Precision mapping and expression analysis of recessive bacterial blight resistance gene xa-45(t) from Oryza glaberrima.

BACKGROUND: Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases of rice leading to huge yield losses in Southeast Asia. The recessive resistance gene xa-45(t) from Oryza glaberrima IRGC102600B, mapped on rice chromosome 8, spans 80 Kb with 9 candidate genes on Nipponbare reference genome IRGSP-1.0. The xa-45(t) gene provides durable resistance against all the ten Xanthomonas pathotypes of Northern India, thus aiding in the expansion of recessive bacterial blight resistance gene pool. Punjab Rice PR127, carrying xa-45(t), was released for wider use in breeding programs. This study aims to precisely locate the target gene among the 9 candidates conferring resistance to bacterial blight disease. METHODS AND RESULTS: Sanger sequencing of all nine candidate genes revealed seven SNPs and an Indel between the susceptible parent Pusa 44 and the resistant introgression line IL274. The genotyping with polymorphic markers identified three recombinant breakpoints for LOC_Os08g42370, and LOC_Os08g42400, 15 recombinants for LOC_Os08g423420 and 26 for LOC_Os08g42440 out of 190 individuals. Relative expression analysis across six time intervals (0, 8, 24, 48, 72, and 96 h) after bacterial blight infection showed over expression of LOC_Os08g42410-specific transcripts in IL274 compared to Pusa 44, with a significant 4.46-fold increase observed at 72 h post-inoculation. CONCLUSIONS: The Indel marker at the locus LOC_Os08g42410 was found co-segregating with the phenotype, suggesting its candidacy towards xa-45(t). The transcript abundance assay provides strong evidence for the involvement of LOC_Os08g42410 in the resistance conferred by the bacterial blight gene xa-45(t).

Effect of irrigation on wild and inbred maize with relation to the antioxidant status of pollens, flag leaves, and developing grains.

The investigation was carried out to evaluate the net effect of limited irrigation on the antioxidant status of pollens, flag leaves, and developing grains of wild and inbred maize lines. Teosinte pollens showed the highest activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-s-transferase (GST), and peroxidase (POX) under stressful conditions while LM 11 showed a significant decrease in APX, CAT, GR, and GST activities. Limited irrigations increased the contents of superoxide and malondialdehyde (MDA) to maximum levels in LM 11 leaves. The pollens, leaves, and developing grains of teosinte had the highest content of total phenols. Proline was maximum in the developing grains of teosinte and CML 32 while lowest in those of LM 11. Principal component analysis showed that LM 11 genotype and the respective antioxidant enzymes were in completely opposite quadrants. Chord analysis showed that CAT activity and total phenol content in pollens, leaves, and developing grains contributed towards most of the variations observed in teosinte and might be responsible for managing the yield attributes of genotype during stress conditions. The pollens and leaves of teosinte, with significant SOD activity, further helped in optimizing plant yield, under stressful conditions. CML 32 occupied intermediate position owing to the unaffected activities of most of the antioxidant enzymes and high content of antioxidants in its tissues. It may be concluded that the overall antioxidant status of tissues decides the tolerance behavior of plants.

Silage maize as a potent candidate for sustainable animal husbandry development-perspectives and strategies for genetic enhancement.

Maize is recognized as the queen of cereals, with an ability to adapt to diverse agroecologies (from 58oN to 55oS latitude) and the highest genetic yield potential among cereals. Under contemporary conditions of global climate change, C4 maize crops offer resilience and sustainability to ensure food, nutritional security, and farmer livelihood. In the northwestern plains of India, maize is an important alternative to paddy for crop diversification in the wake of depleting water resources, reduced farm diversity, nutrient mining, and environmental pollution due to paddy straw burning. Owing to its quick growth, high biomass, good palatability, and absence of anti-nutritional components, maize is also one of the most nutritious non-legume green fodders. It is a high-energy, low-protein forage commonly used for dairy animals like cows and buffalos, often in combination with a complementary high-protein forage such as alfalfa. Maize is also preferred for silage over other fodders due to its softness, high starch content, and sufficient soluble sugars required for proper ensiling. With a rapid population increase in developing countries like China and India, there is an upsurge in meat consumption and, hence, the requirement for animal feed, which entails high usage of maize. The global maize silage market is projected to grow at a compound annual growth rate of 7.84% from 2021 to 2030. Factors such as increasing demand for sustainable and environment-friendly food sources coupled with rising health awareness are fueling this growth. With the dairy sector growing at about 4%-5% and the increasing shortage faced for fodder, demand for silage maize is expected to increase worldwide. The progress in improved mechanization for the provision of silage maize, reduced labor demand, lack of moisture-related marketing issues as associated with grain maize, early vacancy of farms for next crops, and easy and economical form of feed to sustain household dairy sector make maize silage a profitable venture. However, sustaining the profitability of this enterprise requires the development of hybrids specific for silage production. Little attention has yet been paid to breeding for a plant ideotype for silage with specific consideration of traits such as dry matter yield, nutrient yield, energy in organic matter, genetic architecture of cell wall components determining their digestibility, stalk standability, maturity span, and losses during ensiling. This review explores the available information on the underlying genetic mechanisms and gene/gene families impacting silage yield and quality. The trade-offs between yield and nutritive value in relation to crop duration are also discussed. Based on available genetic information on inheritance and molecular aspects, breeding strategies are proposed to develop maize ideotypes for silage for the development of sustainable animal husbandry.

DNA-free genome editing for ZmPLA1 gene via targeting immature embryos in tropical maize.

Doubled haploid (DH) production accelerates the development of homozygous lines in a single generation. In maize, haploids are widely produced by the use of haploid inducer Stock 6, earlier reported in 1959. Three independent studies reported haploid induction in maize which is triggered due to a 4 bp frame-shift mutation in matrilineal (ZmPLA1) gene. The present study was focused on the generation of mutants for ZmPLA1 gene in maize inbred line LM13 through site-directed mutagenesis via CRISPR/Cas9-mediated ribonucleoprotein (RNP) complex method to increase the haploid induction rate. Three single guide RNAs (sgRNAs) for the ZmPLA1 gene locus were used for transforming the 14 days old immature embryos via bombardment. 373 regenerated plants were subjected to mutation detection followed by Sanger's sequencing. Out of three putative mutants identified, one mutant depicted one base pair substitution and one base pair deletion at the target site.

Genome-Wide Meta-Analysis of QTLs Associated with Root Traits and Implications for Maize Breeding.

Root system architecture (RSA), also known as root morphology, is critical in plant acquisition of soil resources, plant growth, and yield formation. Many QTLs associated with RSA or root traits in maize have been identified using several bi-parental populations, particularly in response to various environmental factors. In the present study, a meta-analysis of QTLs associated with root traits was performed in maize using 917 QTLs retrieved from 43 mapping studies published from 1998 to 2020. A total of 631 QTLs were projected onto a consensus map involving 19,714 markers, which led to the prediction of 68 meta-QTLs (MQTLs). Among these 68 MQTLs, 36 MQTLs were validated with the marker-trait associations available from previous genome-wide association studies for root traits. The use of comparative genomics approaches revealed several gene models conserved among the maize, sorghum, and rice genomes. Among the conserved genomic regions, the ortho-MQTL analysis uncovered 20 maize MQTLs syntenic to 27 rice MQTLs for root traits. Functional analysis of some high-confidence MQTL regions revealed 442 gene models, which were then subjected to in silico expression analysis, yielding 235 gene models with significant expression in various tissues. Furthermore, 16 known genes viz., DXS2, PHT, RTP1, TUA4, YUC3, YUC6, RTCS1, NSA1, EIN2, NHX1, CPPS4, BIGE1, RCP1, SKUS13, YUC5, and AW330564 associated with various root traits were present within or near the MQTL regions. These results could aid in QTL cloning and pyramiding in developing new maize varieties with specific root architecture for proper plant growth and development under optimum and abiotic stress conditions.

Transcriptional dynamics of maize leaves, pollens and ovules to gain insights into heat stress-related responses.

Heat stress (HS) is one of the alarming issues today due to global warming and is the foremost detrimental to crop production. Maize is one of the versatile crops grown over different agro-climatic conditions. However, it is significantly sensitive to heat stress, especially during the reproductive phase. The heat stress tolerance mechanism is yet to be elucidated at the reproductive stage. Thus, the present study focused on identifying transcriptional changes in two inbreds, LM 11 (sensitive to HS) and CML 25 (tolerant to HS), under intense heat stress at 42°C during the reproductive stage from three tissues viz. flag leaf, tassel, and ovule. Samples from each inbred were collected after 5 days of pollinations for RNA isolation. Six cDNA libraries were constructed from three separate tissues of LM 11 and CML 25 and sequenced using an Illumina HiSeq2500 platform. A total of 2,164 (1127 up-regulated and 1037 down-regulated) differentially expressed genes (DEGs) were identified with 1151, 451, and 562 DEGs in comparisons of LM 11 and CML 25, corresponding to a leaf, pollen, and ovule, respectively. Functional annotated DEGs associated with transcription factors (TFs) viz. AP2, MYB, WRKY, PsbP, bZIP, and NAM, heat shock proteins (HSP20, HSP70, and HSP101/ClpB), as well as genes related to photosynthesis (PsaD & PsaN), antioxidation (APX and CAT) and polyamines (Spd and Spm). KEGG pathways analyses showed that the metabolic overview pathway and secondary metabolites biosynthesis pathway, with the involvement of 264 and 146 genes, respectively, were highly enriched in response to heat stress. Notably, the expression changes of the most common HS-responsive genes were typically much more significant in CML 25, which might explain why CML 25 is more heat tolerant. Seven DEGs were common in leaf, pollen, and ovule; and involved in the polyamines biosynthesis pathway. Their exact role in maize heat stress response would warrant further studies. These results enhanced our understanding to heat stress responses in maize.

Inheritance and molecular tagging of genes introgressed from Gossypium arboreum to G. hirsutum for leafhopper tolerance.

Cotton cultivation is conquered by transgenic Bt upland cotton hybrids in India. Bt gene does not provide resistance against sucking insect pests. Due to the inherent vulnerability of extant Bt cotton hybrids to sap-sucking insect pests including leafhopper, upland cotton cultivation is seriously threatened by surging populations of these pests. Consistent and extensive screening of upland cotton germplasm over the years has revealed absence of adequate resistance against leafhopper. Here, we report introgression of leafhopper tolerance from a diploid A-genome cotton species, Gossypium arboreum into G. hirsutum. The dominance of leafhopper tolerance was observed over its susceptibility. Genetic analysis revealed that tolerance to leafhopper was inherited in a simple Mendelian fashion and was controlled by two genes, either singly or in combination. Using bulked segregant analysis, two simple-sequence repeat markers, namely NAU 922 and BNL 1705, located on chromosomes A5 and A11 respectively, were tagged with leafhopper tolerance. To the best of our knowledge, this is the first report of molecular tagging of leafhopper tolerance introgressed from G. arboreum into G. hirsutum. A significant negative association was observed between leaf trichome density and leafhopper nymph population.

2Gs and plant architecture: breaking grain yield ceiling through breeding approaches for next wave of revolution in rice (Oryza sativa L.).

Rice is a principal food crop for more than half of the global population. Grain number and grain weight (2Gs) are the two complex traits controlled by several quantitative trait loci (QTLs) and are considered the most critical components for yield enhancement in rice. Novel molecular biology and QTL mapping strategies can be utilized in dissecting the complex genetic architecture of these traits. Discovering the valuable genes/QTLs associated with 2Gs traits hidden in the rice genome and utilizing them in breeding programs may bring a revolution in rice production. Furthermore, the positional cloning and functional characterization of identified genes and QTLs may aid in understanding the molecular mechanisms underlying the 2Gs traits. In addition, knowledge of modern genomic tools aids the understanding of the nature of plant and panicle architecture, which enhances their photosynthetic activity. Rice researchers continue to combine important yield component traits (including 2Gs for the yield ceiling) by utilizing modern breeding tools, such as marker-assisted selection (MAS), haplotype-based breeding, and allele mining. Physical co-localization of GW7 (for grain weight) and DEP2 (for grain number) genes present on chromosome 7 revealed the possibility of simultaneous introgression of these two genes, if desirable allelic variants were found in the single donor parent. This review article will reveal the genetic nature of 2Gs traits and use this knowledge to break the yield ceiling by using different breeding and biotechnological tools, which will sustain the world's food requirements.

BSA-seq Identifies a Major Locus on Chromosome 6 for Root-Knot Nematode (Meloidogyne graminicola) Resistance From Oryza glaberrima.

Root-knot nematode (Meloidogyne graminicola) is one of the emerging threats to rice production worldwide that causes substantial yield reductions. There is a progressive shift of the cropping system from traditional transplanting to direct-seeded water-saving rice production that favored the development of M. graminicola. Scouting and deploying new resistance genes is an economical approach to managing the root-knot nematodes. Here, we report that the inheritance of root-knot nematode resistance in Oryza glaberrima acc. IRGC102206 is governed by a single dominant gene. Traditional mapping coupled with BSA-seq is used to map nematode resistance gene(s) using the BC1F1 population derived from a cross of O. sativa cv. PR121 (S) and O. glaberrima acc. IRGC102206 (R). One major novel genomic region spanning a 3.0-Mb interval on chromosome 6 and two minor QTLs on chromosomes 2 and 4 are the potential genomic regions associated with rice root-knot nematode resistance. Within the QTL regions, 19 putative candidate genes contain 81 non-synonymous variants. The detected major candidate region could be fine mapped to accelerate marker-assisted breeding for root-knot nematode resistance in rice.

A Prospective Review on Selectable Marker-Free Genome Engineered Rice: Past, Present and Future Scientific Realm.

As a staple food crop, rice has gained mainstream attention in genome engineering for its genetic improvement. Genome engineering technologies such as transgenic and genome editing have enabled the significant improvement of target traits in relation to various biotic and abiotic aspects as well as nutrition, for which genetic diversity is lacking. In comparison to conventional breeding, genome engineering techniques are more precise and less time-consuming. However, one of the major issues with biotech rice commercialization is the utilization of selectable marker genes (SMGs) in the vector construct, which when incorporated into the genome are considered to pose risks to human health, the environment, and biodiversity, and thus become a matter of regulation. Various conventional strategies (co-transformation, transposon, recombinase systems, and MAT-vector) have been used in rice to avoid or remove the SMG from the developed events. However, the major limitations of these methods are; time-consuming, leftover cryptic sequences in the genome, and there is variable frequency. In contrast to these methods, CRISPR/Cas9-based marker excision, marker-free targeted gene insertion, programmed self-elimination, and RNP-based delivery enable us to generate marker-free engineered rice plants precisely and in less time. Although the CRISPR/Cas9-based SMG-free approaches are in their early stages, further research and their utilization in rice could help to break the regulatory barrier in its commercialization. In the current review, we have discussed the limitations of traditional methods followed by advanced techniques. We have also proposed a hypothesis, "DNA-free marker-less transformation" to overcome the regulatory barriers posed by SMGs.

Molecular mapping and transfer of a novel brown planthopper resistance gene bph42 from Oryza rufipogon (Griff.) To cultivated rice (Oryza sativa L.).

BACKGROUND: Brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most destructive pests of rice accounting for 52% of annual yield loss. The breakdown of resistance against known BPH biotypes necessitates the identification and deployment of new genes from diverse sources. The current study aimed at mapping and transfer of a novel BPH resistance gene from the wild species of rice O. rufipogon accession CR100441 to the elite rice cultivar against BPH biotype 4. METHODS AND RESULTS: The phenotypic screening against BPH biotype 4 was conducted using the standard seedbox screening technique (SSST). Inheritance study using damage score caused by BPH infestation at the seedling stage indicated the presence of a single major recessive gene with the segregation ratio of susceptible to resistant plants in 3:1 (210:66, χ2c = 0.17 ≤ χ20.05,1 = 3.84). The genotyping of the mapping population was done using polymorphic microsatellite markers between PR122 and O.rufipogon acc.CR100441 spanning all the 12 chromosomes of rice. A total of 537 SSR markers were used to map a BPH resistance gene (designated as bph42) on the short arm of chromosome 4 between RM16282 and RM6659. QTL analysis identified a peak marker RM16335 contributing 29% of the phenotypic variance at 40.76 LOD. CONCLUSIONS: The identified marker co-segregates with the bph42 and hence could be efficiently used for marker-assisted selection (MAS) for the transfer of resistance into elite rice cultivars. The introgression lines with higher yield and BPH resistance were identified and are under advanced yield trails for further varietal release.

Introgressing cry1Ac for Pod Borer Resistance in Chickpea Through Marker-Assisted Backcross Breeding.

The gram pod borer Helicoverpa armigera is a major constraint to chickpea (Cicer arietinum L.) production worldwide, reducing crop yield by up to 90%. The constraint is difficult to overcome as chickpea germplasm including wild species either lacks pod borer resistance or if possessing resistance is cross-incompatible. This study describes conversion of elite but pod borer-susceptible commercial chickpea cultivars into resistant cultivars through introgression of cry1Ac using marker-assisted backcross breeding. The chickpea cultivars (PBG7 and L552) were crossed with pod borer-resistant transgenic lines (BS 100B and BS 100E) carrying cry1Ac that led to the development of BC1F1, BC1F2, BC1F3, BC2F1, BC2F2, and BC2F3 populations from three cross combinations. The foreground selection revealed that 35.38% BC1F1 and 8.4% BC1F2 plants obtained from Cross A (PBG7 × BS 100B), 50% BC1F1 and 76.5% BC1F2 plants from Cross B (L552 × BS 100E), and 12.05% BC2F2 and 82.81% (average) BC2F3 plants derived from Cross C (PBG7 × BS 100E) carried the cry1Ac gene. The bioassay of backcross populations for toxicity to H. armigera displayed up to 100% larval mortality. BC1F1 and BC1F2 populations derived from Cross B and BC2F3 population from Cross C segregated in the Mendelian ratio for cry1Ac confirmed inheritance of a single copy of transgene, whereas BC1F1 and BC1F2 populations obtained from Cross A and BC2F2 population from Cross C exhibited distorted segregation ratios. BC1F1 plants of Cross A and Cross B accumulated Cry1Ac protein ranging from 11.03 to 11.71 µgg-1 in leaf tissue. Cry1Ac-positive BC2F2 plants from Cross C demonstrated high recurrent parent genome recovery (91.3%) through background selection using SSR markers and phenome recovery of 90.94%, amongst these 30% plants, were homozygous for transgene. The performance of BC2F3 progenies derived from homozygous plants was similar to that of the recurrent parent for main agronomic traits, such as number of pods and seed yield per plant. These progenies are a valuable source for H. armigera resistance in chickpea breeding programs.

High-resolution mapping of the quantitative trait locus (QTLs) conferring resistance to false smut disease in rice.

Rice false smut (RFS), an emerging major fungal disease worldwide caused by Ustilaginoidea virens, affects rice grain quality and yield. RFS cause 2.8-49% global yield loss depending upon disease severity and cultivars. In India, the yield loss due to RFS ranged from 2 to 75%. Identification of the genes or quantitative trait loci (QTLs) governing disease resistance would be of utmost importance towards mitigating the economic losses incurred due to RFS. Here, we report mapping of RFS resistance QTLs from a resistant breeding line RYT2668. The mapping population was evaluated for RFS resistance under the field condition in three cropping seasons 2013, 2015, and 2016. A positive correlation among infected panicle/plant, total smut ball/panicle, and disease score was observed in the years 2013, 2015, and the mean data. A total of seven QTLs were mapped on rice chromosomes 2, 4, 5, 7, and 9 using 2326 single nucleotide polymorphism markers. Of these, two QTLs, qRFSr5.3 and qRFSr7.1a, were associated with the infected panicle per plant, one QTL qRFsr9.1 with total smut ball per panicle, and four QTLs qRFSr2.2, qRFSr4.3, qRFSr5.4, and qRFSr7.1b with disease score. Among them, a novel QTL qRFSr9.1 on chromosome 9 exhibits the largest phenotypic effect. The prediction of putative candidate genes within the qRFSr9.1 revealed four nucleotide-binding sites-leucine-rich repeat (NBS-LRR) domain-containing disease resistance proteins. In summary, our findings mark the hotspot region of rice chromosomes carrying genes/QTLs for resistance to the RFS disease.

Chitosan-Urea Nanocomposite for Improved Fertilizer Applications: The Effect on the Soil Enzymatic Activities and Microflora Dynamics in N Cycle of Potatoes (Solanum tuberosum L.).

The impact of polymer-based slow-release urea formulations on soil microbial N dynamics in potatoes has been sparingly deciphered. The present study investigated the effect of a biodegradable nano-polymer urea formulation on soil enzymatic activities and microflora involved in the N cycling of potato (Solanum tuberosum L.). The nano-chitosan-urea composite (NCUC) treatment significantly increased the soil dehydrogenase activity, organic carbon content and available potassium compared to the conventional urea (CU) treatment. The soil ammonical nitrogen (NH4+-N) and nitrate nitrogen (NO3--N) contents and urease activity were significantly decreased in the NCUC-amended soil. The slow urea hydrolysis rate led to low concentrations of NH4+-N and NO3--N in the tested potato soil. Furthermore, these results corroborate the low count of ammonia oxidizer and nitrate reducer populations. Quantitative PCR (q-PCR) studies revealed that the relative abundance of eubacterial (AOB) and archaeal ammonia-oxidizing (AOA) populations was reduced in the NCUC-treated soil compared to CU. The abundance of AOA was particularly lower than AOB, probably due to the more neutral and alkaline conditions of the tested soil. Our results suggest that the biodegradable polymer urea composite had a significant effect on the microbiota associated with soil N dynamics. Therefore, the developed NCUC could be used as a slow N-release fertilizer for enhanced growth and crop yields of potato.

Marker-assisted pyramiding of lycopene-ε-cyclase, ß-carotene hydroxylase1 and opaque2 genes for development of biofortified maize hybrids.

Malnutrition affects growth and development in humans and causes socio-economic losses. Normal maize is deficient in essential amino acids, lysine and tryptophan; and vitamin-A. Crop biofortification is a sustainable and economical approach to alleviate micronutrient malnutrition. We combined favorable alleles of crtRB1 and lcyE genes into opaque2 (o2)-based four inbreds viz. QLM11, QLM12, QLM13, and QLM14 using marker-assisted backcross breeding. These are parents of quality protein maize versions of two elite hybrids viz. Buland and PMH1, grown in India. Gene-based SSRs for o2 and InDel markers for crtRB1 and lcyE were successfully employed for foreground selection in BC1F1, BC2F1, and BC2F2 generations. The recurrent parent genome recovery ranged from 88.9 to 96.0% among introgressed progenies. Kernels of pyramided lines possessed a high concentration of proA (7.14-9.63 ppm), compared to 1.05 to 1.41 ppm in the recurrent parents, while lysine and tryptophan ranged from 0.28-0.44% and 0.07-0.09%, respectively. The reconstituted hybrids (RBuland and RPMH1) showed significant enhancement of endosperm proA (6.97-9.82 ppm), tryptophan (0.07-0.09%), and lysine (0.29-0.43%), while grain yield was at par with their original versions. The dissemination of reconstituted hybrids holds significant promise to alleviate vitamin-A deficiency and protein-energy malnutrition in developing countries.

Genome wide association mapping for heat tolerance in sub-tropical maize.

BACKGROUND: Heat tolerance is becoming increasingly important where maize is grown under spring season in India which coincide with grain filling stage of crop resulting in tassel blast, reduced pollen viability, pollination failure and barren ears that causes devastating yield losses. So, there is need to identify the genomic regions associated with heat tolerance component traits which could be further employed in maize breeding program. RESULTS: An association mapping panel, consisting of 662 doubled haploid (DH) lines, was evaluated for yield contributing traits under normal and natural heat stress conditions. Genome wide association studies (GWAS) carried out using 187,000 SNPs and 130 SNPs significantly associated for grain yield (GY), days to 50% anthesis (AD), days to 50% silking (SD), anthesis-silking interval (ASI), plant height (PH), ear height (EH) and ear position (EPO) were identified under normal conditions. A total of 46 SNPs strongly associated with GY, ASI, EH and EPO were detected under heat stress conditions. Fifteen of the SNPs was found to have common association with more than one trait such as two SNPs viz. S10_1,905,273 and S10_1,905,274 showed colocalization with GY, PH and EH whereas S10_7,132,845 SNP associated with GY, AD and SD under normal conditions. No such colocalization of SNP markers with multiple traits was observed under heat stress conditions. Haplotypes trend regression analysis revealed 122 and 85 haplotype blocks, out of which, 20 and 6 haplotype blocks were associated with more than one trait under normal and heat stress conditions, respectively. Based on SNP association and haplotype mapping, nine and seven candidate genes were identified respectively, which belongs to different gene models having different biological functions in stress biology. CONCLUSIONS: The present study identified significant SNPs and haplotype blocks associated with yield contributing traits that help in selection of donor lines with favorable alleles for multiple traits. These results provided insights of genetics of heat stress tolerance. The genomic regions detected in the present study need further validation before being applied in the breeding pipelines.

Elucidating the morpho-physiological adaptations and molecular responses under long-term waterlogging stress in maize through gene expression analysis.

Waterlogging stress in maize is one of the emerging abiotic stresses in the current climate change scenario. To gain insights in transcriptional reprogramming during late hours of waterlogging stress under field conditions, we aimed to elucidate the transcriptional and anatomical changes in two contrasting maize inbreds viz. I110 (susceptible) and I172 (tolerant). Waterlogging stress reduced dry matter translocations from leaves and stems to ears, resulting in a lack of sink capacity and inadequate grain filling in I110, thus decreased the grain yield drastically. The development of aerenchyma cells within 48 h in I172 enabled hypoxia tolerance. The upregulation of alanine aminotransferase, ubiquitin activating enzyme E1, putative mitogen activated protein kinase and pyruvate kinase in I172 suggested that genes involved in protein degradation, signal transduction and carbon metabolism provided adaptive mechanisms during waterlogging. Overexpression of alcohol dehydrogenase, sucrose synthase, aspartate aminotransferase, NADP dependent malic enzyme and many miRNA targets in I110 indicated that more oxygen and energy consumption might have shortened plant survival during long-term waterlogging exposure. To the best of our knowledge, this is the first report of transcript profiling at late stage (24-96 h) of waterlogging stress under field conditions and provides new visions to understand the molecular basis of waterlogging tolerance in maize.

Genetic enhancement of essential amino acids for nutritional enrichment of maize protein quality through marker assisted selection.

Maize grain protein is deficient in two essential amino acids, lysine and tryptophan, defining it as of low nutritive value. The discovery of opaque2 (o2) gene has led to the development of quality protein maize (QPM) that has enhanced levels of essential amino acids over normal maize. However, the adoption of QPM is still very limited. The present study aims at improving the quality of normal four maize inbred lines (LM11, LM12, LM13 and LM14) of single cross hybrids; Buland (LM11 × LM12) and PMH1 (LM13 × LM14) released in India for different agro-climatic zones by introgressing o2 allele along-with modifiers using marker assisted backcross breeding. Both foreground and background selection coupled with phenotypic selection were employed for selection of o2 specific allele and maximum recovery of the recurrent parent genome (87-90%) with minimum linkage drag across the crosses. The converted QPM lines had < 25% opaqueness which is close to the respective recurrent parents. The QPM versions showed high level of tryptophan content ranging from 0.72 to 1.03 across the four crosses. The newly developed best QPM lines were crossed in original combinations to generate QPM hybrids. The grain yield of improved QPM hybrids was at par and there was significant increase in tryptophan content over the original hybrids.The integrated marker assisted, and phenotypic selection approach holds promise to tackle complex genetics of QPM. The dissemination and adoption of improved QPM versions may help to counteract protein-energy malnutrition in developing countries.

Computational identification of maize miRNA and their gene targets involved in biotic and abiotic stresses.

Plant interactions with biotic and abiotic stresses are complex and entail changes at the transcriptional, cellular and physiological level. MicroRNAs (miRNAs) are small (∼20-24 nt), non-coding RNAs that play a vital role in wide range of biological processes involved in regulation of gene expression through translation inhibition or degradation of their target mRNAs during stress conditions. Therefore, identification of miRNAs and their targets are of immense value in understanding the regulatory networks triggered during stress. Advancement in computational approaches has opened up ways for the prediction of miRNAs and their possible targets with functional pathways. Our objective was to identify miRNA and their potential targets involved in both biotic and abiotic stresses in maize. A total of 2,019,524 downloaded ESTs from dbEST were processed and trimmed by Seq Clean. The program trashed 264,000 and trimmed 284,979 sequences and the resulting 1,755,534 sequences were submitted for clustering and assembled to RepeatMasker and TGICL. A total of 30 miRNAs were found to hybridize with the potential targets of gene families such as CoA ligase, lipoxygenase 1, Terpenoideyclases, Zn finger, transducing, etc. Ten of the identified miRNAs targeted cytochrome c1 family. Zm_miR23 class targeted 11 different genes. The identified targets are involved in the plant growth and development during biotic and abiotic stresses in maize. These miRNAs may be further used for functional analysis. Furthermore, four and two of the miRNA targets were validated in response to waterlogging tolerance and southern leaf blight resistance, respectively, to understand the miRNA-assisted regulation of target miRNAs. The functional annotation of the predicted targets indicated that these stress-responsive miRNAs regulate cellular function; molecular function and biological process in maize at the post-transcriptional level. The present results have paved way towards better understanding the role of miRNAs in the mechanism of stress tolerance in maize.

Genome-Wide Development and Validation of Cost-Effective KASP Marker Assays for Genetic Dissection of Heat Stress Tolerance in Maize.

Maize is the third most important cereal crop worldwide. However, its production is vulnerable to heat stress, which is expected to become more and more severe in coming years. Germplasm resilient to heat stress has been identified, but its underlying genetic basis remains poorly understood. Genomic mapping technologies can fill the void, provided robust markers are available to tease apart the genotype-phenotype relationship. In the present investigation, we used data from an RNA-seq experiment to identify single nucleotide polymorphisms (SNPs) between two contrasting lines, LM11 and CML25, sensitive and tolerant to heat stress, respectively. The libraries for RNA-seq were made following heat stress treatment from three separate tissues/organs, comprising the top leaf, ovule, and pollen, all of which are highly vulnerable to damage by heat stress. The single nucleotide variants (SNVs) calling used STAR mapper and GATK caller pipelines in a combined approach to identify highly accurate SNPs between the two lines. A total of 554,423, 410,698, and 596,868 SNVs were discovered between LM11 and CML25 after comparing the transcript sequence reads from the leaf, pollen, and ovule libraries, respectively. Hundreds of these SNPs were then selected to develop into genome-wide Kompetitive Allele-Specific PCR (KASP) markers, which were validated to be robust with a successful SNP conversion rate of 71%. Subsequently, these KASP markers were used to effectively genotype an F2 mapping population derived from a cross of LM11 and CML25. Being highly cost-effective, these KASP markers provide a reliable molecular marker toolkit to not only facilitate the genetic dissection of the trait of heat stress tolerance but also to accelerate the breeding of heat-resilient maize by marker-assisted selection (MAS).