Rajeev K Varshney FRS

Present study aimed to identify DNA polymorphisms (variants) which can modulate the risk of COVID-19 infection progression to severe condition. TaqMan based SNP genotyping assay was performed for 11 single nucleotide polymorhisms (SNPs) in pro-coagulant and anti-coagulant genes. A total of 33 COVID-19 patients, including dead, severe and moderately infected individuals were compared to 35 healthy controls. Both alleles in the SNP were labeled with two different fluorescent dyes (FAM and VIC) during assay formulation. DNA of study subjects were mixed with SNP assay and TaqMan master mix on 96 well PCR plate according to manufacturer’s protocol and RT-PCR was performed. Allelic discrimination assay gave clear results for presence of specific allele in each sample. Three SNPs were located in the pro-coagulant genes, another three involved in blood clot dissolution while rest five were in the genes encoding natural anti-coagulants. COVID-19 infected patients were further sub-divided into three groups, deceased (n=16), severe (n=10) and moderately infected (n=7). Results of SNP genotyping showed significant difference between COVID-19 patients and controls in two SNPs, rs6133 in Selectin-P (SELP) and rs5361 in Selectin-E (SELE) gene. Also, rs2020921 and rs8176592, in clot dissolution genes, tissue Plasminogen activator (tPA) and tissue factor pathway inhibitor (TFPI) respectively showed significant genotypic and allelic difference in patients of COVID-19 compared to healthy controls. Further three SNPs rs2227589, rs757583846 and rs121918476 in natural anti-coagulant genes anti-thrombin III (ATIII), protein C (PROC) and protein S (PROS) respectively showed statistically significant difference between the study groups. Our findings indicate that gene variants, those involved in coagulation and anti-coagulation may play a major role in determining individual susceptibility to COVID-19. Funding Information: This work was supported by Defence Institute of Physiology and Allied Sciences (DIPAS), Defence Research and Development Organization (DRDO), Delhi-110054. Conflict of Interests: The authors declare that there is no conflict of interest or financial disclosure related to this publication. Ethical Approval: The study parameters and design was approved by the Ethical Committee of Indian Council of Medical Research (ICMR), India. Written informed consent was obtained from all study participants before collection of their blood sample. All experimental protocols were conducted in accordance to the Strengthening of the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.

FAD3 play important roles in modulating membrane fluidity in response to various abiotic stresses. However, a comprehensive analysis of FAD3 in drought, salinity and heat stress tolerance is lacking in soybean. The present study assessed the functional role of fatty acid desaturase 3 to abiotic stress responses in soybean. We used Bean Pod Mottle Virus -based vector to alter expression of Glycine max omega-3 fatty acid desaturase . Higher levels of recombinant BPMV-GmFAD3 transcripts were detected in overexpressing soybean plants. Overexpression of GmFAD3 in soybean resulted in increased levels of jasmonic acid and higher expression of GmWRKY54 as compared to mock-inoculated, vector-infected and FAD3-silenced soybean plants under drought and salinity stress conditions. FAD3 overexpressing plants showed higher levels of chlorophyll content, leaf SPAD value, relative water content, chlorophyll fluorescence, transpiration rate, carbon assimilation rate, proline content and also cooler canopy under drought and salinity stress conditions as compared to mock-inoculated, vector-infected and FAD3-silenced soybean plants. Results from current study revealed that GmFAD3 overexpressing soybean plants exhibited drought and salinity stress tolerance although tolerance to heat stress was reduced. On the other hand, soybean plants silenced for GmFAD3 exhibited tolerance to heat stress, but were vulnerable to drought and salinity stress

Across domains of biological research using genome sequence data, high-quality reference genome sequences are essential for characterizing genetic variation and understanding the genetic basis of phenotypes. However, the construction of genome assemblies for various species is often hampered by complexities of genome organization, especially repetitive and complex sequences, leading to mis-assembly and missing regions. Here, we describe a high-throughput gold standard genome assembly workflow using a large-scale bacterial artificial chromosome (BAC) library with a refined two-step pooling strategy and the Lamp assembler algorithm. This strategy minimizes the laborious processes of physical map construction and clone-by-clone sequencing, enabling inexpensive sequencing of several thousand BAC clones. By applying this strategy with a minimum tiling path BAC clone library for the short arm of chromosome 2D (2DS) of bread wheat, 98% of BAC sequences, covering 92.7% of the 2DS chromosome, were assembled correctly for this species with a highly complex and repetitive genome. We also identified 48 large mis-assemblies in the reference wheat genome assembly (IWGSC RefSeq v1.0) and corrected these large mis-assemblies in addition to filling 92.2% of the gaps in RefSeq v1.0. Our 2DS assembly represents a new benchmark for the assembly of complex genomes with both high accuracy and efficiency.

Aflatoxins, which have been classified as a group-1 carcinogen are the well-known mycotoxins produced by Aspergillus flavus. Aflatoxins have been linked to liver diseases, acute hepatic necrosis, resulting in cirrhosis or hepatocellular carcinomas due to which it incurs a loss of value in international trade for peanuts contaminated with it. The four main aflatoxins are B1, B2, G1, and G2 of which B1 is predominant. In plants, the cell wall is the primary barrier against pathogen invasion. Cell wall fortifications such as deposition of callose, cellulose, lignin, phenolic compounds and structural proteins help to prevent the pathogen infection. Further, the host cell’s ability to rapidly repair and reinforce its cell walls will result in a reduction of the penetration efficiency of the pathogen. Peanut seed coat acts as a physical and biochemical cell wall barrier against both pre and post-harvest pathogen infection. The structure of seed coat and the presence of polyphenol compounds have been reported to inhibit the growth of A. flavus, however, not successfully employed to develop A. flavus resistance in peanut. A comprehensive understanding of peanut seed coat development and biochemistry will provide information to design efficient strategies for the seed coat mediated A. flavus resistance and Aflatoxin contamination.

Nested association mapping (NAM) is a valuable innovation and multi-parental mapping population strategy in peanut genetics which increases the power to map quantitative trait loci and assists in extending the gene pool of elite peanut lines. In the peanut research community, two structured mapping populations were developed using a 2 × 8 (common by unique) factorial nested association mapping design, each with eight founders and a reference line. Here, we demonstrate its usefulness by assessing the phenotypic diversity of two assembled NAM populations (2 × 4). The common parents are Tifrunner and Florida-07 while the four unique parents are N08082oilct, C76-16, NC3033 and SPT06-06. We initially screened the phenotypic characteristics of the RILs including morphological and disease resistance traits. We found that leaf length and width, plant size, main stem height, and leaf spot resistance segregated within the assembled population and exhibited normal distributions. We also calculated the variance and heritability of each trait, and found that plant size had the lowest narrow sense heritability (0.06) while disease resistance had the highest (0.67) in the Tifrunner NAM population. In the Florida-07 population, main stem height had the lowest (0.27) and leaf width had the highest (0.73). Phenotyping of pod and kernel traits is underway along with further genotyping by sequencing. The NAM concept will promote the evaluation of the genetic diversity present in peanut gene pool.

In peanut, limited genetic variation for disease resistance is available in breeding programs necessitating the identification of stable resistance sources for use in cultivar development. ‘Tifrunner’ is a runner cultivar while ‘GT-C20’ is a Spanish-type breeding line with resistance to aflatoxin contamination. We screened a RIL population generated from Tifrunner × GT-C20 for aflatoxin contamination resistance using a laboratory kernel screening assay. We scored visible fungal growth on kernels, and aflatoxin content using a fast methanol extraction method and a fluorometer (verified by TLC and ELISA). This phenotypic screening was repeated three times, each with three biological replicates. We found that surface sporulation ratings were normally distributed across the population. Aflatoxin levels varied among the RILs with a distribution skewed toward lower aflatoxin levels. Quantitative trait locus (QTL) analysis for all replicates identified a total of 16 QTLs, five QTLs for aflatoxin contamination and eleven for fungal growth. Of these, five QTLs were major QTLs with more than 10% phenotypic variation explained (PVE). From these five major QTLs, one was found for aflatoxin contamination with 10.14% PVE, and three QTLs for the fungal growth (PVE = 13.23%, 11.21% and 14.17%, respectively). These novel QTLs will be compared with recently released peanut diploid genome to identify putative resistance genes, and validated for potential applications in breeding.

Aspergillus flavus and aflatoxin contamination in the field are known to be influenced by numerous stress factors, particularly drought and heat stress. However, the purpose of aflatoxin production is unknown. Here, we report transcriptome analyses comprised of 282.6 Gb of sequencing data describing 11,144 of 13,487 (82.6%) annotated A. flavus genes, which provides the gene expression comparisons among different A. flavus isolates and between two culture media, aflatoxin conducive (sucrose) and non-conducive (peptone). We identified genes that are differentially expressed (DEGs) in response to oxidative stress in media with H2O2. Isolates tolerating greater levels of oxidative stress exhibited fewer DEGs in comparison to those with less tolerance (r = -0.6). We found that proteolytic genes were more expressed in the non-conducive medium, while carbohydrate catabolic and glucose transporter genes (e.g. MFS transporters) were more expressed in the conducive medium. Among the observed DEGs, components of polyketide (aflatoxin) and isoprenoid (aflatrem) secondary metabolite production along with kojic acid biosynthesis and monooxygenase genes were prevalent. To our knowledge, this is the first study that explores the molecular responses of different A. flavus isolates to H2O2-induced stress in different culture media. Together, these results demonstrate a potential key role for secondary metabolite production in A. flavus oxidative stress responses.

Aflatoxin contamination by Aspergillus flavus is exacerbated by drought stress in the field. Given that reactive oxygen species (ROS) both accumulate in plant tissues during drought and can stimulate aflatoxin production in vitro, we examined the responses of toxigenic isolates of A. flavus to oxidative stress focusing on secondary metabolite production using whole transcriptome sequencing. We examined two high toxin producing isolates, Tox4 and AF13, and one moderate producer, NRRL3357, in aflatoxin conducive yeast extract sucrose medium amended with various levels of H2O2. The high producers exhibited fewer differentially expressed genes (DEGs) than the moderate producer in response to increasing stress. Altered aminobenzoate degradation expression along with altered conidiation and growth also suggests reduced developmental rates in the isolates caused by oxidative stress. Aflatoxin genes were upregulated in response to increasing stress along with genes encoding aflatrem and kojic acid biosynthesis. Additional changes in primary metabolic genes also indicates adaptations to increasing stress. Given that the number and diversity of DEGs observed correlated with previously observed aflatoxin production and oxidative stress tolerance for each isolate, these data suggest that secondary metabolite production is involved in A. flavus oxidative responses. Continuing proteomic analyses using gel and iTRAQ methods are underway to confirm the transcriptome results.

Pigeonpea [Cajanus cajan (L) Millsp] bi-parental populations segregating for various traits of interest are being developed. The three most advanced populations, named PRIL_A, PRIL_B and PRIL_C (Pigeonpea Recombinant Inbred Line, population A, B and C) have reached F6 generation. PRIL_A: derived from the cross ICPB 2049 x ICPL 99050 segregates for fusarium wilt (FW), 329 lines. PRIL_B: derived from the cross ICPL 20096 x ICPL 332 segregates for FW and sterility mosaic disease (SMD), 342 lines. PRIL_C: derived from the cross ICPL 20097 × ICP 8863 segregates for SMD, 366 lines. Marker genotyping of the parental lines, however, showed low level of genetic variation. After screening over 4,616 (3,000 simple sequence repeats (SSRs) and 1,616 single nucleotide polymorphism (SNPs)) markers on parental genotypes of each mapping population, a total of 159 (104 SSRs and 55 SNPs), 80 (52 SSRs and 28 SNPs) and 157 (143 SSRs and 14 SNPs) markers were found polymorphic for ICPB 2049 vs ICPL 99050, ICPL 20096 vs ICPL 332 and ICPL 20097 vs ICP 8863, respectively. The polymorphic markers will be used for constructing genetic linkage maps. The populations will be screened for FW and SMD in 2012-13, while marker-trait association analysis will also be conducted to understand the genetic basis of resistance to these diseases. Further selection from the above indicated mapping populations during 2011-12, on an effort initiated in 2010, resulted in 28 lines with high yield (up to 2.4 t/ha) and disease resistance