Selection and validation of reliable reference genes for quantitative real-time PCR in Barnyard millet (Echinochloa spp.) under varied abiotic stress conditions.

Quantitative real-time polymerase chain reaction (RT-qPCR) using a stable reference gene is widely used for gene expression research. Barnyard millet (Echinochloa spp.) is an ancient crop in Asia and Africa that is widely cultivated for food and fodder. It thrives well under drought, salinity, cold, and heat environmental conditions, besides adapting to any soil type. To date, there are no gene expression studies performed to identify the potential candidate gene responsible for stress response in barnyard millet, due to lack of reference gene. Here, 10 candidate reference genes, Actin (ACT), α-tubulin (α-TUB), ß-tubulin (ß-TUB), RNA pol II (RP II), elongation factor-1 alpha (EF-1α), adenine phosphoribosyltransferase (APRT), TATA-binding protein-like factor (TLF), ubiquitin-conjugating enzyme 2 (UBC2), ubiquitin-conjugating enzyme E2L5 (UBC5) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were selected from mRNA sequences of E. crus-galli and E. colona var frumentacea. Five statistical algorithms (geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder) were applied to determine the expression stabilities of these genes in barnyard millet grown under four different abiotic stress (drought, salinity, cold and heat) exposed at different time points. The UBC5 and ɑ-TUB in drought, GAPDH in salinity, GAPDH and APRT in cold, and EF-1α and RP II in heat were the most stable reference genes, whereas ß-TUB was the least stable irrespective of stress conditions applied. Further Vn/Vn + 1 analysis revealed two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with Cu-ZnSOD (SOD1) in the plants exposed to different abiotic stress conditions. The results revealed that the relative quantification of the SOD1 gene varied according to reference genes and the number of reference genes used, thus highlighting the importance of the choice of a reference gene in such experiments. This study provides a foundational framework for standardizing RT-qPCR analyses, enabling accurate gene expression profiling in barnyard millet.

Effectiveness and efficiency of electron beam in comparison with gamma rays and ethyl methane sulfonate mutagens in cowpea.

Cowpea is the poor man's crop that lacks variability due to its autogamous nature. Induced mutation serves as a potential source in the induction of variability in crops. On the other hand, the effectiveness and efficiency of mutagens will vary between species and even varieties. In the present study, a novel mutagen electron beam was used in cowpea for the first time along with commonly used mutagens gamma rays (physical) and ethyl methanesulfonate (chemical). The biological damages on eight quantitative characters in M1 generation and chlorophyll mutants in M2 generation were recorded. Two popular varieties viz., P 152 and VBN 1 constituted as the biological material of study. The rate of reduction in biological damage on quantitative characters was directly proportional to the dose of mutagen irrespective of the varieties and mutagens used. Physical mutagens showed the highest biological damage (EB- 37.5% and G- 37.3% overall reduction from control) than chemical mutagen (EMS- 30.4%). Comparing the physical mutagens at similar doses, 200 Gy or 300 Gy of electron beam showed more biological damage than 200 Gy or 300 Gy of gamma rays. Eleven different types of chlorophyll mutants were identified in the M2 generation. Xantha is the most occurred chlorophyll mutants (44.44%), while aurea and yellow viridis have least occurred mutants. Chemical mutagen (EMS) is considered to be the most effective (6.47%) and efficient mutagen (27.09%) based on the chlorophyll mutants and it was followed by an electron beam and gamma rays. Among the physical mutagens, electron beam showed the highest biological damage (37.50% overall reduction from control) and higher effectiveness and efficiency (3.80% and 23.38%) compared to gamma rays (1.87% and 13.38%). Hence, the electron beam can also be used as an effective mutagen in creating variation in cowpea and other crops as it is highly effective, cost less and safe mutagen.

Gene Pyramiding for Achieving Enhanced Resistance to Bacterial Blight, Blast, and Sheath Blight Diseases in Rice.

Bacterial blight, blast, and sheath blight are the commonest diseases causing substantial yield loss in rice around the world. Stacking of broad-spectrum resistance genes/QTLs into popular cultivars is becoming a major objective of any disease resistance breeding program. The varieties ASD 16 and ADT 43 are the two popular, high yielding, and widely grown rice cultivars of South India, which are susceptible to bacterial blight (BB), blast, and sheath blight diseases. The present study was carried out to improve the cultivars (ASD 16 and ADT 43) through introgression of bacterial blight (xa5, xa13, and Xa21), blast (Pi54), and sheath blight (qSBR7-1, qSBR11-1, and qSBR11-2) resistance genes/QTLs by MABB (marker-assisted backcross breeding). IRBB60 (xa5, xa13, and Xa21) and Tetep (Pi54; qSBR7-1, qSBR11-1, and qSBR11-2) were used as donors to introgress BB, blast, and sheath blight resistance into the recurrent parents (ASD 16 and ADT 43). Homozygous (BC3F3 generation), three-gene bacterial blight pyramided (xa5 + xa13 + Xa21) lines were developed, and these lines were crossed with Tetep to combine blast (Pi54) and sheath blight (qSBR7-1, qSBR11-1, and qSBR11-2) resistance. In BC3F3 generation, the improved pyramided lines carrying a total of seven genes/QTLs (xa5 + xa13 + Xa21 + Pi54 + qSBR7-1 + qSBR11-1 + qSBR11-2) were selected through molecular and phenotypic assay, and these were evaluated for resistance against bacterial blight, blast, and sheath blight pathogens under greenhouse conditions. We have selected nine lines in ASD 16 background and 15 lines in ADT 43 background, exhibiting a high degree of resistance to BB, blast, and sheath blight diseases and also possessing phenotypes of recurrent parents. The improved pyramided lines are expected to be used as improved varieties or used as a potential donor in breeding programs. The present study successfully introgressed Pi54, and qSBR QTLs (qSBR7-1, qSBR11-1, and qSBR11-2) from Tetep and major effective BB-resistant genes (xa5, xa13, and Xa21) from IRBB60 into the commercial varieties for durable resistance to multiple diseases.

Improved host-plant resistance to Phytophthora rot and powdery mildew in soybean (Glycine max (L.) Merr.).

Soybean is an important oilseed cum vegetable crop, susceptible to various biotic stresses which is attributed to recent decline in crop productivity. The emergence of virulent biotypes/strains of different plant pathogens necessitates the development of new crop varieties with enhanced host resistance mechanisms. Pyramiding of multiple disease-resistant genes is one of the strategies employed to develop durable disease-resistant cultivars to the prevailing and emerging biotypes of pathogens. The present study, reports the successful introgression of two major R-genes, including Rps2 (Phytophthora rot resistance), Rmd-c (complete-powdery mildew resistance) and effective nodulating gene (rj2) through functional Marker-Assisted Backcross Breeding (MABB) in the genetic background of well-adapted and high yielding soybean varieties, CO 3 and JS 335. We have identified several promising introgressed lines with enhanced resistance to Phytophthora rot and powdery mildew. The improved soybean lines have exhibited medium to high level of resistance against powdery mildew and Phytophthora rot as well as displayed effective nodulation capacity. Our study has proven the generation of resistant genotypes to realize the potential of MABB for achieving host plant resistance in soybean. The improved lines developed can greatly assist the soybean breeding programs in India and other soybean growing countries for evolving disease-resistant varieties.

Barnyard Millet for Food and Nutritional Security: Current Status and Future Research Direction.

Barnyard millet (Echinochloa species) has become one of the most important minor millet crops in Asia, showing a firm upsurge in world production. The genus Echinochloa comprises of two major species, Echinochloa esculenta and Echinochloa frumentacea, which are predominantly cultivated for human consumption and livestock feed. They are less susceptible to biotic and abiotic stresses. Barnyard millet grain is a good source of protein, carbohydrate, fiber, and, most notably, contains more micronutrients (iron and zinc) than other major cereals. Despite its nutritional and agronomic benefits, barnyard millet has remained an underutilized crop. Over the past decades, very limited attempts have been made to study the features of this crop. Hence, more concerted research efforts are required to characterize germplasm resources, identify trait-specific donors, develop mapping population, and discover QTL/gene (s). The recent release of genome and transcriptome sequences of wild and cultivated Echinochloa species, respectively has facilitated in understanding the genetic architecture and decoding the rapport between genotype and phenotype of micronutrients and agronomic traits in this crop. In this review, we highlight the importance of barnyard millet in the current scenario and discuss the up-to-date status of genetic and genomics research and the research gaps to be worked upon by suggesting directions for future research to make barnyard millet a potential crop in contributing to food and nutritional security.