Instructor this video is on the zebra analysisor zebra if youre in different partsof the world besides the us. Mghcvrc mapping resources protocols zfin community wiki. Exon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens. Mutation mapping analysis pipeline for pooled rnaseq jonathon t.
There are scant baseline data on zebrafish pathologic lesions in infectious and noninfectious diseases. Bulked segregant analysis bsa is a technique used to identify genetic markers associated with a mutant phenotype. Schematic representation of bulk segregant analysis bsa with two bulks selected from both f 2 plants and recombinant inbred lines f n derived from the same f 2 plants. Oct 17, 2014 the largest zebra is the grevys zebra, according to the san diego zoo. Zebrafish are remarkably biologically similar to people and share the majority of the same genes as humans, making them an important model for understanding how genes work in health and disease. The identification of these markers is often achieved by a procedure called bulk segregant. Bulk segregant analysis 52 was performed using the male and female pooled gdna samples to screen for potentially sexlinked markers. Forward genetic analysis is one of the principal advantages of the zebrafish model system. Resistant and susceptible dna bulks were constructed following the classification of f2 plants by leaf rust infection type. Bulked segregant analysis definition northwestern university. Often a geneticist is not interested in developing a molecular map, but would rather find a few markers that are closely linked to a specific trait. Its attractive slender body has numerous vertical, evenlyspaced thin orange stripes displaying an intricate pattern. Exploraweb enables easy data upload and parallel processing of different parameter configurations. Danio rerio commonly known as the zebrafish is a tropical fish belonging to the minnow family cyprinidae, commonly kept in aquaria and used for scientific research.
For the majority of these screens over the last few decades, the mutations were mapped using bulk segregant analysis bsa to establish approximate chromosomal locations, followed by fine mapping using microsatellite markers on hundreds or thousands of embryos. Bulked segregant analysis bsa is a technique used to identify genetic markers associated. This allows geneticists to discover genes conferring disease resistance or susceptibility. In its larval stages it is transparent and as it matures to an adult it develops stripes that run along the length of the body and look blue in colour. The zebrafish is a tropical fish native to southeast asia. Bsa requires access to quantitative genetic markers that are polymorphic in the mapping population. Cpsf1 is required for definitive hsc survival in zebrafish. A bulk segregant rnaseq analysis of the f2 generation of interspecific hybrids of channel catfish and blue catfish ictalurus furcatus, which are respectively, relatively susceptible or extremely resistant to e. Poolgenetic mapping by bulk segregant analysis in drosophila.
The zebrafish developmental staging series describes the standard criteria used. Transferrina modulates hepcidin expression in zebrafish. The broken red line indicates the threshold value 3. Zebra fish, any member of either of two unrelated groups of fishes, the freshwater species in the genus brachydanio family cyprinidae. The snp genotype and gene frequencies are shown in table 5. Current methods for administering compounds orally to zebrafish include administering compounds topcoated onto feed, mixed into gelatin diets 1, bioencapsulated into brine shrimp 2, mixed with lipid encapsulated diets 3, wax spray beads 4 and via glutenbased diets. Bulk segregant analysis bsa using microarrays, and extreme array mapping xam have recently been used to rapidly identify genomic regions associated with phenotypes in multiple species.
C the lev vu41 lesion was meiotically mapped to a region of. Starting with a segregating population from a genetic cross, individuals are assayed for the focal trait and two pools bulks of segregants are created by selecting individuals from the tails of the phenotypic. In bsa, dna of progenies corresponding to the phenotypic extremes is extracted and pooled. When dna of ten plants are bulked into one pool, all alleles must be present. The huge cost incurred in genotyping could be considered as a. Bulk segregant analysis and markertrait association reveal. Trait mapping in diverse arthropods by bulked segregant. Wholegenome resequencing from bulkedsegregant analysis. In zebrafish, maternally inherited germ plasm ribonucleoparticles rnps have. Includes content submitted by the community and imported from the zebrafish book, 5th edition.
Therefore, only two pools of extreme lines along with the parents are genotyped for the identification of markers linked with the trait of interest. Bulk segregant analysis and drought resistance in maize 1 fig. Nine to 12 homozygousresistant and 9 to 12 homozygoussusceptible individuals of each f3 progeny were randomly selected to construct resistant and susceptible bulks table 1, respectively. Wholegenome resequencing from bulked segregant analysis reveals gene set based association analyses for the vibrio anguillarum resistance of turbot scophthalmus maximus author links open overlay panel kai zhang a miao han a yuxiang liu a xiaohan lin a xiumei liu a c he zhu a yan he a b quanqi zhang a b jinxiang liu a b. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of.
Bulk segregant analysis bulked segregant analysis bsa was performed as described by michelmore et al. Starting with a segregating population from a genetic cross, individuals are assayed for the focal trait and two pools bulks of segregants are created by selecting individuals from the tails of the phenotypic distribution other. Rapid identification of kidney cyst mutations by whole. We have developed a modification of bsa bsrseq that makes use of rnaseq reads to efficiently map genes even in populations for which no. Rad marker microarrays enable rapid mapping of zebrafish. Bulked segregant analysis bsa is an efficient method to rapidly and efficiently map genes responsible for mutant phenotypes.
It offers scientific data and analysis, and a perspective on the legal and international issues involved. Zebrafish genome found strikingly similar to humans. Forward genetic approaches in zebrafish have provided invaluable information about developmental processes. Perspectives for identification of mutations in the zebrafish. Bulked segregant analysis an overview sciencedirect topics. This limitation has been partially overcome by the. Initial bulk segregant analysis placed the dae locus within a a 0. Simple sequence length polymorphism sslp markers were identified from the zebrafish genetic map shimoda et al. The key design of this method is bulk genotyping of a pool of segregants sharing the same phenotype.
The statistics of bulk segregant analysis using next generation sequencing paul m. The mission of the zebrafish international resource center is to provide a central repository for wildtype and mutant strains of zebrafish and for materials and information about zebrafish research. Kelly3 1department of biology and igsp center for systems biology, duke university, durham, north carolina, united states of america, 2department of biology, duke. M1, m2, m3, and m4 indicate markers located on a hypothetical chromosome containing a gene of interest. A reference cross panel of dna, consisting of 520 f2 progeny 1040 meioses of an ab x india in intercross that has been anchored to a zebrafish genetic linkage map by 102 simple sequence length polymorphisms. This process is very time consuming despite the large clutch sizes of the zebrafish. One hundred and ninetytwo sslp markers from the g4 mapping panel 27 were tested on these pools, revealing linkage of the mutation to markers z21636, z10164, z11876, z17278 and. Bulk segregant mapping protocols isolation of intact genomic dna isolate genomic dna from embryos using our genomic dna isolation protocol. Ijms free fulltext transcriptome analysis based on rna. Mgh cvrc zebrafish server zebrafish information network. Methods and protocols, expert researchers in the field detail. Bulk segregant analysis with molecular markers and its use.
Encyclopedia of genetics, genomics, proteomics and informatics. We have developed a modification of bsa bsrseq that makes use of rnaseq reads to efficiently map genes even in populations for which no polymorphic markers have been. Native to south asia, it is a popular aquarium fish, frequently sold under the trade name zebra danio and thus often called a tropical fish although both tropical and subtropical. The qtlseqr package is quick and produces publication quality figures and tables. The diversity of these loci was high in the largemouth bass population, where the heterozygosity was 0. An r package that performs next generation sequencing bulk segregant analysis was developed. A web service wrapped around our previously published algorithm explora, which exploits linkage disequilibrium ld to increase the power and accuracy of quantitative trait loci identification in bulk segregant analysis bsa.
Efficiency of mas for breeding common bean is still hampered, however, due to the dominance, linkage phase, and loose linkage of previously developed markers. Pdf bulk segregant analysis using single nucleotide. The potential of bulk segregant analysis and rapd technology. Bulk segregant analysis bsa serves as an affordable strategy for mapping large effect qtls by genotyping only the extreme phenotypes instead of the entire mapping population. Fine mapping qtl for drought resistance traits in rice. Bulked segregant transcriptome analysis bsta data processing pipeline. Using nextgeneration sequencing to isolate mutant genes. Parasexual crossings for bulk segregant analysis in aspergillus niger to facilitate mutant identification via whole genome sequencing pages 277287 arentshorst, mark et al. Another time and effort saving approach is bulk segregant analysis bsa michelmoore et al. Mutations in both the medaka and zebrafish rx3 gene, one of the three rx paralogs found in fish, result in a less.
A wide variety of zebra fish options are available to you, there are 431 suppliers who sells zebra fish on, mainly located in asia. The zebrafish danio rerio is a freshwater fish belonging to the minnow family of the order cypriniformes. Despite the popularity of zebrafish as a research model, its sex determination sd mechanism is still unknown. To systematically isolate maternaleffect mutations in the zebrafish, we. Semaphorinplexin signaling guides patterning of the. Bulk segregant analysis 52 was performed using the male and. Germ cell determinant transmission, segregation, and function in. Four bulked dna samples were prepared from progeny of a sugarcane cross, aa157, based on segregation for the fibre trait. Bulked segregant analysis was then used to identify a 2. A guide for the laboratory use of zebrafish brachydanio rerio. With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources. Nonchimeric yac and bac clones were unavailable in the region of interest, so we compared genes in the vicinity of rhodopsin rho in other species. Common bean was one of the first crops that benefited from the development and utilization of molecular markerassisted selection mas for major disease resistance genes. The feasibility of using bulk segregant analysis to identify molecular markers for disease resistance genes in oats was investigated, utilizing random primers in conjunction with polymerase chain reaction technology.
A statistical approach to mutation detection in zebrafish. The coupling of bulked segregant analysis 39 and aflp made it possible to completely saturate target genomic regions relatively quickly. Explain why in principle, when using the bulk segregant analysis procedure with an f2 generation and a dominant marker system, such as rapd or aflp, to identify a marker linked to a dominant acting gene, you will only be able to identify markers linked in coupling phase to the dominant phenotypic allele. Chisquared analysis showed that the genotype frequencies of two snps with cc, ct, and tt or gg, gc, and cc were consistent with the hwe p 0. Also used to label nucleic acid probes for microarray analysis. These experiments, however, require the identification of single feature polymorphisms sfp between the cross parents for each new combination of genotypes, which raises the cost of experiments.
Bulk segregant mapping protocols protocols zfin community. These experiments, however, require the identification of single feature polymorphisms sfp between the cross parents for each new combination of genotypes, which raises the cost. Polygenic sex determination system in zebrafish ncbi. Zebra mussels and aquatic nuisance species is a thorough, wellreferenced, and illustrated look at the growing problem of nonindigenous species pollution. The objective of the present study was to investigate the potential use of bulk segregant analysis michelmore et al. Jan 26, 2012 bfr for putative snps identified in maq120 analysis. Bulk segregant analysis reveals the genetic basis of a. We have previously mapped a qtl linked to leaf rolling and leaf drying in recombinant inbred ri lines derived from two locally adapted indica rice ecotypes viz. Youre asked to choose a body, and direction,number of repeats, locking, opacity, high quality. Forward genetic screens have a long history of uncovering the genetic mutations underlying phenotypes of interest. Two dna pools for use in bulk segregant analysis were created from 44 mutants and 44 siblings as identified by slow myosin antibody labeling at the 26somite stage. The analysis of zebrafish mutants affecting some of the duplicated. Recent improvements in the annotation of the zebrafish genome coupled to a reduction in sequencing costs prompted the. Bulk segregant analysis bsa and resequencing of msa11 snptilling arraybased bulk segregant analysis was performed as previously described 59.
The cost efficiency and ease of data production afforded by nextgeneration sequencing have created new opportunities for rapid mapping of induced. Intermediate resolution mapping of the gavi mutation, using bulk segregant analysis, revealed that the mutation was on linkage group 2, near the transferrina locus c. To score the maltose utilization phenotype, the colonies were inoculated into 96well microplates containing either glucose. The patchytail phenotype is a result of mutation in the zebrafish ortholog of the dag1 gene. The zebrafish book a guide for the laboratory use of zebrafish danio brachydanio rerio by monte westerfield, institute of neuroscience, university of oregon older literature referred to zebrafish as brachydanio rerio. Plsc 731 plant molecular genetics homework 2 due date. Bulk segregant analysis bsa is a gene mapping method firstly developed and implemented in plants michelmore et al. The identification of these markers is often achieved by a procedure called bulk segregant analysis. Initial linkage using bulk segregant analysis placed obd 2 cm telomeric to the final map marker z9637 on lg8. Polygenic sex determination system in zebrafish plos. Springer nature is developing a new tool to find and evaluate protocols. In plants, this technique is highly efficient, as it is relatively easy to grow and screen hundreds or thousands of individuals. The b1127 and b1128 mutations cause premature ossification of craniofacial chondral bones and were. The zebra barred dartfish is also referred to as the zebra dart goby, bar goby, barred dartfish, zebra goby, or chinese zebra goby.
Coupled mutagenesis screens and genetic mapping in zebrafish. Parasexual crossings for bulk segregant analysis in aspergillus niger to facilitate mutant identification via whole genome sequencing mark arentshorst, arthur f. Bfr of 3,172 snps with at least 8fold coverage in the high and low bulk according to maq120 analysis. Programs have been set up on the beckman robot to aliquot the primers, set up the. Bulk segregant analysis all the mixes should be scaled up by the amount necessary for the number of samples to be analyzed. We explored the utility of the array for bulk segregant mapping by attempting to localize four previously unmapped recessive mutations that we had recovered from the ongoing enumediated forward genetic screen at the university of oregon 1719 figure figure2. A bulk segregant transcriptome analysis reveals metabolic and. Zebra technologies relentlessly strives to deliver superior quality products and services to our customers and stakeholders. Zebra fish definition of zebra fish by merriamwebster. This page represents a portion of the mghcvrc zebrafish server content, originally hosted. A simple pipeline for mapping point mutations plant.
Mutant or wildtype sibling adult female dna was pooled for bulk segregant analysis. Males are slender and torpedoshaped usually with a pink or yellow tinge. Wittbrodtrapid chromosomal assignment of medaka mutants by bulked segregant analysis. An f2 population from a cross between barley accession q21861 and the australian barley variety galleon was used to develop rapd markers for resistance to barley leaf rust puccinia hordei. Application of in silico bulked segregant analysis for rapid. Bioinformatics derives knowledge from computer analysis of biological data. Our iso 9001 quality management system, commitment to defect prevention and continuous improvement efforts are evident in everything.
This technique involves forming two groups that display opposing phenotypes for a trait of interest. Suggested markers for bulk segregant analysis markers suggested for firstpass mapping, including protocol and primers list. A an initial bulk segregant analysis implicated marker z228677 on lg22 as being linked to. The b1127 and b1128 mutations cause premature ossification of craniofacial. Apr 23, 2014 zebrafish model for toxicology and toxicologic pathology research shortage of basic data on zebrafish pathology. The use of bulk segregant analysis to identify a rapd marker. See bulk segregant analysis bulk segregant analysis bsa candidate gene and variant identification, 659 materials, 656657 overview, 653654 phenotypically extreme segregant acquisition, 658659 recipes, 659660 recombinant cross progeny, 657658 segregant generation, 657 sequencing and causal loci detection, 659 c calling card. B representative homozygous col8a1a mutant adult e. Within each bulk, the individuals are arbitrary for all traits except the trait or the gene of interest.
Snps are plotted along the xaxis according to the unigene rank based on the rpkm expression values. Pdf polygenic sex determination system in zebrafish. Thus, analysis of zebrafish mutations provides insights into gene functions in other vertebrates, including humans. In particular, genomic and transcriptomic datasets are processed, analysed and, whenever possible, associated with experimental results from various sources, to draw structural, organizational, and functional information relevant to biology. Zebra fish definition is any of various barred fishes. Both the head and body of the zebra dart goby are light green.
This is where zebrafish researchers can share information with the rest of the community. Characterization and investigation of zebrafish models of. Identification of an rapd marker for the crown rust. Medaka and zebrafish, an evolutionary twin study sciencedirect. However, the relative difficulty of mapping and isolating mutations has limited the number of new genetic screens. Random spores were germinated on yes plates to form colonies. However, while the number of markers used can easily be scaled up according to the size of genome, the number of f2 individuals required to order these markers increases in tandem. The statistics of bulk segregant analysis using next. Bulk segregant analysis using single nucleotide polymorphism. Here we applied in silico bulked segregant analysis bsa to the.
A rapid mapping strategy suitable for monogenic qualitative traits. Over the last two decades advances in genotyping technology, and the development of quantitative genetic analytical techniques have made it possible to dissect complex traits and link quantitative variation in traits to allelic variation on chromosomes or quantitative trait loci qtls. Selection of f2 plants through hydroponics screening at higher iron toxic levels for bulk segregant analysis bsa in rice influence of magnesium, calcium, ferrous and zinc nanomaterials on production and multiplication of nomuraea rileyi farlow samson. Bulk segregant analysis bsa was established in 1991 as a method to detect markers in a specific genomic region by comparing two pooled dna samples of individuals from a segregating population.