COTIP: Cotton TILLING Platform, a Resource for Plant Improvement and Reverse Genetic Studies

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.

Ethyl methanesulfonate mutant library construction in Gossypium hirsutum L. for allotetraploid functional genomics and germplasm innovation

“…In the phenotypic analysis of cotton boll mutations, changes in size (small boll) (Figure 2x2), shape (contains two variants, which are flat boll and perfectly round boll; Figure 2q, y2) and color ( Figure 2r) were found compared with the control group. In previous studies, EMS mutants with red cotton had been found (Aslam et al, 2016). However, the boll in our mutants turns red at the top rather than across the whole body.…”

“…And time consuming and costly in cotton species renders physical mutagens impractical. In contrast, a wide range of genetic mutation options are provided by chemical mutagenesis in easy and cost-effective ways (Aslam et al, 2016). Among the chemical mutagenic sources, ethylmethanesulfonate (EMS) is an excellent mutagen that activates the mutagenic potential of plants and has been extensively adopted in plant improvement research (Greene et al, 2003).…”

Identification of novel sources of genetic variation for the improvement of cold germination ability in upland cotton (Gossypium hirsutum)

“…Alongside the natural variation provided by the core GDRS panel, induced genetic variations such as those generated by chemical mutagenesis provide an additional and valuable genetic resource (Auld et al 1998(Auld et al , 2000Bechere et al 2009Bechere et al , 2012Aslam et al 2016) that can be utilized to broaden the genetic base of crops which has been severely limited by the bottleneck effects of domestication and artificial selection such as cotton (Shim et al 2018). In this study, genetic and phenotypic variation for cold germination ability was evaluated across a panel comprised of a subset of the core GDRS and chemically-induced mutant lines generated from the breeding program at Texas Tech University (Auld et al 1998(Auld et al , 2000Bechere et al 2009Bechere et al , 2012.…”

HorTILLUS—A Rich and Renewable Source of Induced Mutations for Forward/Reverse Genetics and Pre-breeding Programs in Barley (Hordeum vulgare L.)

“…Soon TILLING has become a valuable tool in development of desired alleles for crop breeding (reviewed in Kurowska et al, 2011 ; Tadele, 2016 ). The utility of TILLING has been demonstrated in many crop species, including cereals: maize (Till et al, 2004 ), rice (Till et al, 2007 ; Suzuki et al, 2008 ), sorghum (Xin et al, 2008 ), durum (Uauy et al, 2009 ), bread (Slade et al, 2005 ; Uauy et al, 2009 ; Fitzgerald et al, 2010 ; Chen et al, 2012 ), and einkorn wheat (Rawat et al, 2012 ); legumes and oil crops: pea (Dalmais et al, 2008 ), common bean (Porch et al, 2009 ), soybean (Cooper et al, 2008 ), sunflower (Kumar et al, 2013 ), rapeseed (Gilchrist et al, 2013 ); vegetables: tomato (Gady et al, 2009 ; Minoia et al, 2010 ; Piron et al, 2010 ; Okabe et al, 2011 ), pumpkin (Vicente-Dólera et al, 2014 ), cucumber (Fraenkel et al, 2014 ), radish (Kohzuma et al, 2017 ); industrial species: cotton (Aslam et al, 2016 ), flax (Chantreau et al, 2013 ), tobacco (Reddy et al, 2012 ); and staple tropical crops, such as banana (Jankowicz-Cieslak et al, 2012 ).…”