Major chromosome 5H haplotype switch structures the European two-rowed spring barley germplasm of the past 190 years

Selection over 70 years has led to almost complete fixation of a haplotype spanning ~ 250 Mbp of chomosome 5H in European two-rowed spring barleys, possibly originating from North Africa. Plant breeding and selection have shaped the genetic composition of modern crops over the past decades and centuries and have led to great improvements in agronomic and quality traits. Knowledge of the genetic composition of breeding germplasm is essential to make informed decisions in breeding programs. In this study, we characterized the structure and composition of 209 barley cultivars representative of the European two-rowed spring barley germplasm of the past 190 years. Utilizing high-density SNP marker data, we identified a distinct centromeric haplotype spanning a ~ 250 Mbp large region on chromosome 5H which likely was first introduced into the European breeding germplasm in the early to mid-twentieth century and has been non-recombining and under strong positive selection over the past 70 years. Almost all cultivars in our panel that were released after 2000 carry this new haplotype, suggesting that this region carries one or several genes conferring highly beneficial traits. Using the global barley collection of the German Federal ex situ gene bank at IPK Gatersleben, we found the new haplotype at high frequencies in six-rowed spring-type landraces from Northern Africa, from which it may have been introduced into modern European barley germplasm via southern European landraces. The presence of a 250 Mbp genomic region characterized by lack of recombination and high levels of fixation in modern barley germplasm has substantial implications for the genetic diversity of the modern barley germplasm and for barley breeding

Genomic resources for a historical collection of cultivated two-row European spring barley genotypes

Barley genomic resources are increasing rapidly, with the publication of a barley pangenome as one of the latest developments. Two-row spring barley cultivars are intensely studied as they are the source of high-quality grain for malting and distilling. Here we provide data from a European two-row spring barley population containing 209 different genotypes registered for the UK market between 1830 to 2014. The dataset encompasses RNA-sequencing data from six different tissues across a range of barley developmental stages, phenotypic datasets from two consecutive years of field-grown trials in the United Kingdom, Germany and the USA; and whole genome shotgun sequencing from all cultivars, which was used to complement the RNA-sequencing data for variant calling. The outcomes are a filtered SNP marker file, a phenotypic database and a large gene expression dataset providing a comprehensive resource which allows for downstream analyses like genome wide association studies or expression associations.</p

Identification of a Novel ZIC3 Isoform and Mutation Screening in Patients with Heterotaxy and Congenital Heart Disease

Patients with heterotaxy have characteristic cardiovascular malformations, abnormal arrangement of their visceral organs, and midline patterning defects that result from abnormal left-right patterning during embryogenesis. Loss of function of the transcription factor ZIC3 causes X-linked heterotaxy and isolated congenital heart malformations and represents one of the few known monogenic causes of congenital heart disease. The birth incidence of heterotaxy-spectrum malformations is significantly higher in males, but our previous work indicated that mutations within ZIC3 did not account for the male over-representation. Therefore, cross species comparative sequence alignment was used to identify a putative novel fourth exon, and the existence of a novel alternatively spliced transcript was confirmed by amplification from murine embryonic RNA and subsequent sequencing. This transcript, termed Zic3-B, encompasses exons 1, 2, and 4 whereas Zic3-A encompasses exons 1, 2, and 3. The resulting protein isoforms are 466 and 456 amino acid residues respectively, sharing the first 407 residues. Importantly, the last two amino acids in the fifth zinc finger DNA binding domain are altered in the Zic3-B isoform, indicating a potential functional difference that was further evaluated by expression, subcellular localization, and transactivation analyses. The temporo-spatial expression pattern of Zic3-B overlaps with Zic3-A in vivo, and both isoforms are localized to the nucleus in vitro. Both isoforms can transcriptionally activate a Gli binding site reporter, but only ZIC3-A synergistically activates upon co-transfection with Gli3, suggesting that the isoforms are functionally distinct. Screening 109 familial and sporadic male heterotaxy cases did not identify pathogenic mutations in the newly identified fourth exon and larger studies are necessary to establish the importance of the novel isoform in human disease

Natural genetic variation and gene expression patterns underlying lateral shoot (tiller) development in barley (Hordeum vulgare L)

University of Minnesota Ph.D. dissertation.December 2018. Major: Plant and Microbial Biology. Advisor: Gary Muehlbauer. 1 computer file (PDF); xxv, 239 pages + 2 zip files of supplementary tables.The main shoot and other above-ground tissues develop from the shoot apical meristem (SAM), including axillary meristems (AXM) from which lateral branches develop. In barley, lateral branches called tillers contribute directly to grain yield and define shoot architecture. Two studies were completed to gain a better understanding of the genetic control of tiller development. One focused on characterizing natural phenotypic and genetic variation in tiller number throughout development in a large, diverse germplasm collection; and the second focused on identifying meristem-specific genes and characterizing gene expression patterns that varied by meristem type (SAM or AXM) and morphological stage. Results from the first study revealed that correlations between tiller development (tillering) and factors previously shown to influence tiller development, like photoperiod response and spike row-type, varied depending on environment and genetic background. Furthermore, no major trade-offs existed between tiller number and other traits, and natural genetic variation associated with tillering largely overlapped variation associated with days to heading and spike row-type. Results of the second study revealed a set of genes upregulated in most meristems compared to non-meristem tissues, many of which have been characterized in other species and are likely important for general meristem maintenance or function. Results also suggested that gene expression was primarily differentiated by genotype, meristem type, and morphological stage; however, expression profiles of SAM and AXM were very similar at later developmental stages. A small number of genes were only expressed in SAM or AXM, and clustering based on expression across all meristems revealed genes upregulated in AXM that may be important for tiller development, as some, like UNICULME4 and INTERMEDIUM-C (TEOSINTE BRANCHED 1 ortholog) have already been implicated in tiller development. Genes that were upregulated in ligules compared to leaves were also identified, and clustering of these genes revealed some that were expressed more highly in ligules and AXM that may, as in other species, have dual functions in leaf and tiller development

Detection of odontoglossum ringspot virus in inoculated orchid leaf tissue using SYBR green real-time RT-PCR

Odontoglossum ringspot virus (ORSV) is one of the most prevalent orchid viruses that infects greenhouse-grown orchids worldwide. In order to prevent the spread of viruses in greenhouses and to cultivate clones from virus-free mother plants, it is necessary to develop a more sensitive technique for the detection of viruses in orchids. SYBR green real-time RT-PCR is a highly sensitive technique that can specifically detect ORSV in orchid tissue. By harvesting tissue at the inoculation site and at specific distances from the inoculation site at different times past inoculation, this technique can also be used to study the rate of spread of ORSV in orchids. Orchid clones were inoculated with ORSV and other clones were mock-inoculated with molecular grade water. Leaf tissue was harvested from the ORSV-inoculated and mock-inoculated clones at the site of inoculation and at specific distances from this site at 16 h, 24 h, and 72 h past inoculation. Total RNA was extracted from the harvested tissue. Competitive RTPCR was going to be used for the quantification and detection of ORSV in the samples, but attempts at cloning an ORSV fragment into a vector in order to form a competitive standard were unsuccessful. Instead, a highly sensitive qualitative approach called SYBR green real-time RT-PCR was used for the detection of ORSV. ORSV was detected in all virus-inoculated orchids, except for one. Therefore, all of the ORSV inoculated plants except for one were infected with the virus. Unexpectedly, ORSV was also detected in all of the mock-inoculated orchids. Most likely the orchids were previously infected with ORSV, but the viral titer was too low to be detected by commercial techniques. However, there is a small possibility that the orchids were contaminated during experimentation, despite careful technique. The rate of spread of the virus could not be studied because the mock-inoculated samples also contained the virus. Although viral amplification was demonstrated in the mock-inoculated plants, SYBR green real-time RT-PCR is still a sensitive and consistent method for ORSV detection in orchids. With additional controls, this method could prove to be the ideal method for reliable detection of ORSV in commercially-grown orchids.Thesis (M.S.)Department of Biolog

Identification of a novel ZIC3 isoform and mutation screening in patients with heterotaxy and congenital heart disease.

Patients with heterotaxy have characteristic cardiovascular malformations, abnormal arrangement of their visceral organs, and midline patterning defects that result from abnormal left-right patterning during embryogenesis. Loss of function of the transcription factor ZIC3 causes X-linked heterotaxy and isolated congenital heart malformations and represents one of the few known monogenic causes of congenital heart disease. The birth incidence of heterotaxy-spectrum malformations is significantly higher in males, but our previous work indicated that mutations within ZIC3 did not account for the male over-representation. Therefore, cross species comparative sequence alignment was used to identify a putative novel fourth exon, and the existence of a novel alternatively spliced transcript was confirmed by amplification from murine embryonic RNA and subsequent sequencing. This transcript, termed Zic3-B, encompasses exons 1, 2, and 4 whereas Zic3-A encompasses exons 1, 2, and 3. The resulting protein isoforms are 466 and 456 amino acid residues respectively, sharing the first 407 residues. Importantly, the last two amino acids in the fifth zinc finger DNA binding domain are altered in the Zic3-B isoform, indicating a potential functional difference that was further evaluated by expression, subcellular localization, and transactivation analyses. The temporo-spatial expression pattern of Zic3-B overlaps with Zic3-A in vivo, and both isoforms are localized to the nucleus in vitro. Both isoforms can transcriptionally activate a Gli binding site reporter, but only ZIC3-A synergistically activates upon co-transfection with Gli3, suggesting that the isoforms are functionally distinct. Screening 109 familial and sporadic male heterotaxy cases did not identify pathogenic mutations in the newly identified fourth exon and larger studies are necessary to establish the importance of the novel isoform in human disease