Genotyping by low-coverage whole-genome sequencing in intercross pedigrees from outbred founders: a cost efficient approach

ABSTRACTBackgroundExperimental intercrosses between outbred founder populations are powerful resources for mapping loci contributing to complex traits (Quantitative Trait Loci or QTL). Here, we present an approach and accompanying software for high-resolution genotype imputation in such populations using whole-genome high coverage sequence data on founder individuals (∼30×) and low coverage sequence data on intercross individuals (∼0.4×). The method is illustrated in a large F2 pedigree between lines of chickens that have been divergently selected for 40 generations for the same trait (body weight at 8 weeks of age).ResultsDescribed is how hundreds of individuals were whole-genome sequenced in a cost- and time-efficient manner using a Tn5-based library preparation protocol optimized for this application. In total, 7.6M markers segregated in this pedigree and 10.0 to 13.7% were informative for imputing the founder line genotypes within the F0-F2 families. The genotypes imputed from low coverage sequence data were consistent with the founder line genotypes estimated using SNP and microsatellite markers both at individual imputed sites (92%) and across the genome of individual chickens (93%). The resolution of the recombination breakpoints was high with 50% being resolved within <10kb.ConclusionsA method for genotype imputation from low-coverage whole-genome sequencing in outbred intercrosses is described and evaluated. By applying it to an outbred chicken F2 cross it is illustrated that it provides high quality, high-resolution genotypes in a time and cost efficient manner.

Applicability of chromosome-specific SSR wheat markers for the introgression of Triticum urartu in durum wheat breeding programmes

Triticum urartu, the A-genome donor of tetraploid and hexaploid wheats, is a potential source of novel alleles for crop improvement. A fertile amphiploid between T. urartu (2n = 2x = 14; AuAu) and durum wheat cv ‘Yavaros’ (Triticum turgidum ssp. durum; 2n = 4x = 28, AABB) was obtained as a first step to making the genetic variability of the wild ancestor available to durum wheat breeding. The amphiploid was backcrossed with ‘Yavaros’ and the offspring from this cross was selfed. A plant from this progeny (founder line) with 28 chromosomes and active x and y subunits of the Glu-A1 locus of T. urartu was selfed, which resulted in the obtaining of 98 pre-introgression lines (pre-ILs). In this work, a set of 78 wheat chromosome-specific microsatellite markers (simple sequence repeats, SSR), uniformly distributed over the A genome, was used for marker-assisted selection of T. urartu in a durum wheat background. A total of 57 SSRs allowed a clear discrimination between T. urartu and ‘Yavaros’. This set of markers was further used for characterizing the pre-ILs, identifying and defining the T. urartu introgressed regions. The applicability of these markers is discussed.

Overproduction of cardiac S-adenosylmethionine decarboxylase in transgenic mice

The present study was designed to provide a better understanding of the role played by AdoMetDC (S-adenosylmethionine decarboxylase), the key rate-controlling enzyme in the synthesis of spermidine and spermine, in controlling polyamine levels and the importance of polyamines in cardiac physiology. The αMHC (α-myosin heavy chain) promoter was used to generate transgenic mice with cardiac-specific expression of AdoMetDC. A founder line (αMHC/AdoMetDC) was established with a >100-fold increase in AdoMetDC activity in the heart. Transgene expression was maximal by 1 week of age and remained constant into adulthood. However, the changes in polyamine levels were most pronounced during the first week of age, with a 2-fold decrease in putrescine and spermidine and a 2-fold increase in spermine. At later times, spermine returned to near control levels, whereas putrescine and spermidine levels remained lower, suggesting that compensatory mechanisms exist to limit spermine accumulation. The αMHC/AdoMetDC mice did not display an overt cardiac phenotype, but there was an increased cardiac hypertrophy after β-adrenergic stimulation with isoprenaline (‘isoproterenol’), as well as a small increase in spermine content. Crosses of the αMHC/AdoMetDC with αMHC/ornithine decarboxylase mice that have a >1000-fold increase in cardiac ornithine decarboxylase were lethal in utero, presumably due to increase in spermine to toxic levels. These findings suggest that cardiac spermine levels are highly regulated to avoid polyamine-induced toxicity and that homoeostatic mechanisms can maintain non-toxic levels even when one enzyme of the biosynthetic pathway is greatly elevated but are unable to do so when two biosynthetic enzymes are increased.

Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

Megakaryocyte (MK)–specific transgene expression has proved valuable in studying thrombotic and hemostatic processes. Constitutive expression of genes, however, could result in altered phenotypes due to compensatory mechanisms or lethality. To circumvent these limitations, we used the tetracycline/doxycycline (Tet)–off system to conditionally over-express genes in megakaryocytes and platelets in vivo. We generated 3 transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the MK-specific platelet factor 4 promoter (PF4-tTA-VP16). Responder lines were simultaneously generated, each with a bidirectional minimal cytomegalovirus (CMV)–tTA responsive promoter driving prokaryotic β-galactosidase gene, as a cellular reporter, and a gene of interest (in this case, the mitotic regulator Aurora-B). A transactivator founder line that strongly expressed PF4-driven tTA–viral protein 16 (VP16) was crossbred to a responder line. The homozygous double-transgenic mouse line exhibited doxycycline-dependent transgene overexpression in MKs and platelets. Using this line, platelets were conveniently indicated at sites of induced stress by β-galactosidase staining. In addition, we confirmed our earlier report on effects of constitutive expression of Aurora-B, indicating a tight regulation at protein level and a modest effect on MK ploidy. Hence, we generated a new line, PF4-tTA-VP16, that is available for conditionally overexpressing genes of interest in the MK/platelet lineage in vivo.

An Establishment of a System for Conditional Overexpression of Genes in Megakaryocytes and Platelets In Vivo.

Expression of foreign or endogenous genes in vivo is an essential tool for following regulatory processes, including the control of gene expression. Constitutive expression of such genes throughout development often results with lethality due to adverse effects during embryogenesis. Several such examples exist with regard to the megakaryocyte (MK)/platelet lineage. Here, we employed the tetracycline (Tet)-off system to conditionally overexpress genes in MKs and platelets in vivo. We generated three transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the megakaryocyte-specific, platelet factor 4 promoter (PF4-Tet). Three responder lines were simultaneously generated, each with a bidirectional minimal CMV-tTA responsive promoter upstream of prokaryotic ß-Galactosidase gene (ß-Gal) (as a marker), with an insertion site in the opposite side for a gene of interest. A transactivator founder line that strongly expressed tTA was cross-bred to the three other responder lines. The resulting double transgenic mouse lines exhibited tetracycline-dependent transgene overexpression in the megakaryocytic lineage. Lineage specificity was confirmed by immunohistochemistry and western blot analyses. By changing tetracycline concentration in the drinking water, we could turn on/off the expression of the gene at any desirable time during the mouse lifespan, and showed that in adult mice under inducing conditions, ß-Gal expression was restricted to MKs and platelets. Hence, we generated a mouse line, PF4-Tet that is available to conditionally overexpress any gene of interest in the MK/platelet lineage in vivo. This system should be valuable for the study of effects of selected proteins on an array of genes expressed in vivo and on consequent megakarypoiesis and/or thrombosis and hemostasis.

A dose effect of IL-7 on thymocyte development

To study interleukin-7 (IL-7) in early thymocyte development, we generated mice transgenic (Tg) for the IL-7 gene under control of the lck proximal promoter. Founder line TgA, with the lowest level of IL-7 overexpression, showed enhanced αβ T-cell development. In contrast, in the highest overexpressing founder line, TgB, αβ T-cell development was disturbed with a block at the earliest intrathymic precursor stage. This was due to decreased progenitor proliferation as assessed by Ki-67 staining and in vivo bromodeoxyuridine (BrdU) incorporation. Bcl-2 was up-regulated in T-cell–committed progenitors in all Tg lines, and accounted for greater numbers of double positive (DP), CD4 single positive (SP), and CD8SP thymocytes in TgA mice where, in contrast to TgB mice, thymocyte progenitor proliferation was normal. Mixed marrow chimeras using TgB+ and congenic mice as donors, and experiments using anti–IL-7 monoclonal antibody (MAb) in vivo, confirmed the role of IL-7 protein in the observed TgB phenotype. In conclusion, at low Tg overexpression, IL-7 enhanced αβ T-cell development by increasing thymocyte progenitor survival, while at high overexpression IL-7 reduces their proliferation, inducing a dramatic block in DP production. These results show for the first time in vivo a dose effect of IL-7 on αβ T-cell development and have implications for IL-7 in the clinical setting.

Spi-1/PU.1 transgenic mice develop multistep erythroleukemias.

Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages.