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Bulked Segregant analysis

Bulked segregant analysis (BSA) is a technique employed to quickly identify phenotype associated genetic markers. Main workflow of BSA contains selecting two groups of individuals with extremely opposing phenotypes, pooling the DNA of all individuals to form two bulk of DNA, identifying differential sequences between two pools. This technique has been extensively employed in identifying genetic markers strongly associated by targeted genes in plant/animal genomes.

Service Details

Demo Results

Case Study

Service Advantages


Takagi et al., The plant journal, 2013

● Accurate localization: Mixing bulks with 30+30 to 200+200 individuals to minimize background noise; non-synonymous mutatants-based candidate region prediction.

● Comprehensive analysis: In-depth candidate gene function annotation, including NR, SwissProt, GO, KEGG, COG, KOG ,etc.

● Faster Turnaround time: Rapid gene localization within 45 working days.

● Extensive experience: BMK has contributed in thousands of traits localization, covering diverse species like crops, aquatic products, forest, flowers, fruits, etc.

Service Specifications

Segregating progeny of parents with opposing phenotypes.
e.g. F2 progeny, Backcrossing (BC), Recombinant inbred line(RIL)

Mixing pool
For qualitative traits: 30 to 50 individuals(minimum 20)/bulk
For quantitative tratis: top 5% to 10% individuals with either extreme phenotypes in the whole population(minimum 30+30).

Recommended sequencing depth
At least 20X/parent and 1X/offspring individual (e.g. for offspring mixing pool of 30+30 individual, sequencing depth will be 30X per bulk)

Bioinformatics analyses

● Whole genome resequencing
● Data processing
● SNP/Indel calling
● Candidate region screening
● Candidate gene function annotation


Sample Requirements and Delivery

Sample Requirements:


gDNA sample

Tissue sample

Concentration: ≥30 ng/μl

Plants: 1-2 g

Amount: ≥2 μg (Volumn ≥15 μl)

Animals: 0.5-1 g

Purity: OD260/280= 1.6-2.5

Whole blood: 1.5 ml

Service Work Flow

Sample QC

Experiment design

sample delivery

Sample delivery

Pilot experiment

RNA extraction

Library Preparation

Library construction



Data analysis

Data analysis

After sale Services

After-sale services

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  • 1.Association analysis base on Euclidean Distance (ED) to identify candidate region. In the following figure

    X-axis: Chromosome number; Each dot represents an ED value of an SNP. The Black line correspond to fitted ED value. A higher ED value indicates a more significant association between the site and the phenotype. Red dash line represents threshold of significant association.



    2.Association analysis based no SNP-index

    X-axis: Chromosome number; Each dot represents SNP-index value. The black line stands for fitted SNP-index value. The larger the value is, the more significant the association is.



    BMK Case

    The major-effect quantitative trait locus Fnl7.1 encodes a late embryogenesis abundant protein associated with fruit neck length in cucumber 

    Published: Plant Biotechnology Journal, 2020

    Sequencing strategy:

    Parents (Jin5-508, YN): Whole genome resequencing for 34× and 20×.

    DNA pools (50 Long-necked and 50 short-necked): Resequencing for 61× and 52×

    Key results

    In this study, segregating population(F2 and F2:3) was generated by crossing long-neck cucumber line Jin5-508 and short-neck YN. Two DNA pools were constructed by 50 extreme long-neck individuals and 50 extreme short-neck individuals. Major-effect QTL was identified on Chr07 by BSA analysis and traditional QTL mapping. The candidate region was further narrowed down by fine-mapping, gene expression quantification and transgenic experiments, which revealed key gene in controlling neck-length, CsFnl7.1. In addition, polymorphism in CsFnl7.1 promoter region was found to be associated with corresponding expression. Further phylogenetic analysis suggested that Fnl7.1 locus is very likely to be originated from India.


    QTL-mapping in BSA analysis to identify candidate region associated with cucumber neck length


    LOD profiles of cucumber neck-length QTL identified on Chr07


    Xu, X. , et al. “The major-effect quantitative trait locus Fnl7.1 encodes a late embryogenesis abundant protein associated with fruit neck length in cucumber.” Plant Biotechnology Journal 18.7(2020).

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