A NOVEL GAIN OF FUNCTION OF THE \u3ci\u3eIRX1\u3c/i\u3e AND \u3ci\u3eIRX2\u3c/i\u3e GENES DISRUPTS AXIS ELONGATION IN THE ARAUCANA RUMPLESS CHICKEN

Caudal dysplasia describes a range of developmental disorders that affect normal development of the lumbar spinal column, sacrum and pelvis. An important goal of the congenital malformation field is to identify the genetic mechanisms leading to caudal deformities. To identify the genetic cause(s) and subsequent molecular mechanisms I turned to an animal model, the rumpless Araucana chicken breed. Araucana fail to form vertebrae beyond the level of the hips. I performed a genome wide association study to identify candidate genomic regions associated with the rumpless phenotype, compared to tailed Araucana. A candidate region of chromosome 2 containing just two genes, IRX1 and IRX2, was identified. In situ hybridization analysis showed that a gain-of-function mutation resulted in both genes being misexpressed at the onset of secondary neurulation in the caudal organizer progenitor population. The caudal progenitor population has a bipotential fate, contributing cells to both mesoderm and neural lineages. This finding is significant because it is the first identified instance of a gain-of-function mutation resulting in axial truncation. The main question that arises from this novel finding is what is the functional mechanism leading to axial truncation? Possibilities include: the effect on the balance of cell fates within the progenitor population, on proliferation and apoptosis, on cell ingression, and the effect on molecular signaling within caudal tissues. Whereas none of these is mutually exclusive, I wanted to identify the single molecular event that triggers the cascade of downstream changes that results in axial truncation. I functionally examined each potential to determine the sequence of events in affected Araucana embryos. Based on the results of this study, I propose a model of development where initial misexpression of the two proneural Iroquois gene family members directs the bipotential progenitor population toward the neural lineage. This results in premature reduction of the progenitor population due to 1) the withdrawal of neuralized cells from the cell cycle, 2) reduced ingression of new progenitor cells via the ventral ectodermal ridge 3) reduced proliferation rates resulting in a failure to extend the axis that then results in 4) early termination of axial elongation and widespread apoptosis. In conclusion, I have identified a novel genetic basis for axial truncation that sheds light on the molecular mechanisms operating during secondary neurulation and axial elongation

Discovery of genomic variations by whole-genome resequencing of the North American Araucana chicken

Gallus gallus (chicken) is phenotypically diverse, with over 60 recognized breeds, among the myriad species within the Aves lineage. Domestic chickens have been under artificial selection by humans for thousands of years for agricultural purposes. The North American Araucana (NAA) breed arose as a cross between the Chilean “Collonocas” that laid blue eggs and was rumpless and the “Quetros” that had unusual tufts but with tail. NAAs were introduced from South America in the 1940s and have been kept as show birds by enthusiasts since then due to several distinctive traits: laying eggs with blue eggshells, characteristic ear-tufts, a pea comb, and rumplessness. The population has maintained variants for clean-faced and tufted, as well as tailed and rumplessness traits making it advantageous for genetic studies. Genome resequencing of six NAA chickens with a mixture of these traits was done to 71-fold coverage using Illumina HiSeq 2000 paired-end reads. Trimmed and concordant reads were mapped to the Gallus_gallus-5.0 reference genome (galGal5), generated from a female Red Junglefowl (UCD001). To identify candidate genes that are associated with traits of the NAA, their genome was compared with the Korean Araucana, Korean Domestic and White Leghorn breeds. Genomic regions with significantly reduced levels of heterogeneity were detected on five different chromosomes in NAA. The sequence data generated confirm the identity of variants responsible for the blue eggshells, pea comb, and rumplessness traits of NAA and propose one for ear-tufts

Whole Genome Bisulfite Sequencing of Medicago truncatula A17 wild type and lss mutants

Earlier work in our lab identified a spontaneous mutant in Medicago truncatula, resulting in increased nodulation. Molecular genetic evidence indicated the phenotype was due to an unknown lesion resulting in cis-silencing of the SUNN gene. Altered methylation of the promoter was suspected, but analysis of the SUNN promoter by bi-sulfite sequencing at the time of publication revealed no significant methylation differences between the SUNN promoter in wild type and lss plants. Using advances in methylome generation we compared the methylome of wild type and the lss mutant in the larger 810 kB area of the genome where lss maps

Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR

Alternative splicing enables a single gene to produce multiple mRNA isoforms by varying splice site selection. In animals, alternative splicing of mRNA isoforms between cell types is widespread and supports cellular differentiation. In plants, at least 20% of multi-exon genes are alternatively spliced, but the extent and significance of tissue-specific splicing is less well understood, partly because it is difficult to isolate cells of a single type. Pollen is a useful model system to study tissue-specific splicing in higher plants because pollen grains contain only two cell types and can be collected in large amounts without damaging cells. Previously, we identified pollen-specific splicing patterns by comparing RNA-Seq data from Arabidopsis pollen and leaves. Here, we used semi-quantitative PCR to validate pollen-specific splicing patterns among genes where RNA-Seq data analysis indicated splicing was most different between pollen and leaves. PCR testing confirmed eight of nine alternative splicing patterns, and results from the ninth were inconclusive. In four genes, alternative transcriptional start sites coincided with alternative splicing. This study highlights the value of the low-cost PCR assay as a method of validating RNA-Seq results

Many rice genes are differentially spliced between roots and shoots but cytokinin has minimal effect on splicing

Abstract Alternatively spliced genes produce multiple spliced isoforms, called transcript variants. In differential alternative splicing, transcript variant abundance differs across sample types. Differential alternative splicing is common in animal systems and influences cellular development in many processes, but its extent and significance is not as well known in plants. To investigate differential alternative splicing in plants, we examined RNA‐Seq data from rice seedlings. The data included three biological replicates per sample type, approximately 30 million sequence alignments per replicate, and four sample types: roots and shoots treated with exogenous cytokinin delivered hydroponically or a mock treatment. Cytokinin treatment triggered expression changes in thousands of genes but had negligible effect on splicing patterns. However, many genes were differentially spliced between mock‐treated roots and shoots, indicating that our methods were sufficiently sensitive to detect differential splicing between data sets. Quantitative fragment analysis of reverse transcriptase‐PCR products made from newly prepared rice samples confirmed 9 of 10 differential splicing events between rice roots and shoots. Differential alternative splicing typically changed the relative abundance of splice variants that co‐occurred in a data set. Analysis of a similar (but less deeply sequenced) RNA‐Seq data set from Arabidopsis showed the same pattern. In both the Arabidopsis and rice RNA‐Seq data sets, most genes annotated as alternatively spliced had small minor variant frequencies. Of splicing choices with abundant support for minor forms, most alternative splicing events were located within the protein‐coding sequence and maintained the annotated reading frame. A tool for visualizing protein annotations in the context of genomic sequence (ProtAnnot) together with a genome browser (Integrated Genome Browser) were used to visualize and assess effects of differential splicing on gene function. In general, differentially spliced regions coincided with conserved protein domains, indicating that differential alternative splicing is likely to affect protein function between root and shoot tissue in rice

Discovery of genomic variations by whole-genome resequencing of the North American Araucana chicken.

Gallus gallus (chicken) is phenotypically diverse, with over 60 recognized breeds, among the myriad species within the Aves lineage. Domestic chickens have been under artificial selection by humans for thousands of years for agricultural purposes. The North American Araucana (NAA) breed arose as a cross between the Chilean "Collonocas" that laid blue eggs and was rumpless and the "Quetros" that had unusual tufts but with tail. NAAs were introduced from South America in the 1940s and have been kept as show birds by enthusiasts since then due to several distinctive traits: laying eggs with blue eggshells, characteristic ear-tufts, a pea comb, and rumplessness. The population has maintained variants for clean-faced and tufted, as well as tailed and rumplessness traits making it advantageous for genetic studies. Genome resequencing of six NAA chickens with a mixture of these traits was done to 71-fold coverage using Illumina HiSeq 2000 paired-end reads. Trimmed and concordant reads were mapped to the Gallus_gallus-5.0 reference genome (galGal5), generated from a female Red Junglefowl (UCD001). To identify candidate genes that are associated with traits of the NAA, their genome was compared with the Korean Araucana, Korean Domestic and White Leghorn breeds. Genomic regions with significantly reduced levels of heterogeneity were detected on five different chromosomes in NAA. The sequence data generated confirm the identity of variants responsible for the blue eggshells, pea comb, and rumplessness traits of NAA and propose one for ear-tufts