Marker-assisted selection for transfer of QTLs to a promising line for drought tolerance in wheat (Triticum aestivum L.).

Wheat crop is subjected to various biotic and abiotic stresses, which affect crop productivity and yield. Among various abiotic stresses, drought stress is a major problem considering the current global climate change scenario. A high-yielding wheat variety, HD3086, has been released for commercial cultivation under timely sown irrigated conditions for the North Western Plain Zone (NWPZ) and North Eastern Plain Zone NEPZ of India. Presently, HD3086 is one of the highest breeder seed indented wheat varieties and has a stable yield over the years. However, under moisture deficit conditions, its potential yield cannot be achieved. The present study was undertaken to transfer drought-tolerant QTLs in the background of the variety HD3086 using marker-assisted backcross breeding. QTLs governing Biomass (BIO), Canopy Temperature (CT), Thousand Kernel Weight (TKW), Normalized Difference Vegetation Index (NDVI), and Yield (YLD) were transferred to improve performance under moisture deficit conditions. In BC1F1, BC2F1, and BC2F2 generations, the foreground selection was carried out to identify the plants with positive QTLs conferring drought tolerance and linked to traits NDVI, CT, TKW, and yield. The positive homozygous lines for targeted QTLs were advanced from BC2F2 to BC2F4 via the pedigree-based phenotypic selection method. Background analysis was carried out in BC2F5 and obtained 78-91% recovery of the recurrent parent genome in the improved lines. Furthermore, the advanced lines were evaluated for 2 years under drought stress to assess improvement in MABB-derived lines. Increased GWPS, TKW, and NDVI and reduced CT was observed in improved lines. Seven improved lines were identified with significantly higher yields in comparison to HD3086 under stress conditions.

Marker assisted improvement for leaf rust and moisture deficit stress tolerance in wheat variety HD3086.

There is a significant yield reduction in the wheat crop as a result of different biotic and abiotic stresses, and changing climate, among them moisture deficit stress and leaf rust are the major ones affecting wheat worldwide. HD3086 is a high-yielding wheat variety that has been released for commercial cultivation under timely sown irrigated conditions in the Indo-Gangetic plains of India. Variety HD3086 provides a good, stable yield, and it is the choice of millions of farmers in India. It becomes susceptible to the most prevalent pathotypes 77-5 and 77-9 of Puccinia triticina (causing leaf rust) in the production environment and its potential yield cannot be realized under moisture deficit stress. The present study demonstrates the use of a marker-assisted back cross breeding approach to the successful transfer of leaf rust resistance gene Lr24 and QTLs linked to moisture deficit stress tolerance in the background of HD3086. The genotype HI1500 was used as a donor parent that possesses leaf rust-resistant gene Lr24, which confers resistance against the major pathotypes found in the production environment. It possesses inbuilt tolerance under abiotic stresses with superior quality traits. Foreground selection for gene Lr24 and moisture deficit stress tolerance QTLs linked to Canopy temperature (CT), Normal Differential Vegetation Index (NDVI) and Thousand Kernel Weight (TKW) in different generations of the backcrossing and selection. In BC2F2, foreground selection was carried out to identify homozygous lines based on the linked markers and were advanced following pedigree based phenotypic selection. The selected lines were evaluated against P. triticina pathotypes 77-5 and 77-9 under controlled conditions. Recurrent parent recovery of the selected lines ranged from 78-94%. The identified lines were evaluated for their tolerance to moisture stress under field conditions and their resistance to rust under artificial epiphytotic conditions for two years. In BC2F5 generation, eight positive lines for marker alleles were selected which showed resistance to leaf rust and recorded an improvement in component traits of moisture deficit stress tolerance such as CT, NDVI, TKW and yield compared to the recurrent parent HD3086. The derived line is named HD3471 and is nominated for national trials for testing and further release for commercial cultivation.

Optimization of medium composition to increase the expression of recombinant human interferon-ß using the Plackett-Burman and central composite design in E. coli SE1.

Recombinant human interferon-ß (rhIFN-ß) is therapeutically important and new commercially viable approaches are needed for its increased production. In this study, a codon-optimized gene encoding for rhIFN-ß(C17S) protein was designed and expressed in E. coli SE1. As a first step of medium optimization, growth of E. coli as a function of different media components was studied. Subsequently, to optimize the media composition, a response surface methodology (RSM) was used. Our results show that optimized medium (15.0 g/L tryptone, 12.3 g/L meat extract, 1.0 g/L MgSO4 and 0.5 g/L thiamine along with minimal medium) obtained in this study provide better growth of recombinant cells and the expression level of recombinant protein was ~ 1.7-fold more than Luria-Bertani medium. The optimized medium may be utilized for the large-scale production of rhIFN-ß.

Application of Genomics Tools in Wheat Breeding to Attain Durable Rust Resistance.

Wheat is an important source of dietary protein and calories for the majority of the world's population. It is one of the largest grown cereal in the world occupying over 215 M ha. Wheat production globally is challenged by biotic stresses such as pests and diseases. Of the 50 diseases of wheat that are of economic importance, the three rust diseases are the most ubiquitous causing significant yield losses in the majority of wheat production environments. Under severe epidemics they can lead to food insecurity threats amid the continuous evolution of new races of the pathogens, shifts in population dynamics and their virulence patterns, thereby rendering several effective resistance genes deployed in wheat breeding programs vulnerable. This emphasizes the need to identify, characterize, and deploy effective rust-resistant genes from diverse sources into pre-breeding lines and future wheat varieties. The use of genetic resistance has been marked as eco-friendly and to curb the further evolution of rust pathogens. Deployment of multiple rust resistance genes including major and minor genes in wheat lines could enhance the durability of resistance thereby reducing pathogen evolution. Advances in next-generation sequencing (NGS) platforms and associated bioinformatics tools have revolutionized wheat genomics. The sequence alignment of the wheat genome is the most important landmark which will enable genomics to identify marker-trait associations, candidate genes and enhanced breeding values in genomic selection (GS) studies. High throughput genotyping platforms have demonstrated their role in the estimation of genetic diversity, construction of the high-density genetic maps, dissecting polygenic traits, and better understanding their interactions through GWAS (genome-wide association studies) and QTL mapping, and isolation of R genes. Application of breeder's friendly KASP assays in the wheat breeding program has expedited the identification and pyramiding of rust resistance alleles/genes in elite lines. The present review covers the evolutionary trends of the rust pathogen and contemporary wheat varieties, and how these research strategies galvanized to control the wheat killer genus Puccinia. It will also highlight the outcome and research impact of cost-effective NGS technologies and cloning of rust resistance genes amid the public availability of common and tetraploid wheat reference genomes.

Harnessing genetic potential of wheat germplasm banks through impact-oriented-prebreeding for future food and nutritional security.

The value of exotic wheat genetic resources for accelerating grain yield gains is largely unproven and unrealized. We used next-generation sequencing, together with multi-environment phenotyping, to study the contribution of exotic genomes to 984 three-way-cross-derived (exotic/elite1//elite2) pre-breeding lines (PBLs). Genomic characterization of these lines with haplotype map-based and SNP marker approaches revealed exotic specific imprints of 16.1 to 25.1%, which compares to theoretical expectation of 25%. A rare and favorable haplotype (GT) with 0.4% frequency in gene bank identified on chromosome 6D minimized grain yield (GY) loss under heat stress without GY penalty under irrigated conditions. More specifically, the 'T' allele of the haplotype GT originated in Aegilops tauschii and was absent in all elite lines used in study. In silico analysis of the SNP showed hits with a candidate gene coding for isoflavone reductase IRL-like protein in Ae. tauschii. Rare haplotypes were also identified on chromosomes 1A, 6A and 2B effective against abiotic/biotic stresses. Results demonstrate positive contributions of exotic germplasm to PBLs derived from crosses of exotics with CIMMYT's best elite lines. This is a major impact-oriented pre-breeding effort at CIMMYT, resulting in large-scale development of PBLs for deployment in breeding programs addressing food security under climate change scenarios.

Antibiotic-free expression system for the production of human interferon-beta protein.

Recombinant human interferon-ß (rhIFN-ß), a therapeutic protein, is produced using both prokaryotic and eukaryotic expression systems. However, instability of recombinant plasmid during cultivation of Escherichia coli results in low yield of the recombinant proteins. In addition, use of antibiotics during the cultivation imposes a major concern. In this study, we have compared the expression yield of rhIFN-ß in E. coli BL21 (DE3) and E coli SE1 cells. Gene-encoding rhIFN-ß was expressed in E. coli BL21 (DE3) and SE1 cells and the cultivation of recombinant E. coli cells was done in a laboratory scale bioreactor. Our results suggest that, compared to BL21(DE3) cells, the SE1 cells expressing rhIFN-ß protein can be cultivated in the medium without antibiotic and provide increased stability of recombinant plasmid and higher expression yield of rhIFN-ß protein. This system can be used for the production of rhIFN-ß proteins for biomedical applications.

Dissection of genomic features and variations of three pathotypes of Puccinia striiformis through whole genome sequencing.

Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici, is one of the important diseases of wheat. We used NGS technologies to generate a draft genome sequence of two highly virulent (46S 119 and 31) and a least virulent (K) pathotypes of P. striiformis from the Indian subcontinent. We generated ~24,000-32,000 sequence contigs (N50;7.4-9.2 kb), which accounted for ~86X-105X sequence depth coverage with an estimated genome size of these pathotypes ranging from 66.2-70.2 Mb. A genome-wide analysis revealed that pathotype 46S 119 might be highly evolved among the three pathotypes in terms of year of detection and prevalence. SNP analysis revealed that ~47% of the gene sets are affected by nonsynonymous mutations. The extracellular secreted (ES) proteins presumably are well conserved among the three pathotypes, and perhaps purifying selection has an important role in differentiating pathotype 46S 119 from pathotypes K and 31. In the present study, we decoded the genomes of three pathotypes, with 81% of the total annotated genes being successfully assigned functional roles. Besides the identification of secretory genes, genes essential for pathogen-host interactions shall prove this study as a huge genomic resource for the management of this disease using host resistance.

Draft Genome of the Wheat Rust Pathogen (Puccinia triticina) Unravels Genome-Wide Structural Variations during Evolution.

Leaf rust is one of the most important diseases of wheat and is caused by Puccinia triticina, a highly variable rust pathogen prevalent worldwide. Decoding the genome of this pathogen will help in unraveling the molecular basis of its evolution and in the identification of genes responsible for its various biological functions. We generated high quality draft genome sequences (approximately 100- 106 Mb) of two races of P. triticina; the variable and virulent Race77 and the old, avirulent Race106. The genomes of races 77 and 106 had 33X and 27X coverage, respectively. We predicted 27678 and 26384 genes, with average lengths of 1,129 and 1,086 bases in races 77 and 106, respectively and found that the genomes consisted of 37.49% and 39.99% repetitive sequences. Genome wide comparative analysis revealed that Race77 differs substantially from Race106 with regard to segmental duplication (SD), repeat element, and SNP/InDel characteristics. Comparative analyses showed that Race 77 is a recent, highly variable and adapted Race compared with Race106. Further sequence analyses of 13 additional pathotypes of Race77 clearly differentiated the recent, active and virulent, from the older pathotypes. Average densities of 2.4 SNPs and 0.32 InDels per kb were obtained for all P. triticina pathotypes. Secretome analysis demonstrated that Race77 has more virulence factors than Race 106, which may be responsible for the greater degree of adaptation of this pathogen. We also found that genes under greater selection pressure were conserved in the genomes of both races, and may affect functions crucial for the higher levels of virulence factors in Race77. This study provides insights into the genome structure, genome organization, molecular basis of variation, and pathogenicity of P. triticina The genome sequence data generated in this study have been submitted to public domain databases and will be an important resource for comparative genomics studies of the more than 4000 existing Puccinia species.

Effect of different preservative treatments on the shelf-life of sorghum malt based fermented milk beverage.

Studies were conducted to extend the shelf life of sorghum based fermented milk beverage (referred to SSL) developed at our Institute. In the first approach preservatives namely nisin (N) @ 400RU/ml, MicroGARD (M) @ (1%), potassium sorbate (PS) @ (0.15%) were added to the beverage before packaging and in the second approach thermization (65 °C/5 min) of the packaged samples containing above mentioned preservatives was adopted. The shelf life was assessed on the basis of sensory, physico-chemical and microbiological parameters. Total plate count and lactic acid bacterial count increased till 7th day of storage and afterwards decreased significantly (p < 0.01). Yeasts and molds count increased significantly (p < 0.01) throughout the storage period and the rate of increase was lowest in samples containing PS. Samples containing PS, N and M were found sensorily acceptable up to 35, 28 and 21 days, respectively. Though the thermization treatment helped in improving the microbiological and chemical quality of the beverage, the sensory and physical quality was adversely affected. Addition of PS without thermization was found to be the best preservation technique for enhancing the shelf life of SSL at refrigeration conditions.

Effect of dietary fibers on physico-chemical, sensory and textural properties of Misti Dahi.

Misti dahi, a popular ethnic delicacy of eastern India analogous to caramel coloured set style sweetened yoghurt, besides several therapeutic virtues, contains high fat and sugar. Alike people elsewhere in the world, people in India too are now becoming health conscious and are aware of the relation between diet and health. Hence, high fat and sugar contents are causes of concern for the successful marketing of misti dahi in India. With a view to enhance the health attributes of misti dahi and improve marketability, three commercial dietary fiber preparations (inulin, soy fiber and oat fiber) were incorporated and their effect on the product's quality in terms of physicochemical, sensory and textural quality was assessed. Standard method was followed for the preparation of fiber fortified misti dahi (FFMD). Among the three dietary fibers, inulin significantly decreased viscosity and instrumental firmness and increased lightness (L*), redness (a*), yellowness (b*), syneresis and work of shear values of FFMD. Oat fiber settled at the bottom and gave a poor appearance. Soy fiber did not affect the flavor of FFMD. Although overall acceptability scores of inulin and soy fiber containing FFMD were significantly lower than control, they were still above the minimum acceptable score. Based on the results obtained in the present study, it was concluded that acceptable quality FFMD could be prepared using inulin and soy fiber at 1.5 % level of fortification.

Optimization of Rabadi-like fermented milk beverage using pearl millet.

Rabadi, prepared by fermenting pearl millet (Pennisetum typhoideum (L.)) (PM) flour with butter milk, is a traditional popular beverage of North-Western states of India. A process for PM based Rabadi-like fermented milk beverage was attempted. Skim milk and flour of 24 h germinated PM grains (FGG-24 h) were used as sources of solids. FGG-24 h was mixed in skim milk before fermentation and level of flour and water were determined using Response Surface Methodology (RSM) with central composite rotatable design (CCRD). The product developed using 5.3% flour and 72% water on the basis of curd gave the most acceptable product. For further stabilization during storage, pectin and/or carboxy methyl cellulose were tried at different levels and a level of 0.6% pectin was selected. The standardized product was packaged in glass bottles and stored under refrigeration (5-7 °C). The shelflife of the product was 7 days.