Expression profiling of transgenes(Cry1Ac and Cry2A) in cotton genotypes under different genetic backgrounds

Transgenic cotton carrying the CrylAc gene has revolutionized insect pest control since its adoption,although the development of resistance in insect pests has reduced its efficacy.After 10 years of cultivating Bacillus thuringiensis(Bt)cotton with a single Cry1 Ac gene,growers are on the verge of adopting Bt cotton that carries the double gene(Cry1 Ac+Cry2 A)due to its better effectiveness against insect pests.Thus,the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.The expression levels of the Cry1 Ac and Cry2 A genes were evaluated in the leaves of 10 genotypes(2 parents and 8 Fhybrids)at 30 days after sowing(DAS),while samples of leaves,bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.The Fhybrids were developed through reciprocal crosses between two Bt(CKC-1,CKC-2)and two non-Bt(MNH-786,FH-942)parents.The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay(ELISA).The results showed that the MNH786 xCKC-1 hybrid had the highest concentrations of Cry1 Ac gene at30 DAS(3.08μg g^(-1))and 110 DAS(1.01μg g^(-1))in leaves.In contrast,the CKC-2 xMNH-786 hybrid showed the lowest concentrations of Cry1 Ac gene at 30 DAS(2.30μg g^(-1))and 110 DAS(0.86μg g^(-1)).The Fhybrid FH-942×CKC-2 showed the highest concentrations of Cry2 A gene at 30 DAS(8.39μg g^(-1))and 110 DAS(7.74μg g^(-1))in leaves,while the CKC-1 xMNH-786 hybrid expressed the lowest concentrations of Cry2 A gene at 30 DAS(7.10μg g^(-1))and 110 DAS(8.31μg g^(-1)).A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS,whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.When the expression pattern was compared between various plant parts in genotype CKC-2,it was found that leaves had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))at 70 DAS,followed by bolls(Cry1 Ac(1.66μg g^(-1))and Cry2 A(8.15μg g^(-1)))and flowers(Cry1 Ac(1.07μg g^(-1))and Cry2 A(7.99μg g^(-1))).The genotype CKC-2 had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))in the upper canopy but less accumulation(2.66μg g^(-1)of Cry1 Ac,8.09μg g^(-1)of Cry2 A)in the lower canopy at 70 DAS.Similarly,at 110 DAS,the expression levels of Cry1 Ac and Cry2 A in upper and lower canopy leaves were 1.52 and 7.92μg 9,and 0.99 and 7.54μg 9,respectively.Hence,the current study demonstrates that different genotypes showed variable expression for both of the Cry1 Ac and Cry2 A genes during plant growth due to different genetic backgrounds.The Cry2 A gene had three-fold higher expression than Cry1 Ac with significant differences in expression in different plant parts.The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1 Ac and Cry2 A for sustainable cotton production worldwide.

Polymorphic information and genetic diversity in Brassica speciesrevealed by RAPD markers

Randomly amplified polymorphic DNA(RAPD)is a tremendously convenient approach used to discriminate between Brassica species owing to its accuracy and speed.RAPD primers generate adequate genetic information that can be used in the primer-marker system.In this work,twenty RAPD-PCR based markers were executed to generate polymorphic data,like polymorphic information content(PIC),mean resolving power(MRP),resolving power(RP),effective multiplex ratio(EMR),and marker index(MI)for the first time and genetic distance among and between six Brassica species were calculated.Our results indicated that 20 primers produced a total of 231 scored band and generated 87%polymorphic bands.Average PIC,MRP,RP,MI,and EMR values were 0.088,0.65,6.7,0.78,and 8.9,respectively.PIC showed an overall negative correlation with MRP,RP,MI,and EMR,whereas MRP,RP,and EMR,were positively correlated with each other.Genetic identities ranged from 41.99%(between Brassica napus and Brassica oleracea)to 68.83%(between Brassica campestris and Brassica oleracea).Dendrogram results showed no clustering between species except between Brassica campestris and Brassica nigra.Nevertheless,these results will be helpful to acquire useful information about the markers and their use to determine the genomic structures of Brassica species.Further,based on genetic distance and polymorphic information,new hybrids can be developed for effective oilseed production.