Abstract
Cotton is cultivated worldwide for its white fiber, of which around 90% is tetraploid upland cotton (Gossypium hirsutum L.) carrying both A and D genome. Since centuries, yield increasing efforts for the cotton crop by conventional breeding approaches have caused an extensive erosion of natural genetic variability. Mutation based improvement strategies provide an effective way of creating new allelic variations. Targeting Induced Local Lesions IN Genomes (TILLING) provides a mutation based reverse genetic strategy to create and evaluate induced genetic variability at DNA level. Here, we report development and testing of TILLING populations of allotetraploid cotton (G. hirsutum) for functional genomic studies and mutation based enrichment of cotton genetic resources. Seed of two cotton cultivars “PB-899 and PB-900” were mutagenized with 0.3 and 0.2% (v/v) ethyl methanesulfonate, respectively. The phenotyping of M1 and M2 populations presented numerous mutants regarding the branching pattern, leaf morphology, disease resistance, photosynthetic lesions and flower sterility. Molecular screening for point mutations was performed by TILLING PCR aided CEL1 mismatch cleavage. To estimate the mutation frequency in the mutant genomes, five gene classes were TILLed in 8000 M2 plants of each var. “PB-899” and “PB-900.” These include actin (GhACT), Pectin Methyl Esterase (GhPME), sucrose synthase (GhSUS), resistance gene analog, and defense response gene (DRGs). The var. PB-899 was harboring 47% higher mutation induction rate than PB-900. The highest rate of mutation frequency was identified for NAC-TF5 (EU706348) of DRGs class, ranging from 1/58 kb in PB-899 to 1/105 kb in PB-900. The mutation screening assay revealed the presence of significant proportion of induced mutations in cotton TILLING populations such as 1/153 kb and 1/326 kb in var. “PB-899” and “PB-900,” respectively. The establishment of a cotton TILLING platform (COTIP) and data obtained from the resource TILLING population suggest its effectiveness in widening the genetic bases of cotton for improvement and utilizing it for subsequent reverse genetic studies of various genes.
Highlights
Cotton is primarily grown for its white fiber around the globe with an added advantage of its seeds as the major oil production source
Based on our previously described kill curve analysis of ethyl methanesulfonate (EMS) mutagenesis in Gossypium species, 0.3% (v/v) and 0.2% (v/v) aqueous solution of EMS were estimated as effective doses for seed treatment of var
The behavior of mutagens varied between species and among cultivars within a species, to estimate the maximum likelihood of effectiveness of EMS mutagenesis, we selected two closely related cotton cultivars of cotton
Summary
Cotton is primarily grown for its white fiber around the globe with an added advantage of its seeds as the major oil production source. According to the global econometrics, the polyploid AD genome of G. hirsutum contributes more than 90% to the world yarn production It is the best candidate in Gossypium genus to explore the silent variability related to its resistance mechanism and fiber quality. The continuous biased manipulation of available variability in cotton germplasm through conventional breeding approaches has resulted in the great loss of genetic potential added with increased vulnerability to many biological hazards In this scenario, widening the genetic base by mutation can diversify and create novel changes in the functional genes. TILLING has been proved a successful reverse genetic approach to induce and exploit genetic variation especially in crop plants It comprises two major steps of development of EMS mutagenized population followed by detection of base pair substitutions in targeted genes. This is an enriched resource for reverse genetic analysis of various gene families and annotating the important genes, related to yield and fiber quality traits
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
