CD Genomics Perspective: Practicing Genetic Marker Identification by Bulk Segreg

Posted by kiko on January 24th, 2022

Introduction to Bulked Segregant Analysis

BSA (bulked segregant analysis) is a method for identifying genetic markers linked to a mutant phenotype. Geneticists can use this information to find genes that confer disease resistance or susceptibility. Forming two groups with opposing phenotypes for a trait of interest is the goal of this technique. Individuals in one group, for example, are disease resistant whereas those in the second group are not. The DNA of all individuals in each group is then pooled to create two bulked DNA samples.

These two bulked samples can then be analyzed using Restriction fragment length polymorphism (RFLP) or RAPD to detect similarities and differences in the genome\'s various loci. Except for loci associated with the mutation, both groups will have a random distribution of alleles in all of the genome\'s loci. A consistent difference between the two bulked samples on a locus most likely indicates that the locus is linked to the mutation of interest.

Individuals in the two testing groups are usually produced in animals by crossing two siblings who are heterozygous for the mutation of interest. It\'s necessary to use siblings to ensure that the alleles that cause the mutation are the same among the individuals.

To identify the genes associated with the trait of interest, there must be a minimum amount of heterozygosity in the various loci of the groups. Due to the inbred nature of most laboratory strains, outcrossing the homozygous mutated individual with a polymorphic strain is required to generate effective testing groups. To create testing groups, the offspring are crossed with each other.

Analysis Techniques for Bulked Segregant Analysis

Southern blotting can be used to examine bulk DNA samples. RFLP and RAPD analysis both require the use of restriction enzymes or PCR amplification on DNA. The restriction digest sites and the sequences on which PCR primers attach are the loci that are examined in these techniques. These sites are typically found all over the genome. Once linked loci have been identified, they can be mapped and linkage distances calculated.

Quantitative Trait Loci Sequencing (QTL-Seq)

Bulked segregant analysis, as used in QTL-seq, is a powerful and efficient way to find agronomically important loci in crop plants. To find quantitative trait loci, QTL-seq was adapted from MutMap. This method locates a gene that has undergone natural mutations.

Mutmap

The MutMap-Gap methodology identifies the mutation site in the reference genome\'s gap regions. This method locates a gene that has been artificially mutated.

Applications of Bulked Segregant Analysis

Early applications almost exclusively used molecular markers and bulked segregant analysis to map genes with relatively large effects for agronomically important traits like grain yield, drought tolerance, and heat tolerance in rice, water-stress tolerance in wheat, and salt tolerance in Egyptian cotton.

Many agronomical traits, including resistance genes to fungal pathogens, have been mapped using bulked segregant analysis (BSA) in several crops. It was suggested that the inheritance of ABS resistance in citrus is managed by a central recessive allele using BSA in diploid progenies.

References

1. Zhao J, Yang Y, Yang D, Cheng Y, Jiao M, Zhan G, Zhang H, Wang J, Zhou K, Huang L, Kang Z. Characterization and genetic analysis of rice mutant crr1 exhibiting compromised non-host resistance to Puccinia striiformis f. sp. tritici (Pst). Frontiers in plant science. 2016 Nov 30;7:1822.

2. Ramirez?Gonzalez RH, Segovia V, Bird N, Fenwick P, Holdgate S, Berry S, Jack P, Caccamo M, Uauy C. RNA?S eq bulked segregant analysis enables the identification of high?resolution genetic markers for breeding in hexaploid wheat. Plant biotechnology journal. 2015 Jun;13(5):613-24.