Comprehensive RNAseq analysis for identification of genes expressed under heat stress in lentil.

Lentils are highly sensitive to abrupt increases in temperature during the mid to late reproductive stages, leading to severe biomass and seed yield reduction. Therefore, we carried out an RNAseq analysis between IG4258 (heat tolerant) and IG3973 (heat sensitive) lentil genotypes at the reproductive stage under both normal and heat stress conditions in the field. It resulted in 209,549 assembled transcripts and among these 161,809 transcripts had coding regions, of which 94,437 transcripts were annotated. The differential gene expression analysis showed upregulation of 678 transcripts and downregulation of 680 transcripts between the tolerant and sensitive genotypes at the early reproductive stage. While 76 transcripts were upregulated and 47 transcripts were downregulated at the late reproductive stage under heat stress conditions. The validation of 12 up-or downregulated transcripts through RT-PCR corresponded well with the expression analysis data of RNAseq, with a correlation of R2  = 0.89. Among these transcripts, the DN364_c1_g1_i9 and DN2218_c0_g1_i5 transcripts encoded enzymes involved in the tryptophan pathway, indicating that tryptophan biosynthesis plays a role under heat stress in lentil. Moreover, KEGG pathways enrichment analysis identified transcripts associated with genes encoding proteins/regulating factors related to different metabolic pathways including signal transduction, fatty acid biosynthesis, rRNA processing, ribosome biogenesis, gibberellin (GA) biosynthesis, and riboflavin biosynthesis. This analysis also identified 6852 genic-SSRs leading to the development of 4968 SSR primers that are potential genomic resources for molecular mapping of heat-tolerant genes in lentil.

Effect of elevated carbon-dioxide on plant growth, physiology, yield and seed quality of chickpea (Cicer arietinum L.) in Indo-Gangetic plains.

In the present scenario of climate change with constantly increasing CO2 concentration, there is a risk of altered crop performance in terms of growth, yield, grain nutritional value and seed quality. Therefore, an experiment was conducted in open top chamber (OTCs) during 2017-18 and 2018-19 to assess the effect of elevated atmospheric carbondioxide (e[CO2]) (600 ppm) on chickpea (cv. JG 14) crop growth, biomass accumulation, physiological function, seed yield and its quality in terms of germination and vigour. The e[CO2] treatment increased the plant height, leaf and stem biomass over ambient CO2 (a[CO2]) treatment. The e[CO2] increased seed yield by 11-18% which was attributed to an increase in the number of pods (6-10%) and seeds plant-1 (8-9%) over a[CO2]. However, e[CO2] reduced the seed protein (7%), total phenol (13%) and thiobarbituric acid reactive substances (12%) and increased the starch (21%) and water uptake rate as compared to seeds harvested from a[CO2] environment. Exposing chickpea plant to e[CO2] treatment had no impact on germination and vigour of the harvested seeds. Also, the physical attributes, total soluble sugar and antioxidant enzymes activities of harvested seeds were comparable in a[CO2] and e[CO2] treatment. Hence, the experimental findings depict that e[CO2] upto 600 ppm could add to the growth and productivity of chickpea in a sub-tropical climate with an implication on its nutritional quality of the produce.

Transgenic chickpea (Cicer arietinum L.) harbouring AtDREB1a are physiologically better adapted to water deficit.

BACKGROUND: Chickpea (Cicer arietinum L.) is the second most widely grown pulse and drought (limiting water) is one of the major constraints leading to about 40-50% yield losses annually. Dehydration responsive element binding proteins (DREBs) are important plant transcription factors that regulate the expression of many stress-inducible genes and play a critical role in improving the abiotic stress tolerance. Transgenic chickpea lines harbouring transcription factor, Dehydration Responsive Element-Binding protein 1A from Arabidopsis thaliana (AtDREB1a gene) driven by stress inducible promoter rd29a were developed, with the intent of enhancing drought tolerance in chickpea. Performance of the progenies of one transgenic event and control were assessed based on key physiological traits imparting drought tolerance such as plant water relation characteristics, chlorophyll retention, photosynthesis, membrane stability and water use efficiency under water stressed conditions. RESULTS: Four transgenic chickpea lines harbouring stress inducible AtDREB1a were generated with transformation efficiency of 0.1%. The integration, transmission and regulated expression were confirmed by Polymerase Chain Reaction (PCR), Southern Blot hybridization and Reverse Transcriptase polymerase chain reaction (RT-PCR), respectively. Transgenic chickpea lines exhibited higher relative water content, longer chlorophyll retention capacity and higher osmotic adjustment under severe drought stress (stress level 4), as compared to control. The enhanced drought tolerance in transgenic chickpea lines were also manifested by undeterred photosynthesis involving enhanced quantum yield of PSII, electron transport rate at saturated irradiance levels and maintaining higher relative water content in leaves under relatively severe soil water deficit. Further, lower values of carbon isotope discrimination in some transgenic chickpea lines indicated higher water use efficiency. Transgenic chickpea lines exhibiting better OA resulted in higher seed yield, with progressive increase in water stress, as compared to control. CONCLUSIONS: Based on precise phenotyping, involving non-invasive chlorophyll fluorescence imaging, carbon isotope discrimination, osmotic adjustment, higher chlorophyll retention and membrane stability index, it can be concluded that AtDREB1a transgenic chickpea lines were better adapted to water deficit by modifying important physiological traits. The selected transgenic chickpea event would be a valuable resource that can be used in pre-breeding or directly in varietal development programs for enhanced drought tolerance under parched conditions.

Physiological Traits for Shortening Crop Duration and Improving Productivity of Greengram (Vigna radiata L. Wilczek) Under High Temperature.

Greengram is an important protein-rich food legume crop. During the reproductive stage, high temperatures cause flower drop, induce male sterility, impair anthesis, and shortens the grain-filling period. Initially, 116 genotypes were evaluated for 3 years in two locations, and based on flowering, biomass, and yield attributes, they were grouped into four major clusters. A panel of 17 contrasting genotypes was selected for their heat tolerance in high-temperature greenhouses. The seedlings of the selected genotypes were exposed to heat shock in the range 37°C-52°C and their recovery after heat shock was assessed at 30°C. The seedlings of EC 398889 turned completely green and rejuvenated, while those of LGG 460 failed to recover, therefore, EC 398889 and LGG 460 were identified as heat-tolerant and heat-sensitive genotypes, respectively. Except for EC 398889, the remaining genotypes could not survive after heat shock. Fresh seeds of EC 398889 and LGG 460 were planted in field and pollen fertility and sucrose-synthase (SuSy) activity in grains were assessed at high temperatures. The pollen germination and SuSy activity were normal even at temperatures beyond 40°C in EC 398889 and high SuSy activity enabled faster grain filling than in LGG 460. The precise phenotyping demonstrated significant differences in the light-temperature response of photosynthesis, chlorophyll fluorescence imaging of quantum yield (Fv/Fm), and electron transport rate (ETR) between heat-tolerant (EC 398889) and heat-sensitive (LGG 460) genotypes. Molecular profiling of selected accessions showed polymorphism with 11 SSR markers and the markers CEDG147, CEDG247, and CEDG044 distinguished tolerant and sensitive groups of accessions.

Physiological and molecular characterisation for high temperature stress in Lens culinaris.

In the present study, 11 lentil (Lens culinaris Medik) genotypes including heat tolerant and heat sensitive genotypes identified after a screening of 334 accessions of lentil for traits imparting heat tolerance, were characterised based on physiological traits and molecular markers. Results showed a higher reduction in pollen viability among sensitive genotypes (up to 52.3%) compared with tolerant genotypes (up to 32.4%) at 43°C. Higher photosynthetic electron transport rate was observed among heat tolerant genotypes and two heat tolerant lentil genotypes, IG 4258 (0.43) and IG 3330 (0.38) were having highest Fv/Fm values. However, membrane stability was significantly higher in only one heat tolerant genotype, ILL 10712, indicating that different mechanisms are involved to control heat tolerance in lentil. The molecular characterisation of lentil genotypes with 70 polymorphic SSR and genic markers resulted into distinct clusters in accordance with their heat stress tolerance. A functional marker ISM11257 (intron spanning marker) amplifying an allele of 205bp in size was present only among heat tolerant genotypes, and could be further used in a breeding program to identify heat tolerant lentil genotypes. The findings of this study will contribute to the development of heat tolerant lentil cultivars.

Correction: Genetic Dissection of Drought and Heat Tolerance in Chickpea through Genome-Wide and Candidate Gene-Based Association Mapping Approaches.

[This corrects the article DOI: 10.1371/journal.pone.0096758.].

Integrative genomic and network analysis identified novel genes associated with the development of advanced cervical squamous cell carcinoma.

BACKGROUND: CSCC is one of the most common cancer affecting women globally. Though it is caused by the infection of hrHPV but long latency period for malignant outcome in only a subset of hrHPV infected women indicates involvement of additional alterations, primarily CNVs. Here, we showed how CNVs played a crucial role in development of advanced tumors (stage III/IV) in Indian patients. METHODS: Initially, high-resolution CGH-SNP microarray analysis pointed out frequent CNVs followed by significantly altered genes. After comparison with TCGA dataset, expressions of the genes were checked in three CSCC datasets to identify key genes followed by Ingenuity® Pathway analysis. Then node effect property analysis was applied on the constructed PPI network to rank the key proteins. Finally, validations in independent samples were performed. RESULTS: For the first time, frequent chromosomal amplifications at 3q13.13-3q29, 1p36.11-1p31.1, 1q21.1-1q44 and 5p15.33-5p12 followed by common deletions at 11q14.1-11q25, 2q34-2q37.3, 4p16.3-4p12 and 13q13.3-13q14.3 were identified in Indian CSCC patients. Integrative analysis found 78 key genes including several novel ones, which were mostly associated with 'Cancer' and may regulate DNA repair and metabolic pathways. Analysis showed PARP1 and ATR were among the top ranking protein interactors. CONCLUSIONS: Frequent amplification and over-expression of ATR and PARP1 were further confirmed in cervical lesions, indicating their association with poor prognosis of advanced CSCC patients. GENERAL SIGNIFICANCE: Our novel approach identified precise CNVs along with several novel genes within these loci and showed that PARP1 and ATR, having biologically significant interactions, may be involved in development of advanced CSCC.

Genetic dissection of drought and heat tolerance in chickpea through genome-wide and candidate gene-based association mapping approaches.

To understand the genetic basis of tolerance to drought and heat stresses in chickpea, a comprehensive association mapping approach has been undertaken. Phenotypic data were generated on the reference set (300 accessions, including 211 mini-core collection accessions) for drought tolerance related root traits, heat tolerance, yield and yield component traits from 1-7 seasons and 1-3 locations in India (Patancheru, Kanpur, Bangalore) and three locations in Africa (Nairobi, Egerton in Kenya and Debre Zeit in Ethiopia). Diversity Array Technology (DArT) markers equally distributed across chickpea genome were used to determine population structure and three sub-populations were identified using admixture model in STRUCTURE. The pairwise linkage disequilibrium (LD) estimated using the squared-allele frequency correlations (r2; when r2<0.20) was found to decay rapidly with the genetic distance of 5 cM. For establishing marker-trait associations (MTAs), both genome-wide and candidate gene-sequencing based association mapping approaches were conducted using 1,872 markers (1,072 DArTs, 651 single nucleotide polymorphisms [SNPs], 113 gene-based SNPs and 36 simple sequence repeats [SSRs]) and phenotyping data mentioned above employing mixed linear model (MLM) analysis with optimum compression with P3D method and kinship matrix. As a result, 312 significant MTAs were identified and a maximum number of MTAs (70) was identified for 100-seed weight. A total of 18 SNPs from 5 genes (ERECTA, 11 SNPs; ASR, 4 SNPs; DREB, 1 SNP; CAP2 promoter, 1 SNP and AMDH, 1SNP) were significantly associated with different traits. This study provides significant MTAs for drought and heat tolerance in chickpea that can be used, after validation, in molecular breeding for developing superior varieties with enhanced drought and heat tolerance.

Genetic and epigenetic changes of HPV16 in cervical cancer differentially regulate E6/E7 expression and associate with disease progression.

OBJECTIVE: The study was aimed at understanding the complex interactions of genetic and epigenetic events in expression of HPV16 E6/E7 and progression of cervical carcinoma. For this, expression of E6/E7 was done in 36 samples, along with the physical status, methylation and LCR sequence variations. Later, the genetic and epigenetic studies were extended to 239 samples to find out the association of these factors with progression of cervical cancer. METHODS: E6/E7 expression was quantified by real-time PCR. Physical status of HPV16 was determined by mutiplex-PCR of whole E2 ORF using overlapping primers and E6 ORF and validated by real-time PCR. Methylation status of P97 promoter/enhancer was analyzed by methylation sensitive restriction analysis (MSRA). Viral lineage and variations in LCR was ascertained by sequencing LCR/E6/E7 ORFs. RESULTS: Samples with episomal unmethylated virus showed comparatively high expression of E6/E7 than episomal methylated, integrated unmethylated and integrated methylated forms of HPV16. Variations in the LCR, particularly in the binding sites of negatively regulating transcription factors, also contribute to high expression of E6/E7. The integrated form significantly increases with decrease of episomal form during tumor progression. Methylation of the promoter/enhancer gradually decreased with tumor progression and is inversely correlated to integration. Two novel variants were observed in E6 gene in European- and North-American-1-lineages. Log-rank test revealed better prognosis of the patients with episomal methylated HPV16 compared to the other forms. CONCLUSION: Our results show higher expression of E6/E7 in samples with episomal unmethylated virus having sequence variations in LCR.

Alterations of ATM and CADM1 in chromosomal 11q22.3-23.2 region are associated with the development of invasive cervical carcinoma.

To understand the importance of chr11q22.3-23.2 region in the development of cervical cancer, we have studied the genetic and epigenetic alterations of the candidate genes ATM, PPP2R1B, SDHD and CADM1 in cervical intraepithelial neoplasia (CIN) and cervical carcinoma (CACX) samples. Our study revealed low expression and high alterations (methylation/deletion) (55-59%) of ATM and CADM1 genes along with poor patient outcome. The alterations of ATM and CADM1 are associated with the progression of tumor from CIN to Stage I/II, thus implying their role in early invasiveness. The two genes, PPP2R1B and SDHD, lying in between ATM and CADM1, have low frequency of alterations, and majority of the alterations are in CACX samples, indicating that their alterations might be associated with disease progression. Expressions (mRNA/protein) of the genes showed concordance with their molecular alterations. Significant co-alteration of ATM and CADM1 points to their synergic action for the development of CACX. Mutation is, however, a rare phenomenon for inactivation of ATM. Association between the alteration of ATM and CHEK1 and poor survival of the patients having co-alterations of ATM and CHEK1 points to the DNA damage response pathway disruption in development of CACX. Thus, our data suggest that inactivation of ATM-CHEK1-associated DNA damage response pathway and CADM1-associated signaling network might have an important role in the development of CACX.

Inactivation of CHEK1 and EI24 is associated with the development of invasive cervical carcinoma: clinical and prognostic implications.

To understand the importance of frequent deletion of chromosomal 11q23.3-24.3 region in cervical carcinogenesis, alterations (deletion/methylation/mutation/expression) of the candidate genes LOH11CR2A, EI24 and CHEK1 located in the region were analyzed in 29 cervical intraepithelial neoplasia (CIN), 112 cervical carcinoma (CACX) samples and two CACX cell lines. The deletion frequency of these genes was low in CIN than in CACX [CIN: CHEK1: 28%, EI24: 21%, LOH11CR2A: 15% and CACX: CHEK1: 51%, EI24: 41%, LOH11CR2A: 36%]. Similar trend was seen in promoter methylation of these genes [CIN: CHEK1: 10%, EI24: 3%, LOH11CR2A: 3% and CACX: CHEK1: 55%, EI24: 31%, LOH11CR2A: 14%]. Mutations of the genes are a rare event. Overall alterations (deletion and methylation) of CHEK1 and EI24 were associated with progression of CACX. Quantitative mRNA expression analysis showed reduced expression of the three genes in concordance to their molecular alterations. A shorter isoform of CHEK1 lacking exon 8, hence impaired in substrate binding capacity, was found in two samples. Immunohistochemical analysis showed nuclear expression of Chek1, p-Chek1 and Ei24 in tumor tissues, whereas the cell lines exhibited both nuclear and cytoplasmic expression of Chek1 and Ei24, as is also evident from Western blot analysis suggesting differential localization of the proteins. Alterations of CHEK1 and EI24 coupled with tumor stage and early sexual debut (≤ 19 years) predicted worst prognosis. Thus, our data suggest that inactivation of EI24 and CHEK1 through two independent mechanisms contributes to the development of CACX.

Cervical Cancer Working Group report.

Disease burden of cervical cancer in Asia was summarized. Human papillomavirus 16 is the most oncogenic human papillomavirus type. Korea's national cervical cancer screening program targets women aged 30 or over, with coverage of almost 80%. Japan has a long history (50 years) of cervical cancer screening, and cytological screening programs have reduced the incidence/mortality of cervical cancer by 70%. But, recent cervical cancer screening coverage is ∼24%. Modeling suggested that vaccination of all 12-year-old girls would reduce cervical cancer cases by 73% in Japan. India has no cervical cancer screening program, as well as a serious lack of awareness in the general population, medical professionals and policy-makers. A realistic, affordable approach would be a low-volume, once-in-a-lifetime human papillomavirus-based screening program. In Australia, the national cervical cancer program has been very successful in reducing the incidence and mortality of cervical cancer. Australia was the first country to implement free, national human papillomavirus immunization (April 2007), expected to reduce human papillomavirus 16 infections by 56% in 2010 and 92% in 2050. A comparison of the UK and Japan was demonstrated that in the UK, cervical cancer screening and human papillomavirus vaccination uptakes are high because the government provides adequate education/funding. The Japanese government needs to put more emphasis on women's health and preventative medicine. Our conclusion and recommendations are that heightened public awareness of cervical cancer prevention, focusing on screening and vaccination will lead to improved survival and a better quality of life.

Deletions in chromosome 4 differentially associated with the development of cervical cancer: evidence of slit2 as a candidate tumor suppressor gene.

The aim of this study was to locate the candidate tumor suppressor genes (TSGs) loci in the chromosomal 4p15-16, 4q22-23 and 4q34-35 regions associated with the development of uterine cervical carcinoma (CA-CX). Deletion mapping of the regions by microsatellite markers identified six discrete areas with high frequency of deletions, viz. 4p16.2 (D1: 40%), 4p15.31 (D2: 35-38%), 4p15.2 (D3: 37-40%), 4q22.2 (D4: 34%), 4q34.2-34.3 (D5: 37-59%) and 4q35.1 (D6: 40-50%). Significant correlation was noted among the deleted regions D1, D2 and D3. The deletions in D1, D2, D5 and D6 regions are suggested to be associated with the cervical intraepithelial neoplasia (CIN), and deletions in the D2, D3, D5 and D6 regions seems to be associated with progression of CA-CX. The deletions in the D2 and D6 regions showed significant prognostic implications (P = 0.001; 0.02). The expression of the candidate TSG SLIT2 mapped to D2 region gradually reduced from normal cervix uteri -->CIN --> CA-CX. SLIT2 promoter hypermethylation was seen in 28% CIN samples and significantly increased with tumor progression (P = 0.04). Significant correlation was seen between SLIT2 deletion and its promoter methylation (P = 0.001), indicating that both these phenomena could occur simultaneously to inactivate this gene. Immunohistochemical analysis showed reduced expression of SLIT2 in cervical lesions and CA-CX cell lines. Although no mutation was detected in the SLIT2 promoter region (-432 to + 55 bp), CC and AA haplotypes were seen in -227 and -195 positions, respectively. Thus, it indicates that inactivation of SLIT2-ROBO1 signaling pathway may have an important role in CA-CX development.

Grand challenges in global health and the practical prevention program? Asian focus on cancer prevention in females of the developing world.

In response to the request for 'Breakthrough Questions' for 'Grand Challenges in Global Health' recently published in Nature, the Asian Pacific Organization for Cancer Prevention should focus its attention on what projects are of the highest priority for integration with its Practical Prevention Program (PPP). The most common female cancers in most of the countries of Asia are carcinoma of the breast, followed by the uterine cervix. While the incidences of breast adenocarcinomas are still generally lower than in the Western world they are rapidly increasing, and squamous cell carcinomas of the cervix are a major problem. Clearly there are many areas which would reward research. One factor which appears of major relevance in the mammary gland case is the diet, and particularly the phytoestrogens included in 'tofu', along with physical exercise. The age at which these could be operating needs to be elucidated, with reference to timing of menarche and menopause, and also breast mammographic density, another predictor of likelihood of neoplasia. In the cervix, the predominant influence is well established to be persistent infection with a high risk 'oncogenic' type of human papilloma virus (HPV). Vaccines therefore hold much promise, but a better understanding of the mechanisms underlying spontaneous clearance of both infection and cervical intraepithelial neoplasia (CIN) of different grades is also essential for optimal intervention. The roles of smoking and antioxidant intake in particular deserve emphasis. In Asia, with the considerable variation evident in both breast and cervical cancer incidence rates, as well as in cultural and other environmental factors, we are in a very favourable position to meet two specific challenges: 1). elucidation of how diet in adolescence determines susceptibility to neoplasia of the mammary glands; and 2). determination of what governs persistence of HPV infection. Realisation of these pivotal research aims, with especial emphasis on the context of the PPP, is our shared goal.

Cervical Cancer: Etiology, Pathogenesis, Treatment, and Future Vaccines.

Cervical cancer is a sexually transmitted disease caused by the human papillomavirus (HPV), especially HPV-16 and -18. Of the half million new cases of cervical cancer reported yearly, 20% occur in India. Mass cancer screening programs to detect and treat cervical cancer and its precursor lesions are not available in India and most other developing countries because of the lack of resources. Curative and palliative treatments are not the same for all patients with cervical cancer because the result depends on the immunological response of the patient. This article describes the natural history of cervical carcinogenesis and the rational behind various modalities of prevention and treatment for the practising gynecological oncologist. Prophylactic vaccines against HPV-16 and -18 and therapeutic vaccines against cervical cancers should be able to overcome the logistical problems that now exist to screen, diagnose and treat cervical cancer and its precursor lesions.

Response of Cervical Intra-epithelial Lesions to Vitamin E Supplementation - A Preliminary Report.

Carcinoma of the uterine cervix is preceded by well characterized pre-cancerous lesions which if left untreated may progress to invasive carcinoma. In the present study women in the age group of 35-55 years with cervical intraepithelial lesions (CIN I & II) were treated with vitamin E and advised to come for follow up after every three months for one year. This preliminary report shows vitamin E can restrict and regress CIN I & II lesions with elevation in circulating vitamin E levels. Improvement in immune status as reflected in mitogenic responses of peripheral blood lymphocytes was also noted.