Pallavi Sinha

Next‐generation sequencing for identification of candidate genes for Fusarium wilt and sterility mosaic disease in pigeonpea (Cajanus cajan)

Summary To map resistance genes for Fusarium wilt (FW) and sterility mosaic disease (SMD) in pigeonpea, sequencing‐based bulked segregant analysis (Seq‐BSA) was used. Resistant (R) and susceptible (S) bulks from the extreme recombinant inbred lines of ICPL 20096 × ICPL 332 were sequenced. Subsequently, SNP index was calculated between R‐ and S‐bulks with the help of draft genome sequence and reference‐guided assembly of ICPL 20096 (resistant parent). Seq‐BSA has provided seven candidate SNPs for FW and SMD resistance in pigeonpea. In parallel, four additional genotypes were re‐sequenced and their combined analysis with R‐ and S‐bulks has provided a total of 8362 nonsynonymous (ns) SNPs. Of 8362 nsSNPs, 60 were found within the 2‐Mb flanking regions of seven candidate SNPs identified through Seq‐BSA. Haplotype analysis narrowed down to eight nsSNPs in seven genes. These eight nsSNPs were further validated by re‐sequencing 11 genotypes that are resistant and susceptible to FW and SMD. This analysis revealed association of four candidate nsSNPs in four genes with FW resistance and four candidate nsSNPs in three genes with SMD resistance. Further, In silico protein analysis and expression profiling identified two most promising candidate genes namely C.cajan_01839 for SMD resistance and C.cajan_03203 for FW resistance. Identified candidate genomic regions/SNPs will be useful for genomics‐assisted breeding in pigeonpea.

Association of nad7a Gene with Cytoplasmic Male Sterility in Pigeonpea

Cytoplasmic male sterility (CMS) has been exploited in the commercial pigeonpea [Cajanus cajan (L.) Millsp.] hybrid breeding system; however, the molecular mechanism behind this system is unknown. To understand the underlying molecular mechanism involved in A4 CMS system derived from C. cajanifolius (Haines) Maesen, 34 mitochondrial genes were analyzed for expression profiling and structural variation analysis between CMS line (ICRISAT Pigeonpea A line, ICPA 2039) and its cognate maintainer (ICPB 2039). Expression profiling of 34 mitochondrial genes revealed nine genes with significant fold differential gene expression at P ≤ 0.01, including one gene, nad4L, with 1366‐fold higher expression in CMS line as compared with the maintainer. Structural variation analysis of these mitochondrial genes identified length variation between ICPA 2039 and ICPB 2039 for nad7a (subunit of nad7 gene). Sanger sequencing of nad4L and nad7a genes in the CMS and the maintainer lines identified two single nucleotide polymorphisms (SNPs) in upstream region of nad4L and a deletion of 10 bp in nad7a in the CMS line. Protein structure evaluation showed conformational changes in predicted protein structures for nad7a between ICPA 2039 and ICPB 2039 lines. All above analyses indicate association of nad7a gene with the CMS for A4 cytoplasm in pigeonpea. Additionally, one polymerase chain reaction (PCR) based Indel marker (nad7a_del) has been developed and validated for testing genetic purity of A4 derived CMS lines to strengthen the commercial hybrid breeding program in pigeonpea.

Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.

Genomics-assisted breeding for boosting crop improvement in pigeonpea (Cajanus cajan)

Pigeonpea is an important pulse crop grown predominantly in the tropical and sub-tropical regions of the world. Although pigeonpea growing area has considerably increased, yield has remained stagnant for the last six decades mainly due to the exposure of the crop to various biotic and abiotic constraints. In addition, low level of genetic variability and limited genomic resources have been serious impediments to pigeonpea crop improvement through modern breeding approaches. In recent years, however, due to the availability of next generation sequencing and high-throughput genotyping technologies, the scenario has changed tremendously. The reduced sequencing costs resulting in the decoding of the pigeonpea genome has led to the development of various genomic resources including molecular markers, transcript sequences and comprehensive genetic maps. Mapping of some important traits including resistance to Fusarium wilt and sterility mosaic disease, fertility restoration, determinacy with other agronomically important traits have paved the way for applying genomics-assisted breeding (GAB) through marker assisted selection as well as genomic selection (GS). This would accelerate the development and improvement of both varieties and hybrids in pigeonpea. Particularly for hybrid breeding programme, mitochondrial genomes of cytoplasmic male sterile (CMS) lines, maintainers and hybrids have been sequenced to identify genes responsible for cytoplasmic male sterility. Furthermore, several diagnostic molecular markers have been developed to assess the purity of commercial hybrids. In summary, pigeonpea has become a genomic resources-rich crop and efforts have already been initiated to integrate these resources in pigeonpea breeding.

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

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 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.

Identification and Validation of Selected Universal Stress Protein Domain Containing Drought-Responsive Genes in Pigeonpea (Cajanus cajan L.).

Pigeonpea is a resilient crop, which is relatively more drought tolerant than many other legume crops. To understand the molecular mechanisms of this unique feature of pigeonpea, 51 genes were selected using the Hidden Markov Models (HMM) those codes for proteins having close similarity to universal stress protein domain. Validation of these genes was conducted on three pigeonpea genotypes (ICPL 151, ICPL 8755, and ICPL 227) having different levels of drought tolerance. Gene expression analysis using qRT-PCR revealed 6, 8, and 18 genes to be ≥2-fold differentially expressed in ICPL 151, ICPL 8755, and ICPL 227, respectively. A total of 10 differentially expressed genes showed ≥2-fold up-regulation in the more drought tolerant genotype, which encoded four different classes of proteins. These include plant U-box protein (four genes), universal stress protein A-like protein (four genes), cation/H(+) antiporter protein (one gene) and an uncharacterized protein (one gene). Genes C.cajan_29830 and C.cajan_33874 belonging to uspA, were found significantly expressed in all the three genotypes with ≥2-fold expression variations. Expression profiling of these two genes on the four other legume crops revealed their specific role in pigeonpea. Therefore, these genes seem to be promising candidates for conferring drought tolerance specifically to pigeonpea.

Indel-seq: a fast-forward genetics approach for identification of trait-associated putative candidate genomic regions and its application in pigeonpea (Cajanus cajan)

Abstract Identification of candidate genomic regions associated with target traits using conventional mapping methods is challenging and time‐consuming. In recent years, a number of single nucleotide polymorphism (SNP)‐based mapping approaches have been developed and used for identification of candidate/putative genomic regions. However, in the majority of these studies, insertion–deletion (Indel) were largely ignored. For efficient use of Indels in mapping target traits, we propose Indel‐seq approach, which is a combination of whole‐genome resequencing (WGRS) and bulked segregant analysis (BSA) and relies on the Indel frequencies in extreme bulks. Deployment of Indel‐seq approach for identification of candidate genomic regions associated with fusarium wilt (FW) and sterility mosaic disease (SMD) resistance in pigeonpea has identified 16 Indels affecting 26 putative candidate genes. Of these 26 affected putative candidate genes, 24 genes showed effect in the upstream/downstream of the genic region and two genes showed effect in the genes. Validation of these 16 candidate Indels in other FW‐ and SMD‐resistant and FW‐ and SMD‐susceptible genotypes revealed a significant association of five Indels (three for FW and two for SMD resistance). Comparative analysis of Indel‐seq with other genetic mapping approaches highlighted the importance of the approach in identification of significant genomic regions associated with target traits. Therefore, the Indel‐seq approach can be used for quick and precise identification of candidate genomic regions for any target traits in any crop species.

Rapid Screening of MDR-TB in Cases of Extra Pulmonary Tuberculosis Using Geno Type MTBDRplus.

Background Drug resistance in tuberculosis is a major public health challenge in developing countries. The limited data available on drug resistance in extra pulmonary tuberculosis stimulated us to design our study on anti-tuberculosis drug resistance pattern in cases of extra pulmonary tuberculosis in a tertiary referral hospital of North India. We performed Geno Type MTBDRplus assay in comparison with conventional drug susceptibility testing by proportion method to study the mutation patterns in rpoB, katG and inhA genes. Methods A total of 510 extra pulmonary samples were included in this study. After the smear microscopy, all the specimens were subjected for culture on Lowenstein Jensen (LJ) media. Phenotypic drug susceptibility testing (DST) was performed on LJ media for all the MTB isolates and compared with the results of Geno Type MTBDRplus assay which was performed with the DNA isolated from the culture by conventional method. Results Of 510 specimens cultured, the total culture positivity obtained was 11.8% (60) encompassing 54 (10.6%) Mycobacterium tuberculosis and 6 (1.2%) non-tubercular mycobacteria (NTM). DST results by Geno Type MTBDRplus assay and solid culture methods were compared in 51 MTB isolates excluding the two Rif indeterminate and one invalid test. Geno Type MTBDRplus accurately identified 13 of 14 rifampicin-resistant strains, 14 of 15 isoniazid-resistant strains and 13 of 14 as multi drug resistant tuberculosis (MDR-TB) in comparison with conventional method. Sensitivity and specificity were 92.86% and 97.30% respectively for detection of RIF resistance, 93.33% and 94.44% respectively for detection of INH resistance, 92.86% and 97.30% respectively for detection of MDR-TB, while the overall concordance of Geno Type MTBDRplus assay with conventional DST was 94.11%. The turn-around time for performing Geno Type MTBDRplus assay test was 48 hours. Conclusion The problem of MDR in extra pulmonary tuberculosis (EPTB) cannot be overlooked and due attention on patients should be given. Routine use of Geno Type MTBDRplus assay for the diagnosis of MDR-EPTB can substantially reduce the time between diagnosis and drug therapy. Culture along with Geno Type MTBDRplus assay could be a solution for rapid and accurate diagnosis of MDR-TB in low bacillary non sputum specimens.

Marker-Assisted Improvement of the Elite Maintainer Line of Rice, IR 58025B for Wide Compatibility (S5n) Gene

The degree of heterosis in different hybrid rice varieties is reported to be at the highest in indica/japonica cross combination, however, there is a problem of sterility and semi-sterility in such inter sub specific hybrids. To overcome this problem, it is essential to develop parental lines having wide compatibility (S5n) gene. In this study, a functional marker S5-InDel was used for marker-assisted backcrossing (MABB) to introgress S5n gene from Dular into the genetic background of a widely grown recurrent parent IR 58025B, a maintainer line of wild-abortive (WA) cytoplasmic male sterile line, IR 58025A. Further, a closely linked marker nksbadh2 was used for the identification of plants devoid of aroma in backcross population to develop hybrids with no aroma. The stringent phenotypic selection followed by background selection of BC3F4 identified plants with 94.51–98.90% of the recurrent parent genome recovery of lines carrying S5n gene. Subsequently, at 10 promising BC3F5 lines possessing S5n gene with high yielding and long-slender grain type were validated for their maintainer behavior through test crosses with IR 58025A. Also the improved lines showed significantly improved spikelet fertility performance while crossed with japonica and javanica testers in comparison to the original recurrent parent. The improved lines developed in the present study, are being converted to CMS lines through marker-assisted backcross breeding to facilitate precise and improved hybrid breeding program in rice.

Isolation and characterization of Mycobacterium tuberculosis from extra pulmonary specimens in a tertiary referral hospital of north India

Materials and Methods: A total of 510 different extra pulmonary samples were collected during the period of August 2014 to July 2015. All the samples were subjected to Ziehl Neelsen staining. Samples were decontaminated by NALC-NaOH method and inoculated on a pair of Lowenstein-Jensen (LJ) media and one p-Nitrobenzoic acid (PNB). Cultures were incubated at 37 ̊C and observed for 8weeks for any growth.