Fine mapping of the linkage group 2 drought tolerance QTL in Pearl millet [Pennisetum glaucum (L.) R. Br.]

Srivastava RK1*, Vadez V1, Kholova J1, Lachagari VBR2, Mahendrakar MD1, Katiyar P1, Lekkala SP2, Praveen M1, Gupta R1, Blumwald E3

1 ICRISAT, Patancheru, Hyderabad, India; 2 SciGenom Labs Pvt Ltd, Kochi, India; 3 Department of Plant Sciences, University of California, Davis, CA, USA* Corresponding author: [email protected]

Introduction• Pearl millet [Pennisetum glaucum (L.) R. Br.] is an

important cereal crop capable of growing in semi-arid, arid and marginal regions of the world

• Drought is a major abiotic constraint affecting pearl millet production globally. (Yadav et al., 2002. Theor Appl Genet, 104: 67–83).

• Previously identified and validated major Linkage Group 2 (LG2) drought tolerance (DT) QTL (Bidinger et al., 2005. Field Crops Research, 94:14–32) contributing to hybrid grain and stover yield potential to terminal drought stress is being fine mapped.

• We assessed ddRAD (Peterson et al., 2012. PLoS ONE 7(5): e37135) sequencing over RAD-sequencing for fine mapping population.

Materials and Methods• Double digestion approaches were evaluated for

application in pearl millet NILs (H77/833-2-P10 & ICMR 01029-P1) population was done using SphI and MluCI Restriction enzymes protocols for RADTag generation on Illumina HiSeq2500.

• Clean-up of the digested product using Ampure beads and ligated P1 (barcoded) and P2 adaptors was done using T4 DNA ligase

• PCR amplification was performed to enrich and add the Illumina specific adapters and flowcell annealing sequences.

• Final pooling and sequencing was performed after QC check on bioanalyzer.

• Bioinformatic analysis was done using pipelinse Uclust (ver. 1.2), Bowtie2 (ver. 2.1.0), Samtools (ver. 0.1.18), as described in Figure 1.

Results• We generated ~400 Mb data (Figure 2 and Table 1 ) for

each sample using both RAD and ddRAD techniques for NILs.

• ~91% reads aligned to the reference in ddRAD and 78-86% aligned in RAD sequence data.

• A total 290 out of 6,067 F2 mapping population was generated by crossing H77/833-2-P10 & ICMR 01029-P1 near-isogenic lines (NILs) differing for the LG2 DT QTL

• A total of 52,028 SNPs identified from 888.85 million reads at a read depth of 10 in fine mapping population, by all of 6,821 SNPs were used for mapping.

• Linkage map was constructed using QTL IciMapping 4.1 software at a LOD threshold of 3.0. A total of 189 SNPs anchored to seven SSRs were mapped to the LG2 DT QTL.

• The length of linkage group (LG) was 639.72 cM (Haldane units) with an average inter-marker distance of 6.73 cM (Figure 3).

Fine Mapping of the Linkage Group 2 Drought Tolerance QTL in Pearl Millet [Pennisetum glaucum (L.) R. Br.]

Figure 3. LG 2 DT QTL map anchored to SSRs in circled, rest SNPs.

Conclusion• A modified double digest restriction site associated

DNA (ddRAD) technique using SphI and MluCI enzyme combination were used.

• The current DT QTL map spans over 639.72 cM (Haldane units) with an average inter-marker distance of 6.73 cM (Haldane units)

• ddRAD technique is being further employed for genotyping rest of the fine mapping population.

Acknowledgement• This paper presents result from a commissioned project

supported by the USAID, entitled “Development of Abiotic Stress Tolerant Millet for Africa and South Asia”.

• This work has been published as part of the CGIAR Research Program on Dryland Cereals. ICRISAT is a member of CGIAR consortium.

Figure 2. Summary of reads generated on Illumina HiSeq 2500 for the NILs.

Figure 1 .Bioinformatic pipeline used for data analysis.

Fine Mapping of the Linkage Group 2 Drought Tolerance QTL in Pearl Millet [Pennisetum glaucum (L.) R. Br.]

Srivastava RK1*, Vadez V1, Kholova J1, Lachagari VBR2, Mahendrakar MD1, Katiyar P1, Lekkala SP2, Praveen M1, Gupta R1, BlumwaldE3

1 ICRISAT, Patancheru, Hyderabad, India; 2 SciGenom Labs Pvt Ltd, Kochi, India; 3 Department of Plant Sciences, University of California, Davis, CA, USA

Figure 1 .Bioinformatic pipeline used for data analysis

Pearl millet [Pennisetum glaucum (L.) R. Br.] is an important cereal crop capable of growing in semi-arid, arid and marginal regions of the world

Drought is a major abiotic constraint affecting pearl millet production globally. (Yadav et al., 2002. Theor Appl Genet, 104: 67–83).

Previously identified and validated major Linkage Group 2 (LG2) drought tolerance (DT) QTL (Bidinger et al., 2005. Field Crops Research, 94:14–32) contributing to hybrid grain and stover yield potential to terminal drought stress is being fine mapped.

We assessed ddRAD (Peterson et al., 2012. PLoS ONE 7(5): e37135) sequencing over RAD-sequencing for fine mapping population

Introduction

Double digestion approaches were evaluated for application in pearl millet NILs (H77/833-2-P10 & ICMR 01029-P1) population was done using SphI and MluCI Restriction enzymes protocols for RADTaggeneration on Illumina HiSeq2500.

Clean-up of the digested product using Ampure beads and ligated P1 (barcoded) and P2 adaptors was done using T4 DNA ligase

PCR amplification was performed to enrich and add the Illuminaspecific adapters and flowcell annealing sequences.

Final pooling and sequencing was performed after QC check on bioanalyzer.

Bioinformatic analysis was done using pipelinse Uclust (ver. 1.2), Bowtie2 (ver. 2.1.0), Samtools (ver. 0.1.18), as described in Figure 1.

Materials and Methods

Figure 2. Summary of reads generated on Illumina HiSeq 2500 for the NILs.

Results

We generated ~400 Mb data (Figure 2 and Table 1 ) for each sample using both RAD and ddRAD techniques for NILs.

~91% reads aligned to the reference in ddRAD and 78-86% aligned in RAD sequence data.

A total 290 out of 6,067 F2 mapping population was generated by crossing H77/833-2-P10 & ICMR 01029-P1 near-isogenic lines (NILs) differing for the LG2 DT QTL

A total of 52,028 SNPs identified from 888.85 million reads at a read depth of 10 in fine mapping population, by all of 6,821 SNPs were used for mapping.

Linkage map was constructed using QTL IciMapping 4.1 software at a LOD threshold of 3.0. A total of 189 SNPs anchored to seven SSRs were mapped to the LG2 DT QTL.

The length of linkage group (LG) was 639.72 cM (Haldane units) with an average inter-marker distance of 6.73 cM(Figure 3).

A modified double digest restriction site associated DNA (ddRAD) technique using SphI and MluCI enzyme combination were used.

The current DT QTL map spans over 639.72 cM (Haldane units) with an average inter-marker distance of 6.73 cM(Haldane units)

ddRAD technique is being further employed for genotyping rest of the fine mapping population.

Conclusion

Figure 3. LG 2 DT QTL map anchored to SSRs in circles.

Acknowledgements

This paper presents result from a commissioned project supported by the USAID, entitled “Development of Abiotic Stress Tolerant Millet for Africa and South Asia”.

This work has been published as part of the CGIAR Research Program on DrylandCereals. ICRISAT is a member of CGIAR consortium

Fine Mapping of the Linkage Group 2 Drought Tolerance QTL in Pearl Millet [Pennisetum glaucum (L.) R. Br.]

Srivastava RK1*, Vadez V1, Kholova J1, Lachagari VBR2, Mahendrakar MD1, Katiyar P1, Lekkala SP2, Praveen M1, Gupta R1, BlumwaldE3

1 ICRISAT, Patancheru, Hyderabad, India; 2 SciGenom Labs Pvt Ltd, Kochi, India; 3 Department of Plant Sciences, University of California, Davis, CA, USA

Figure 1 .Bioinformatic pipeline used for data analysis

Pearl millet [Pennisetum glaucum (L.) R. Br.] is an important cereal crop capable of growing in semi-arid, arid and marginal regions of the world

Drought is a major abiotic constraint affecting pearl millet production globally. (Yadav et al., 2002. Theor Appl Genet, 104: 67–83).

Previously identified and validated major Linkage Group 2 (LG2) drought tolerance (DT) QTL (Bidinger et al., 2005. Field Crops Research, 94:14–32) contributing to hybrid grain and stover yield potential to terminal drought stress is being fine mapped.

We assessed ddRAD (Peterson et al., 2012. PLoS ONE 7(5): e37135) sequencing over RAD-sequencing for fine mapping population

Introduction

Double digestion approaches were evaluated for application in pearl millet NILs (H77/833-2-P10 & ICMR 01029-P1) population was done using SphI and MluCI Restriction enzymes protocols for RADTaggeneration on Illumina HiSeq2500.

Clean-up of the digested product using Ampure beads and ligated P1 (barcoded) and P2 adaptors was done using T4 DNA ligase

PCR amplification was performed to enrich and add the Illuminaspecific adapters and flowcell annealing sequences.

Final pooling and sequencing was performed after QC check on bioanalyzer.

Bioinformatic analysis was done using pipelinse Uclust (ver. 1.2), Bowtie2 (ver. 2.1.0), Samtools (ver. 0.1.18), as described in Figure 1.

Materials and Methods

Figure 2. Summary of reads generated on Illumina HiSeq 2500 for the NILs.

Results

We generated ~400 Mb data (Figure 2 and Table 1 ) for each sample using both RAD and ddRAD techniques for NILs.

~91% reads aligned to the reference in ddRAD and 78-86% aligned in RAD sequence data.

A total 290 out of 6,067 F2 mapping population was generated by crossing H77/833-2-P10 & ICMR 01029-P1 near-isogenic lines (NILs) differing for the LG2 DT QTL

A total of 52,028 SNPs identified from 888.85 million reads at a read depth of 10 in fine mapping population, by all of 6,821 SNPs were used for mapping.

Linkage map was constructed using QTL IciMapping 4.1 software at a LOD threshold of 3.0. A total of 189 SNPs anchored to seven SSRs were mapped to the LG2 DT QTL.

The length of linkage group (LG) was 639.72 cM (Haldane units) with an average inter-marker distance of 6.73 cM(Figure 3).

A modified double digest restriction site associated DNA (ddRAD) technique using SphI and MluCI enzyme combination were used.

The current DT QTL map spans over 639.72 cM (Haldane units) with an average inter-marker distance of 6.73 cM(Haldane units)

ddRAD technique is being further employed for genotyping rest of the fine mapping population.

Conclusion

Figure 3. LG 2 DT QTL map anchored to SSRs in circles.

Acknowledgements

This paper presents result from a commissioned project supported by the USAID, entitled “Development of Abiotic Stress Tolerant Millet for Africa and South Asia”.

This work has been published as part of the CGIAR Research Program on DrylandCereals. ICRISAT is a member of CGIAR consortium

Table1: RADTag and ddRADTagdata summary generated on Illumina HiSeq 2500 for the NILs.

Sample Name

No. of raw reads

No. of bases (Mb) GC (%)

% of data >= Q30

Raw reads Length (bp)

P1 ddRAD 3,018,210 301.82 48.34 88.31 100 x 2

P2 ddRAD 3,990,900 399.09 48.75 89.26 100 x 2

P1 RAD 4,886,676 488.66 47.07 84.01 100 x 2

P2 RAD 4,665,114 466.51 48.25 83.88 100 x 2

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This work has been undertaken as part of the Feb

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