productive tiller/plant and test weight. Path coefficient analysis showed that the productive tiller/plant and test weight contribute to grain yield/ plant through direct effect. The parent off-spring regression was high for all the characters under study suggesting improvement of these characters by mere selection. Based on divergence study, 204 lines were categorized in seven clusters whereas parents were grouped in different clusters. Molecular restricted selection using specific SSR markers with depicting high correlation with aroma could offer great promise to select high yielding rice among high aroma lines. A total of 54 randomly selected F5&nbsp;plants were subjected to SSR marker analysis using SSR markers. The F5&nbsp;plants had an allele from either of the two parental lines (homozygous condition) or alleles from both the parental rice varieties (heterozygous condition). At some SSR loci, new/recombinant alleles were observed, which indicate the active recombination between genomes of two rice varieties and can be used for linkage mapping once complete homozygosity is achieved. SSR allelic profile based on two dimensional principal component analysis demonstrated high level of diversity among parents and F5&nbsp;plants spread between them. &nbsp; Key words:&nbsp;Oryza sativa&nbsp;L., basmati, microsatellite, phenotyping, rice, recombinant inbred lines (RILs).

Matching Maternal Isodisomy in Mucinous Carcinomas and Associated Ovarian Teratomas Provides Evidence of Germ Cell Derivation for Some Mucinous Ovarian Tumors

The tissue derivation of mucinous ovarian carcinoma remains a mystery; however, rare tumors are associated with mature teratoma. Two decades ago, studies of chromosomal heteromorphisms and DNA polymorphisms proved that ovarian teratomas arise during female gametogenesis. We sought to exploit the relationship between mucinous carcinoma and associated teratoma to provide molecular evidence for tissue of origin. Seventeen cases of mucinous ovarian carcinoma were studied, 6 of which had associated mature teratoma. DNA was extracted from the mucinous carcinoma, teratoma, and normal dissected tissue from formalin-fixed, paraffin-embedded sections. Twelve polymorphic microsatellite markers were used to allelotype each sample. Alleles from the teratomas and carcinomas were scored as homozygous (1 allele present in the tumor when normal tissue was heterozygous), heterozygous (2 alleles present matching normal tissue), or noninformative (normal tissue was homozygous). Of the 6 carcinoma/teratoma pairs, 2 showed complete matching homozygosity for informative markers (isodisomy), whereas 2 showed matching heterozygosity. One case did not have the corresponding teratoma available for comparison but demonstrated complete homozygosity and was presumed to be isodisomic. The remaining case had a teratoma homozygous for 7 of 10 informative markers, whereas the matching carcinoma was homozygous for only 2 of these markers. Carcinomas without associated teratoma demonstrated variable zygosity. Microsatellite polymorphism analysis demonstrates that mucinous ovarian carcinomas usually clonally match associated teratomas when present and often show evidence of complete isodisomy, indicating that at least some mucinous carcinomas arise from female gametes and thus are of germ cell origin. The zygosity patterns in mucinous carcinomas without teratoma suggest that these tumors may arise through a different mechanism.

Verification of mitotic gynogenesis in ornamental (koi) carp (Cyprinus carpioL.) using microsatellite DNA markers

The Japanese ornamental (koi) carp is a popular decorative fish all over the world. In koi, clones have not yet been obtained, although production of fish with identical colour patterns could be of commercial interest. Mitotic gynogenetic progenies are essential for subsequent production of clones in fish. However, resulting late-shocked progenies may be contaminated with meiotic gynogens from spontaneous suppression of the second meiotic division in eggs. In this study, microsatellite DNA markers were used to confirm mitotic gynogenetic origin of obtained late-shocked progenies. Recombination rate (y) and mapping distance relative to centromere (M-C) of 10 microsatellite loci were determined based on percentage of heterozygotes in meiotic gynogenetic progenies. The range of y varied from 0.01 to 0.96 and the M-C map ranged from 0.5 to 48 cM. The mean value of y over the 10 loci was 0.481. Six loci, which had y 0.47 and higher, were used as markers in two late-shocked gynogenetic progenies. Complete homozygosity was revealed at all six microsatellite loci indicating mitotic gynogenetic origin of analysed progenies.

Parthenogenic Haploids in Melon: Generation and Molecular Characterization of a Doubled Haploid Line Population

Melon (Cucumis melo) is one of the principal vegetable crops for fresh market, for which a large number of breeding programs, oriented to generate inbred pure lines and hybrids, is established worldwide. The process to obtain and select these lines has been highly accelerated by the use of biotechnological techniques such as the generation of doubled haploid line (DHL) populations and molecular markers. Moreover, the use of DHLs in genetic studies is a useful tool because of their complete homozygosity and the permanent availability of plant material perpetuated by seed. In this work, the parthenogenetic response of 17 melon genotypes and the F1 hybrid PI 161375 × Spanish cultivar Piel de Sapo (PS) was studied considering three stages along the in vitro DHL generation process. The response of the analyzed melon cultivars was heterogeneous through the DHL generation with different limiting steps for each genotype. The response of the PI 161375 × PS hybrid was more similar to the male (PS) than the female parent (PI 161375), although the response of the maternal genotype was higher for some stages. This points to the important role of alleles from both parents in the different steps of the DHL generation process, and it could explain the identification of six genomic regions with distorted allelic segregation skewed toward PS or PI 161375. This hybrid was used to generate a population of 109 DHLs, the gametophytic origin of which was confirmed by flow cytometry and molecular markers.

Patterns of sequence polymorphism in the fleshless berry locus in cultivated and wild Vitis vinifera accessions

BackgroundUnlike in tomato, little is known about the genetic and molecular control of fleshy fruit development of perennial fruit trees like grapevine (Vitis vinifera L.). Here we present the study of the sequence polymorphism in a 1 Mb grapevine genome region at the top of chromosome 18 carrying the fleshless berry mutation (flb) in order, first to identify SNP markers closely linked to the gene and second to search for possible signatures of domestication.ResultsIn total, 62 regions (17 SSR, 3 SNP, 1 CAPS and 41 re-sequenced gene fragments) were scanned for polymorphism along a 3.4 Mb interval (85,127-3,506,060 bp) at the top of the chromosome 18, in both V. vinifera cv. Chardonnay and a genotype carrying the flb mutation, V. vinifera cv. Ugni Blanc mutant. A nearly complete homozygosity in Ugni Blanc (wild and mutant forms) and an expected high level of heterozygosity in Chardonnay were revealed. Experiments using qPCR and BAC FISH confirmed the observed homozygosity. Under the assumption that flb could be one of the genes involved into the domestication syndrome of grapevine, we sequenced 69 gene fragments, spread over the flb region, representing 48,874 bp in a highly diverse set of cultivated and wild V. vinifera genotypes, to identify possible signatures of domestication in the cultivated V. vinifera compartment. We identified eight gene fragments presenting a significant deviation from neutrality of the Tajima's D parameter in the cultivated pool. One of these also showed higher nucleotide diversity in the wild compartments than in the cultivated compartments. In addition, SNPs significantly associated to berry weight variation were identified in the flb region.ConclusionsWe observed the occurrence of a large homozygous region in a non-repetitive region of the grapevine otherwise highly-heterozygous genome and propose a hypothesis for its formation. We demonstrated the feasibility to apply BAC FISH on the very small grapevine chromosomes and provided a specific probe for the identification of chromosome 18 on a cytogenetic map. We evidenced genes showing putative signatures of selection and SNPs significantly associated with berry weight variation in the flb region. In addition, we provided to the community 554 SNPs at the top of chromosome 18 for the development of a genotyping chip for future fine mapping of the flb gene in a F2 population when available.

Thrombotic genetic risk factors and warfarin pharmacogenetic variants in São Miguel's healthy population (Azores).

BackgroundThe Azorean population presents the highest standardized mortality rate for cardiovascular diseases (CVD) when compared to mainland Portugal and other populations. Since thrombosis is a common cause of CVD, we assessed four polymorphisms in three thrombotic risk genes – F5 (G1691A), F2 (G20210A) and MTHFR (C677T, A1298C), in 469 healthy blood donors from São Miguel Island (Azores). We also analysed the CYP2C9 (C430T, A1075C) and VKORC1 (G1639A) variants in fifty-eight individuals with predisposition to thrombosis (possessing at least one variation in F5 or F2 genes and one in MTHFR) to evaluate their warfarin drug response genetic profiles.ResultsAmong the 469 individuals, the data showed that thrombotic risk allele frequencies – 1691A (4.9%), 20210A (1.8%), 677T (41.7%) and 1298C (24.8%) – were similar to other Caucasians, but significantly different from mainland Portuguese (χ2, p < 0.001). The combined analysis of these variants identified twenty-two different genetic profiles (genotype order: F5, F2, MTHFR C677T and A1298C). Complete homozygosity for all wild-type alleles (GG GG CC AA) was present in 11.7%, being GG GG CT AA (22.4%) the most frequent profile. The results also demonstrated that 12.4% (58 out of 469) of São Miguel islanders have increased genetic predisposition to thrombosis. Subsequently, we evaluated these individuals for their warfarin response genetic profiles. The data showed that seven out of fifty-eight individuals are poor metabolizers (two with CYP2C9*2/*2 and five with CYP2C9*2/*3 genotypes). VKORC1 polymorphism analysis identified twelve individuals (20.7%) with AA genotype, who probably will require lower doses of warfarin. The joint analysis of CYP2C9 and VKORC1 revealed that 79.3% (46 out of 58) of the individuals carry at least one polymorphism in these genes. Within these, twenty-five individuals (43.1%) need intermediate and/or low doses of warfarin, if treatment is started.ConclusionThe present study demonstrated, for the first time, that São Miguel, and possibly the Azores population, shows significant differences on allele frequencies of thrombotic risk factors when compared to mainland Portugal. This research constitutes a primary approach for future studies on CVD, as well as for the implementation of warfarin dosing protocols using the patient's genotypic information.

Leukocyte adhesion deficiency syndrome: a controversy solved

Leukocyte adhesion deficiency syndrome: a controversy solved

Reação de progênies de pimentão à infecção por Meloidogyne incógnita, raças 1 e 3

This study was carried out to screen sweet pepper genotypes both resistant and homozygous to races 1 and 3 of Meloidogyne incognita ; as well as to establish a criterion for characterization of resistant plants. The genotypes evaluated were: PIX-021E0818, PIX-021E0824, PIX-021E1232, PIX-021E1235, PIX-021E1540, PIX-021E1542, PIX-022E23 and PIX-023D09 (with Me1 or Me3 alleles to M. incognita resistance) and Linha 004, Agronomico-8, Ikeda and Magda (susceptible to nematodes) in two experiments, with one race each in a completely randomized design with 4 replicates and 16 plants per plot. Inoculation with nematodes was made at 30 eggs/cm 3 of substrate proportion and the evaluation for number of root knots and egg masses, as well as egg masses index was performed 60 days after the inoculation. The resistant plant percentage was evaluated and no genotype showed complete homozygosity to Me1 or Me3 alleles. The genotypes PIX-021E1235, PIX-021E0818, PIX-022E23 and PIX-023D09 showed a high resistance to M. incognita races 1 and 3. The best criterion for characterization of resistant plants was number of egg masses

Designing an optimal marker-based pedigree selection strategy for parent building in barley in the presence of repulsion linkage, using computer simulation

Pyramiding multiple desirable genes is an important method for the development of improved breeding materials and/or new cultivars. When the number of genes to be pyramided is many, or the genes are tightly linked in repulsion, it is practically impossible to recover the desirable recombinants in a single generation using a realistic population size, and repeated selection at several generations is required. The availability of markers tightly linked to the desirable genes makes it possible to conduct effective individual selection at early generations. This reduces the number of lines tested in the later generations and increases the desirable genotype frequency in the selected progeny. Computer simulation was used to develop such a marker-based pedigree selection strategy for the development of a barley line that contains 6 desired genes from 3 parental breeding lines (HS078 (H): 221222; PI366444 (P): 212222; Sloop Vic. (S): 122111; with 1 and 2 representing desirable and undesirable alleles, respectively), using the top cross H/P//S. The 6 genes targetted contribute to photoperiod sensitivity, Russian wheat aphid resistance, leaf rust resistance, boron tolerance, earliness per se, and cereal cyst nematode resistance. Under the assumption that perfect markers were available for all the 6 genes, a TC1 population of 300 plants was required to obtain 3 or more lines of the best genotype ‘211222/122111’, in which 3 loci were fixed for the desirable alleles, while the remaining 3 were kept as heterozygous. When single seed descent was used from the TC2 generation until complete homozygosity, the probability of obtaining lines of the desirable genotype (fixed for the desirable alleles at all 6 loci) was low due to the tight repulsion linkage between some of the genes. About 4000 individuals would be required to ensure with 99% probability the recovery of at least 1 line with the desirable genotype. The total number of lines that would need to be genotyped would be at least 5000. When the pedigree method was used in all test-cross generations, many schemes resulted in more lines of the fixed desirable genotype by genotyping fewer lines. The various options were compared using the genetic simulation software module QuLine, based on the QU-GENE simulation platform. The optimum scheme in terms of high success rate and relatively low genotyping costs consisted of the following steps: (1) in TC1 genotyping of 300 individuals allows for 3 or more individuals with the genotype ‘211222/122111’ to be identified; (2) in the TC2 individuals that are fixed for 3 loci and segregating for the remaining 3, loci can be selected from among 500 TC2 plants; (3) in the TC3, 50 or more individuals per TC3 line are genotyped for the 3 segregating loci, and individuals fixed for 5 loci and segregating for the 6th locus can be detected (genotyping is only needed for the segregating loci); (4) 25 individuals per TC4 line are genotyped for the single remaining segregating locus and several individuals of the desirable genotype (111111/111111) are finally selected. The desirable line is then obtained by collecting selfed seed from the selected TC4 plants. Using this scheme, on average, 320 desired TC5 lines were obtained by genotyping fewer than 2000 lines. When markers were tightly linked to the target genes but not diagnostic (perfect), not only was more genotyping required, but also appropriate phenotyping at the end of the marker selection process was necessary to confirm the presence of all the target genes. Under the assumption that recombination between marker and target gene was 5%, the best selection scheme identified, on average, 30 fixed desirable lines by genotyping 8000 lines and phenotyping 700 TC5 lines. If double haploid lines were produced from the F1 generation between H and P, and marker and phenotypic screening were conducted, followed by crossing of the individual with the target 2 loci in desired homozygous allelic status with parent S, the total amount of genotyping and phenotyping could be halved. This study showed that genetic simulation allows for numerous strategies to be compared using real data, and to develop an optimal crossing and selection strategy to combine desired alleles in the most effective and efficient way. This approach could likewise be used in other marker-assisted breeding programs.

Genetic verification of induced gynogenesis and microsatellite–centromere mapping in the barfin flounder, Verasper moseri

Primer sets were newly developed for 34 polymorphic microsatellite loci in the barfin flounder Verasper moseri. Mendelian inheritance was confirmed by examining the genotypic segregation in two normal diploid full-sib families. All the 34 loci showed genotypic segregation according to the Mendelian manner of inheritance; in some cases, null alleles were assumed. The genotypes at 27 loci were also examined in four meiotic gynogenetic diploid lines produced by fertilizing eggs with UV-irradiated sperm, followed by inhibition of the second meiotic division by cold shock. The absence of paternal alleles verified the success of gynogenetic development in all four meiotic gynogenetic diploid lines; the proportion of heterozygous progeny of a heterozygous mother, i.e., the frequency of second division segregation (y), was used to estimate the map distance of each microsatellite locus in relation to the centromere. Marker–centromere distances were estimated to be in the range of 0 to approximately 50 centiMorgan (cM) under the assumption of complete interference. Using eight diagnostic loci located at the telomeric region of the chromosome, complete homozygosity was confirmed in 1 mitotic gynogenetic diploid line that was produced by suppressing the first cleavage via hydrostatic pressure shock.

Origin and outcome of pregnancies affected by androgenetic/biparental chimerism

Androgenetic diploid cells confined to the placenta have recently been reported in several cases of normally developed fetuses in association with placental mesenchymal dysplasia (PMD). We investigated two singleton, mildly growth-restricted, female pregnancies ascertained on the basis of PMD. One case had liver hemangiomas and both infants had multiple skin hemangiomas. Post-natal development was normal. Molecular marker analysis confirmed the diagnosis of androgenetic and normal mixed cell populations in the placenta. Both cases derived from a single maternal genome (M1) and two distinct paternal genomes (P1 and P2). In one case, the androgenetic cell population contained both paternal genomes (P1P2), with one shared in common with the biparental (M1P1) population. In the second case, the androgenetic lineage showed complete homozygosity (P2P2) for a paternal genome not common to the biparental cell population. These new PMD cases help to define the range of possible clinical presentations of androgenetic/biparental mosaicism or chimerism. Placentas with androgenetic/biparental chimeric cell populations may derive from a single tri-pronuclear (3PN) zygote in which one or more parental genomes are not equally apportioned to the daughter cells in the first cell division.

CUCUMBER (CUCUMIS SATIVUS L.) DIHAPLOID LINE PRODUCTION USING IN VITRO RESCUE OF IN VIVO INDUCED PARTHENOGENIC EMBRYOS

Homozygous dihaploid lines (DHLs) from new cucumber (Cucumis sativus L.) accessions, source of resistance to diseases, would be useful to accelerate breeding for new resistant varieties. DHLs have been generated by in vitro rescue of in vivo induced parthenogenic embryos. The protocol developed involves the following: 1) induction of parthenogenic embryos by pollination with pollen irradiated at 0.5 KGy with a Co 60 γ-ray source; 2) in vitro rescue of putative parthenogenic embryos identified by their morphology and localized under a dissecting scope or X-ray radiography; 3) discrimination of undesirable zygotic individuals from the homozygous plants using cucumber and melon SSR markers; 4) determination of ploidy level from homozygous plants by flow cytometry; 5) in vitro chromosome doubling of haploids; and 6) acclimation and selfing of lines. Codominant markers and flow cytometry confirmed the gametophytic origin of plants regenerated by parthenogenesis, since all homozygous lines were haploids. No spontaneous doubled haploid plants were rescued. Chromosome doubling of haploid plants was accomplished by an in vitro treatment with 500 μM colchicine. Rescue of diploid or chimerical plants was confirmed by flow cytometry, previous to their acclimation and plant growth in the greenhouse. Selfing of colchicine-treated haploid plants allowed the perpetuation by seed of homozygous lines. Seeds were harvested from almost all lines after selfing in the greenhouse. The high rate of seed production facilitated the recovery of inbred lines. Despite some limiting factors, parthenogenesis is routinely used in a cucumber-breeding program to achieve complete homozygosity in one generation, and accelerate breeding for new commercial varieties. DHLs have been used as parental lines for the production of commercial hybrids.

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25 degrees C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25-27 degrees C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.