Bulked segregant analysis definition northwestern university. The use of bulk segregant analysis to identify a rapd marker. Therefore, only two pools of extreme lines along with the parents are genotyped for the identification of markers linked with the trait of interest. Four bulked dna samples were prepared from progeny of a sugarcane cross, aa157, based on segregation for the fibre trait. A bulk segregant transcriptome analysis reveals metabolic and. Resistant and susceptible dna bulks were constructed following the classification of f2 plants by leaf rust infection type. Primer combinations are indicated on the top right corner of the peak profiles. The zebrafish wt and mutant cdna of the ccbl gene were amplified. The objective of the present study was to investigate the potential use of bulk segregant analysis michelmore et al. Skretting is now offering our premium marine hatchery diet gemma micro, through our new webshop.
You paste in a list of ensembl gene identifiers, and a reference set of gene identifiers default is the entire genome, and you quickly get back a list of all the go terms. 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. Variant sites are nonrandom, with an excess of specific novel t and arich motifs. Mesabtricaine diluted in fish water recipe in the zebrafish book. 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. Hardcopies of the 4th edition of the zebrafish book can be obtained for a nominal fee from the zebrafish international resource center, 5274 university of oregon, eugene, or 97403 usa.
However, managing zebrafish mutant lines derived from mutagenesis. We further demonstrate bulked segregant analysis using the. Oct 17, 2014 the largest zebra is the grevys zebra, according to the san diego zoo. The potential of bulk segregant analysis and rapd technology. 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. Utrs and intronic gene regions in the 1590 mutant embryo panel that we generated. Bfr for putative snps identified in maq120 analysis.
Moleculargenetic mapping of zebrafish mutants with variable. In plants, this technique is highly efficient, as it is relatively easy to grow and screen hundreds or thousands of individuals. Louis, missouri, united states of america, 2center for plant environmental stress physiology, purdue university, west. Males are slender and torpedoshaped usually with a. C the lev vu41 lesion was meiotically mapped to a region of chromosome 9 between the. Pdf polygenic sex determination system in zebrafish. The identification of these markers is often achieved by a procedure called bulk segregant.
Bulk segregant analysis bsa is a gene mapping method firstly developed and implemented in plants michelmore et al. Gemma micro provides complete nutrition for all life stages. Here you can test for statistical enrichment or impoverishment of gene ontology go annotation terms in a list of genes of interest. Within each bulk, the individuals are arbitrary for all traits except the trait or the gene of interest. M1, m2, m3, and m4 indicate markers located on a hypothetical chromosome containing a gene of interest. If your order contains this item, you will only see ups next day air as a shipping option during checkout the zebra crayfish cherax peknyi is a fairly recently discovered and uncommon species of crayfish native to papua new guinea. A forward genetic screen is one of the best methods for revealing the function of genes. Males are slender and torpedoshaped usually with a pink or yellow tinge.
Youre asked to choose a body, and direction,number of repeats, locking, opacity, high quality. Bfr of 3,172 snps with at least 8fold coverage in the high and low bulk according to maq120 analysis. B representative homozygous col8a1a mutant adult e. Bulk segregant analysis bulked segregant analysis bsa was performed as described by michelmore et al. Bulk segregant analysis all the mixes should be scaled up by the amount necessary for the number of samples to be analyzed. In standard bulked segregant mapping, f2 individuals from a polymorphic.
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. One can enlist n number of biological disciplines in which. 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. This page represents a portion of the mghcvrc zebrafish server content, originally hosted. Use of the islet1 isl1gfp transgenic line enabled precise realtime observations of the growth cone behaviour of the vp and vii motoneurons within ba1 and ba2. Transferrina modulates hepcidin expression in zebrafish. The diversity of these loci was high in the largemouth bass population, where the heterozygosity was 0. Hardcopies of the 4th edition of the zebrafish book can be obtained for a nominal fee from the zebrafish international resource center, 5274. Using our firstpass marker panel in bulkedsegregant analysis, we were able to identify. Bulked segregant analysis to detect qtl related to heat. 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.
Instructor this video is on the zebra analysisor zebra if youre in different partsof the world besides the us. 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. Zebrafish and rotifer food feed skretting gemma skretting. Our investigator who needs this service is only able to obtain 2 5ul of blood. In zebrafish embryos, the axons of the posterior trigeminal vp and facial vii motoneurons project stereotypically to a small number of target muscles derived from the first and second branchial arches ba1, ba2. We identified linkage with nrd m805 in a 3cm interval between z21155 and z6365 on linkage group 16. Plsc 731 plant molecular genetics homework 2 due date. The cost efficiency and ease of data production afforded by nextgeneration sequencing have created new opportunities for rapid mapping of induced mutations. Another time and effort saving approach is bulk segregant analysis bsa michelmoore et al. When dna of ten plants are bulked into one pool, all alleles must be present. Anyone know of a reference lab that can and will do cbc and biochemistry work for zebrafish. Application of in silico bulked segregant analysis for rapid. This technique involves forming two groups that display opposing phenotypes for a trait of interest. 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.
To systematically isolate maternaleffect mutations in the zebrafish, we designed. Background despite the popularity of zebrafish as a research model, its sex determination sd mechanism is still unknown. A guide for the laboratory use of zebrafish danio rerio. A rapid mapping strategy suitable for monogenic qualitative traits. 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. This allows geneticists to discover genes conferring disease resistance or susceptibility. Using ssr markers 485 ssr marker and heat tolerance association analysis the two polymorphic markers identified based on the analysis of bulked extremes were used to screen the 279 f2 individuals to establish the association. The key design of this method is bulk genotyping of a pool of segregants sharing the same phenotype. 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. Bulk segregant analysis using single nucleotide polymorphism microarrays anthony becker1, daiyin chao2, xu zhang3, david e.
Rapid positional cloning of zebrafish mutations by linkage and. On behalf of the organisation for economic cooperation and development oecd, the. Jan 26, 2012 bfr for putative snps identified in maq120 analysis. One can enlist n number of biological disciplines in which this smallcreature finds acceptance. Second, a technique called bulk segregant analysis was introduced as an initial mapping device for new mutants 48. Potential markers were then subjected to an additional round of analysis on the individuals that formed the pooled samples for confirmation. The huge cost incurred in genotyping could be considered as a. Efficiency of mas for breeding common bean is still hampered, however, due to the dominance, linkage phase, and loose linkage of previously developed markers. Snps are plotted along the xaxis according to the unigene rank based on the rpkm expression values. 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. Bulk segregant mapping protocols protocols zfin community. This crayfish is also commonly known as the asian tiger.
We positioned half of the polymorphisms on zebrafish genetic and physical. Genetic mapping by bulk segregant analysis indicated that the zfdfl8a12 mutation was located on linkage group 19 between z11989 and z3816 figure 3 a top. 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. In this procedure, marker segregation is analysed in pooled groups of wildtype. Bulk segregant analysis was performed using the male and female pooled gdna samples to screen for potentially sexlinked markers. A simple pipeline for mapping point mutations plant physiology. Headwaters of the zebrafish emergence of a new model. 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. Zebrafishnatural habitats, animal behaviour and ecological importance. Bulk segregant analysis with molecular markers and its use. Analysis of sslp markers was used to fine map the mutation. Most cytogenetic studies failed to find dimorphic sex chromosomes and no primary sex determining switch has been identified even though the assembly of zebrafish genome sequence is near to completion and a high resolution genetic map is available.
Anyone know of a reference lab that can and will do cbc and biochemistry work for zebra fish. Medaka and zebrafish, an evolutionary twin study sciencedirect. Bulked segregant transcriptome analysis bsta data processing pipeline. Pdf bulk segregant analysis using single nucleotide. 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 snp genotype and gene frequencies are shown in table 5. Zebrafish danio rerio is increasingly used as a model organism in researchworldwide. Bulk segregant analysis with sslp markers was performed to identify the linkage group. Bulk segregation analysis mapped the ldd731 allele to. 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. Zebra crayfish aquatic arts aquatic arts unique fish. Bulked segregant analysis an overview sciencedirect topics. Sexlinked fluomep markers obtained by bulk segregant analysis performed on pooled male and female samples of guppy poecilia reticulata.
Bulk segregant analysis reveals the genetic basis of a. Recently, several zebrafish groups have reported using novel sequencing. Here we applied in silico bulked segregant analysis. Salt2, ivan baxter1,4 1donald danforth plant sciences center, st. Bulk segregant analysis using single nucleotide polymorphism. A point mutation of zebrafish ccbl gene in the ring finger domain. In bsa, dna of progenies corresponding to the phenotypic extremes is extracted and pooled. A mapping zebrafish mutants with weak or variable penetrance using a. Mghcvrc mapping resources protocols zfin community wiki. Coupled mutagenesis screens and genetic mapping in zebrafish.
Genetic mapping by bulk segregant analysis indicated that the zfdfl8a12 mutation was located on linkage group 19 between z11989 and z3816 figure 3a top. Programs have been set up on the beckman robot to aliquot the primers, set up the. In this study, we isolated the zebrafish ldd731 line through genetic screening to identify novel. 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. Mapping crosses followed by bulk segregant analysis bsa can take up to six. The zebrafish danio rerio is a freshwater fish belonging to the minnow family of the order cypriniformes. Zfin community wiki general information zfin community wiki. 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. Bulk segregant analysis and markertrait association reveal. The zebrafish book details these and other methods61. We established a fine map of the region by scoring other sslp and snp markers from 3.
We further demonstrate bulked segregant analysis using the anchored snps as a method. Within each bulk, the individuals are arbitrary for. 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. Bulk segregant mapping protocols isolation of intact genomic dna isolate genomic dna from embryos using our genomic dna isolation protocol. Here we applied in silico bulked segregant analysis bsa to the. Fine mapping qtl for drought resistance traits in rice. Thus, analysis of zebrafish mutations provides insights into gene functions in other vertebrates, including humans. Bulk segregant analysis bsa and resequencing of msa11 snptilling arraybased bulk segregant analysis was performed as previously described 59. Bulked segregant analysis bsa identifies dominant to identify each. Bulk segregant analysis and drought resistance in maize 1 fig. A major zebrafish polymorphism resource for genetic mapping.
Bulked segregant analysis bsa is a technique used to identify genetic markers associated with a mutant phenotype. Bulked segregant analysis was then used to identify a 2. Louis, missouri, united states of america, 2center for plant environmental stress. Bulked segregant analysis to detect qtl related to heat tolerance in rice oryza sativa l. The broken red line indicates the threshold value 3.
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