Genome-wide characterization and expression analysis of Erf gene family in cotton.

BACKGROUND: AP2/ERF transcription factors are important in a variety of biological activities, including plant growth, development, and responses to biotic and abiotic stressors. However, little study has been done on cotton's AP2/ERF genes, although cotton is an essential fibre crop. We were able to examine the tissue and expression patterns of AP2/ERF genes in cotton on a genome-wide basis because of the recently published whole genome sequence of cotton. Genome-wide analysis of ERF gene family within two diploid species (G. arboreum & G. raimondii) and two tetraploid species (G. barbadense, G. hirsutum) was performed. RESULTS: A total of 118, 120, 213, 220 genes containing the sequence of single AP2 domain were identified in G. arboreum, G. raimondii, G. barbadense and G. hirsutum respectively. The identified genes were unevenly distributed across 13/26 chromosomes of A and D genomes of cotton. Synteny and collinearity analysis revealed that segmental duplications may have played crucial roles in the expansion of the cotton ERF gene family, as well as tandem duplications played a minor role. Cis-acting elements of the promoter sites of Ghi-ERFs genes predict the involvement in multiple hormone responses and abiotic stresses. Transcriptome and qRT-PCR analysis revealed that Ghi-ERF-2D.6, Ghi-ERF-12D.13, Ghi-ERF-6D.1, Ghi-ERF-7A.6 and Ghi-ERF-11D.5 are candidate genes against salinity tolerance in upland cotton. CONCLUSION: Overwhelmingly, the present study paves the way to better understand the evolution of cotton ERF genes and lays a foundation for future investigation of ERF genes in improving salinity stress tolerance in cotton.

Heterologous expression of cry3Bb1 and cry3 genes for enhanced resistance against insect pests in cotton.

Transgenic technology played a crucial role in developing insect-resistant plants resulting in the reduced application of pesticides. This article reports the expression of two cry proteins (Cry3Bb1 and Cry3) in cotton for enhanced resistance against chewing insect pests. The aforementioned genes were synthetically developed and were cloned under appropriate regulatory sequences followed by transformation into Eagle-2 genotype (Gossypium hirsutum) of cotton through shoot apex-cut Agro-infiltration. The transgene integration was validated by polymerase chain reaction using primers flanking the aforementioned cry genes. Transgene expression was assessed by qRT-PCR using GADPH as a reference gene. The relative fold expression analyses revealed the highest expression of the transgene(s) in M1 plants, which is a 4.5-fold expression (Cry3 + Cry3Bb1) followed by M3 (fold expression, 3.0) (Cry3Bb1) and M2 (fold expression, 2.5) (Cry3) transformants of cotton. The confirmed transgenic plants were exposed to insect pests, pink bollworm (Pectinophora gossypiella), and army bollworm (Helicoverpa armigera). Bioassay results revealed that 60% mortality was observed against pink bollworm, and 75% mortality was observed against army bollworm in transgenic plants containing both Cry3Bb1 and Cry3 genes (M1 transgenic plants). In M2 transgenic plants containing only the Cry3Bb1 gene, the mortality was observed to be 40% in the pink bollworm population, whereas 45% mortality was observed in the army bollworm population. In the case of M3 transgenic plants containing single gene-Cry3, the mortality was 20% in the pink bollworm population, whereas 30% mortality was observed in the army bollworm population. Almost no mortality was observed in non-transgenic Eagle-2 control plants. Hence, the developed cotton transformants have improved resistance against chewing insect pests.

Unraveling Heat Tolerance in Upland Cotton (Gossypium hirsutum L.) Using Univariate and Multivariate Analysis.

The ever-changing global environment currently includes an increasing ambient temperature that can be a devastating stress for organisms. Plants, being sessile, are adversely affected by heat stress in their physiology, development, growth, and ultimately yield. Since little is known about the response of biochemical traits to high-temperature ambiance, we evaluated eight parental lines (five lines and three testers) and their 15 F1 hybrids under normal and high-temperature stress to assess the impact of these conditions over 2 consecutive years. The research was performed under a triplicate randomized complete block design including a split-plot arrangement. Data were recorded for agronomic, biochemical, and fiber quality traits. Mean values of agronomic traits were significantly reduced under heat stress conditions, while hydrogen peroxide, peroxidase, total soluble protein, superoxide dismutase, catalase (CAT), carotenoids, and fiber strength displayed higher mean values under heat stress conditions. Under both conditions, high genetic advance and high heritability were observed for seed cotton yield (SCY), CAT, micronaire value, plant height, and chlorophyll-a and b content, indicating that an additive type of gene action controls these traits under both the conditions. For more insights into variation, Pearson correlation analysis and principal component analysis (PCA) were performed. Significant positive associations were observed among agronomic, biochemical, and fiber quality-related traits. The multivariate analyses involving hierarchical clustering and PCA classified the 23 experimental genotypes into four groups under normal and high-temperature stress conditions. Under both conditions, the F1 hybrid genotype FB-SHAHEEN × JSQ WHITE GOLD followed by Ghuari-1, CCRI-24, Eagle-2 × FB-Falcon, Ghuari-1 × JSQ White Gold, and Eagle-2 exhibited better performance in response to high-temperature stress regarding the agronomic and fiber quality-related traits. The mentioned genotypes could be utilized in future cotton breeding programs to enhance heat tolerance and improve cotton yield and productivity through resistance to environmental stressors.

Pyramiding of cry toxins and methanol producing genes to increase insect resistance in cotton.

The idea of enhanced methanol production from cell wall by pectin methyl esterase enzymes (PME) combined with expression of cry genes from Bacillus thuringiensis as a strategy to improve insect pest control in cotton is presented. We constructed a cassette containing two cry genes (cry1Fa and Cry32Aa) and two pme genes, one from Arabidopsis thaliana (AtPME), and other from Aspergillus. niger (AnPME) in pCAMBIA1301 plant expression vector using CAMV-35S promoter. This construction was transformed in Eagle-2 cotton variety by using shoot apex-cut Agrobacterium-mediated transformation. Expression of cry genes and pme genes was confirmed by qPCR. Methanol production was measured in control and in the cry and pme transformed plants showing methanol production only in transformed plants, in contrast to the non-transgenic cotton plants. Finally, insect bioassays performed with transgenic plants expressing cry and pme genes showed 100% mortality for Helicoverpa armigera (cotton bollworm) larvae, 70% mortality for Pectinophora gossypiella (pink bollworm) larvae and 95% mortality of Earias fabia, (spotted bollworm) larvae, that was higher than the transgenic plants expressing only cry genes that showed 84%, 49% and 79% mortality, respectively. These results demonstrate that Bt. cry-genes coupled with pme genes are an effective strategy to improve the control of different insect pests.