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EcoTILLING-Based Association Mapping Efficiently Delineates Functionally Relevant Natural Allelic Variants of Candidate Genes Governing Agronomic Traits in Chickpea.

Bajaj D, Srivastava R, Nath M, Tripathi S, Bharadwaj C, Upadhyaya HD, Tyagi AK, Parida SK - Front Plant Sci (2016)

Bottom Line: The associated individual and combination of all SNPs explained 10-15 and 31% phenotypic variation for seed weight, respectively.The EcoTILLING-based large-scale allele mining and genotyping strategy implemented for association mapping is found much effective for a diploid genome crop species like chickpea with narrow genetic base and low genetic polymorphism.The seed weight-associated natural allelic variants and candidate TF genes delineated have potential to accelerate marker-assisted genetic improvement of chickpea.

View Article: PubMed Central - PubMed

Affiliation: Govt. of India, Plant Genomics and Molecular Breeding Lab, Department of Biotechnology, National Institute of Plant Genome Research New Delhi, India.

ABSTRACT
The large-scale mining and high-throughput genotyping of novel gene-based allelic variants in natural mapping population are essential for association mapping to identify functionally relevant molecular tags governing useful agronomic traits in chickpea. The present study employs an alternative time-saving, non-laborious and economical pool-based EcoTILLING approach coupled with agarose gel detection assay to discover 1133 novel SNP allelic variants from diverse coding and regulatory sequence components of 1133 transcription factor (TF) genes by genotyping in 192 diverse desi and kabuli chickpea accessions constituting a seed weight association panel. Integrating these SNP genotyping data with seed weight field phenotypic information of 192 structured association panel identified eight SNP alleles in the eight TF genes regulating seed weight of chickpea. The associated individual and combination of all SNPs explained 10-15 and 31% phenotypic variation for seed weight, respectively. The EcoTILLING-based large-scale allele mining and genotyping strategy implemented for association mapping is found much effective for a diploid genome crop species like chickpea with narrow genetic base and low genetic polymorphism. This optimized approach thus can be deployed for various genomics-assisted breeding applications with optimal expense of resources in domesticated chickpea. The seed weight-associated natural allelic variants and candidate TF genes delineated have potential to accelerate marker-assisted genetic improvement of chickpea.

No MeSH data available.


Related in: MedlinePlus

Optimization and validation of pool-based EcoTILLING approach coupled with agarose gel detection assay for large-scale mining of novel allelic variants from diverse coding and regulatory sequence components of TF genes by genotyping in a 192 desi and kabuli germplasm lines belonging to a seed weight association panel. (A) A representative gel illustrating the optimization followed by PCR amplification of seven primer-pairs (Pr1-Pr7) designed targeting various coding and regulatory regions of seven TF genes in the genomic DNA of a desi chickpea accession (ICC 4958) to produce single reproducible amplicons of each primer for EcoTILLING analysis. (B,C) The representative gels depicting the screening of allelic variants from the eight representative micropools (P1–P8) and two superpools (SP1 and SP2) made from the genomic DNA of 192 desi and kabuli germplasm lines (including ICC 4958 as control) employing an agarose gel-based EcoTILLING assay as defined in the Figure 1. The absence (B) and presence (C) of one non-synonymous SNP allelic variant in the pools and superpools based on cleavage/digestion patterns of 1500 bp fragments amplified from the target CDS region of a mTERF TF gene was apparent in 2.5% agarose gel. The occurrence of 1500 bp homoduplex uncut PCR amplicons as well as mismatch-specific CEL I cleavage of 1500 bp heteroduplex PCR amplified fragments into two varied amplicons of 760 and 740 bp fragment sizes due to the effect of single nucleotide polymorphism (SNP-allele) was observed in the four pools (P5–P8) and one superpool (SP2). M: 1 kb DNA ladder size standard. (D) The sequencing of 1500 bp amplified PCR product of an mTERF TF gene followed by multiple alignment of their high-quality sequences ascertained the presence of one coding SNP (C to G) exhibiting missense non-synonymous amino acid substitution [aspargine (AAC) to lysine (AAG)] in the four pools (P5–P8), one superpool (SP2) and seven individual desi and kabuli accessions as per expectation based on agarose gel-based EcoTILLING assay. The sequenced region carrying the non-synonymous SNP is indicated with a dotted box. The detail information of genes used for validation is mentioned in the Table S2.
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Figure 2: Optimization and validation of pool-based EcoTILLING approach coupled with agarose gel detection assay for large-scale mining of novel allelic variants from diverse coding and regulatory sequence components of TF genes by genotyping in a 192 desi and kabuli germplasm lines belonging to a seed weight association panel. (A) A representative gel illustrating the optimization followed by PCR amplification of seven primer-pairs (Pr1-Pr7) designed targeting various coding and regulatory regions of seven TF genes in the genomic DNA of a desi chickpea accession (ICC 4958) to produce single reproducible amplicons of each primer for EcoTILLING analysis. (B,C) The representative gels depicting the screening of allelic variants from the eight representative micropools (P1–P8) and two superpools (SP1 and SP2) made from the genomic DNA of 192 desi and kabuli germplasm lines (including ICC 4958 as control) employing an agarose gel-based EcoTILLING assay as defined in the Figure 1. The absence (B) and presence (C) of one non-synonymous SNP allelic variant in the pools and superpools based on cleavage/digestion patterns of 1500 bp fragments amplified from the target CDS region of a mTERF TF gene was apparent in 2.5% agarose gel. The occurrence of 1500 bp homoduplex uncut PCR amplicons as well as mismatch-specific CEL I cleavage of 1500 bp heteroduplex PCR amplified fragments into two varied amplicons of 760 and 740 bp fragment sizes due to the effect of single nucleotide polymorphism (SNP-allele) was observed in the four pools (P5–P8) and one superpool (SP2). M: 1 kb DNA ladder size standard. (D) The sequencing of 1500 bp amplified PCR product of an mTERF TF gene followed by multiple alignment of their high-quality sequences ascertained the presence of one coding SNP (C to G) exhibiting missense non-synonymous amino acid substitution [aspargine (AAC) to lysine (AAG)] in the four pools (P5–P8), one superpool (SP2) and seven individual desi and kabuli accessions as per expectation based on agarose gel-based EcoTILLING assay. The sequenced region carrying the non-synonymous SNP is indicated with a dotted box. The detail information of genes used for validation is mentioned in the Table S2.

Mentions: For large-scale mining and genotyping of gene-based SNP alleles by EcoTILLING in chickpea, a set of 1248 TF genes annotated from desi and kabuli genomes were acquired. The selected TFs include 819 desi and kabuli TF genes and 429 TF-encoding transcripts of desi accession (ICC 4958), which were specifically selected from the previous studies of Jhanwar et al. (2012) and Kujur et al. (2015), respectively based on the presence of at least one SNP in the CDS (coding sequence) and regulatory sequences of these genes. The multiple forward and reverse primer combination (at least two primer-pairs per TF gene) with expected amplification product size of 1000–1500 bp (per primer) targeting the diverse CDS and 2000-bp upstream/downstream regulatory regions (URRs/DRRs) of 1248 TF genes were designed. The amplification of each target gene regions was optimized (specifically the annealing temperature) with different combination of primer-pairs using the genomic DNA of one desi chickpea accession (ICC 4958) as per the detail PCR protocol described by Jhanwar et al. (2012) and Kujur et al. (2013). Based on these analyses, 1890 (75.7% of 2496 primer-pairs designed in total) primers designed from the 1133 TF genes exhibited reproducible single amplicons (by eliminating the non-specific amplified fragments and duplicate loci) in ICC 4958 using 2.5% agarose gel (Figure 2A). The fragments amplified specific to diverse coding (CDS) and non-coding URR/DRR sequence components of TF genes using the optimized primers were further assayed through agarose gel-based EcoTILLING approach for allele mining.


EcoTILLING-Based Association Mapping Efficiently Delineates Functionally Relevant Natural Allelic Variants of Candidate Genes Governing Agronomic Traits in Chickpea.

Bajaj D, Srivastava R, Nath M, Tripathi S, Bharadwaj C, Upadhyaya HD, Tyagi AK, Parida SK - Front Plant Sci (2016)

Optimization and validation of pool-based EcoTILLING approach coupled with agarose gel detection assay for large-scale mining of novel allelic variants from diverse coding and regulatory sequence components of TF genes by genotyping in a 192 desi and kabuli germplasm lines belonging to a seed weight association panel. (A) A representative gel illustrating the optimization followed by PCR amplification of seven primer-pairs (Pr1-Pr7) designed targeting various coding and regulatory regions of seven TF genes in the genomic DNA of a desi chickpea accession (ICC 4958) to produce single reproducible amplicons of each primer for EcoTILLING analysis. (B,C) The representative gels depicting the screening of allelic variants from the eight representative micropools (P1–P8) and two superpools (SP1 and SP2) made from the genomic DNA of 192 desi and kabuli germplasm lines (including ICC 4958 as control) employing an agarose gel-based EcoTILLING assay as defined in the Figure 1. The absence (B) and presence (C) of one non-synonymous SNP allelic variant in the pools and superpools based on cleavage/digestion patterns of 1500 bp fragments amplified from the target CDS region of a mTERF TF gene was apparent in 2.5% agarose gel. The occurrence of 1500 bp homoduplex uncut PCR amplicons as well as mismatch-specific CEL I cleavage of 1500 bp heteroduplex PCR amplified fragments into two varied amplicons of 760 and 740 bp fragment sizes due to the effect of single nucleotide polymorphism (SNP-allele) was observed in the four pools (P5–P8) and one superpool (SP2). M: 1 kb DNA ladder size standard. (D) The sequencing of 1500 bp amplified PCR product of an mTERF TF gene followed by multiple alignment of their high-quality sequences ascertained the presence of one coding SNP (C to G) exhibiting missense non-synonymous amino acid substitution [aspargine (AAC) to lysine (AAG)] in the four pools (P5–P8), one superpool (SP2) and seven individual desi and kabuli accessions as per expectation based on agarose gel-based EcoTILLING assay. The sequenced region carrying the non-synonymous SNP is indicated with a dotted box. The detail information of genes used for validation is mentioned in the Table S2.
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: Optimization and validation of pool-based EcoTILLING approach coupled with agarose gel detection assay for large-scale mining of novel allelic variants from diverse coding and regulatory sequence components of TF genes by genotyping in a 192 desi and kabuli germplasm lines belonging to a seed weight association panel. (A) A representative gel illustrating the optimization followed by PCR amplification of seven primer-pairs (Pr1-Pr7) designed targeting various coding and regulatory regions of seven TF genes in the genomic DNA of a desi chickpea accession (ICC 4958) to produce single reproducible amplicons of each primer for EcoTILLING analysis. (B,C) The representative gels depicting the screening of allelic variants from the eight representative micropools (P1–P8) and two superpools (SP1 and SP2) made from the genomic DNA of 192 desi and kabuli germplasm lines (including ICC 4958 as control) employing an agarose gel-based EcoTILLING assay as defined in the Figure 1. The absence (B) and presence (C) of one non-synonymous SNP allelic variant in the pools and superpools based on cleavage/digestion patterns of 1500 bp fragments amplified from the target CDS region of a mTERF TF gene was apparent in 2.5% agarose gel. The occurrence of 1500 bp homoduplex uncut PCR amplicons as well as mismatch-specific CEL I cleavage of 1500 bp heteroduplex PCR amplified fragments into two varied amplicons of 760 and 740 bp fragment sizes due to the effect of single nucleotide polymorphism (SNP-allele) was observed in the four pools (P5–P8) and one superpool (SP2). M: 1 kb DNA ladder size standard. (D) The sequencing of 1500 bp amplified PCR product of an mTERF TF gene followed by multiple alignment of their high-quality sequences ascertained the presence of one coding SNP (C to G) exhibiting missense non-synonymous amino acid substitution [aspargine (AAC) to lysine (AAG)] in the four pools (P5–P8), one superpool (SP2) and seven individual desi and kabuli accessions as per expectation based on agarose gel-based EcoTILLING assay. The sequenced region carrying the non-synonymous SNP is indicated with a dotted box. The detail information of genes used for validation is mentioned in the Table S2.
Mentions: For large-scale mining and genotyping of gene-based SNP alleles by EcoTILLING in chickpea, a set of 1248 TF genes annotated from desi and kabuli genomes were acquired. The selected TFs include 819 desi and kabuli TF genes and 429 TF-encoding transcripts of desi accession (ICC 4958), which were specifically selected from the previous studies of Jhanwar et al. (2012) and Kujur et al. (2015), respectively based on the presence of at least one SNP in the CDS (coding sequence) and regulatory sequences of these genes. The multiple forward and reverse primer combination (at least two primer-pairs per TF gene) with expected amplification product size of 1000–1500 bp (per primer) targeting the diverse CDS and 2000-bp upstream/downstream regulatory regions (URRs/DRRs) of 1248 TF genes were designed. The amplification of each target gene regions was optimized (specifically the annealing temperature) with different combination of primer-pairs using the genomic DNA of one desi chickpea accession (ICC 4958) as per the detail PCR protocol described by Jhanwar et al. (2012) and Kujur et al. (2013). Based on these analyses, 1890 (75.7% of 2496 primer-pairs designed in total) primers designed from the 1133 TF genes exhibited reproducible single amplicons (by eliminating the non-specific amplified fragments and duplicate loci) in ICC 4958 using 2.5% agarose gel (Figure 2A). The fragments amplified specific to diverse coding (CDS) and non-coding URR/DRR sequence components of TF genes using the optimized primers were further assayed through agarose gel-based EcoTILLING approach for allele mining.

Bottom Line: The associated individual and combination of all SNPs explained 10-15 and 31% phenotypic variation for seed weight, respectively.The EcoTILLING-based large-scale allele mining and genotyping strategy implemented for association mapping is found much effective for a diploid genome crop species like chickpea with narrow genetic base and low genetic polymorphism.The seed weight-associated natural allelic variants and candidate TF genes delineated have potential to accelerate marker-assisted genetic improvement of chickpea.

View Article: PubMed Central - PubMed

Affiliation: Govt. of India, Plant Genomics and Molecular Breeding Lab, Department of Biotechnology, National Institute of Plant Genome Research New Delhi, India.

ABSTRACT
The large-scale mining and high-throughput genotyping of novel gene-based allelic variants in natural mapping population are essential for association mapping to identify functionally relevant molecular tags governing useful agronomic traits in chickpea. The present study employs an alternative time-saving, non-laborious and economical pool-based EcoTILLING approach coupled with agarose gel detection assay to discover 1133 novel SNP allelic variants from diverse coding and regulatory sequence components of 1133 transcription factor (TF) genes by genotyping in 192 diverse desi and kabuli chickpea accessions constituting a seed weight association panel. Integrating these SNP genotyping data with seed weight field phenotypic information of 192 structured association panel identified eight SNP alleles in the eight TF genes regulating seed weight of chickpea. The associated individual and combination of all SNPs explained 10-15 and 31% phenotypic variation for seed weight, respectively. The EcoTILLING-based large-scale allele mining and genotyping strategy implemented for association mapping is found much effective for a diploid genome crop species like chickpea with narrow genetic base and low genetic polymorphism. This optimized approach thus can be deployed for various genomics-assisted breeding applications with optimal expense of resources in domesticated chickpea. The seed weight-associated natural allelic variants and candidate TF genes delineated have potential to accelerate marker-assisted genetic improvement of chickpea.

No MeSH data available.


Related in: MedlinePlus