Dataset Information

Selection and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) under Heat and Salt Stress Conditions.

ABSTRACT: To identify stable housekeeping genes as a reference for expression analysis under heat and salt stress conditions in pigeonpea, the relative expression variation for 10 commonly used housekeeping genes (EF1?, UBQ10, GAPDH, 18Sr RNA, 25Sr RNA, TUB6, ACT1, IF4?, UBC, and HSP90) was studied in root, stem, and leaves tissues of Asha (ICPL 87119), a leading pigeonpea variety. Three statistical algorithms geNorm, NormFinder, and BestKeeper were used to define the stability of candidate genes. Under heat stress, UBC, HSP90, and GAPDH were found to be the most stable reference genes. In the case of salinity stress, GAPDH followed by UBC and HSP90 were identified to be the most stable reference genes. Subsequently, the above identified genes were validated using qRT-PCR based gene expression analysis of two universal stress-resposive genes namely uspA and uspB. The relative quantification of these two genes varied according to the internal controls (most stable, least stable, and combination of most stable and least stable housekeeping genes) and thus confirmed the choice as well as validation of internal controls in such experiments. The identified and validated housekeeping genes will facilitate gene expression studies under heat and salt stress conditions in pigeonpea.

SUBMITTER: Sinha P

PROVIDER: S-EPMC4865767 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Publications

Selection and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) under Heat and Salt Stress Conditions.

Sinha Pallavi P Saxena Rachit K RK Singh Vikas K VK Krishnamurthy L L Varshney Rajeev K RK

Frontiers in plant science 20151221

To identify stable housekeeping genes as a reference for expression analysis under heat and salt stress conditions in pigeonpea, the relative expression variation for 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18Sr RNA, 25Sr RNA, TUB6, ACT1, IF4α, UBC, and HSP90) was studied in root, stem, and leaves tissues of Asha (ICPL 87119), a leading pigeonpea variety. Three statistical algorithms geNorm, NormFinder, and BestKeeper were used to define the stability of candidate genes. Under h ...[more]

PMID: 27242803

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Project description:Genome assembly of short reads from large plant genomes remains a challenge in computational biology despite major developments in next generation sequencing. Of late several draft assemblies have been reported in sequenced plant genomes. The reported draft genome assemblies of Cajanus cajan have different levels of genome completeness, a large number of repeats, gaps, and segmental duplications. Draft assemblies with portions of genome missing are shorter than the referenced original genome. These assemblies come with low map accuracy affecting further functional annotation and the prediction of gene components as desired by crop researchers. Genome coverage, i.e., the number of sequenced raw reads mapped onto a certain location of the genome is an important quality indicator of completeness and assembly quality in draft assemblies. The present work aimed to improve the coverage in reported de novo sequenced draft genomes (GCA_000340665.1 and GCA_000230855.2) of pigeonpea, a legume widely cultivated in India. The two recently sequenced assemblies, A1 and A2 comprised 72% and 75% of the estimated coverage of the genome, respectively. We employed an assembly reconciliation approach to compare the draft assemblies and merge them, filling the gaps by employing an algorithm size sorting mate-pair library to generate a high quality and near complete assembly with enhanced contiguity. The majority of gaps present within scaffolds were filled with right-sized mate-pair reads. The improved assembly reduced the number of gaps than those reported in draft assemblies resulting in an improved genome coverage of 82.4%. Map accuracy of the improved assembly was evaluated using various quality metrics and for the presence of specific trait-related functional genes. Employed pair-end and mate-pair local libraries helped us to reduce gaps, repeats, and other sequence errors resulting in lengthier scaffolds compared to the two draft assemblies. We reported the prediction of putative host resistance genes against Fusarium wilt disease by their performance and evaluated them both in wet laboratory and field phenotypic conditions.

Dataset Information

Selection and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) under Heat and Salt Stress Conditions.

Publications

Selection and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) under Heat and Salt Stress Conditions.

Similar Datasets

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