Mutant selection in the self-incompatible plant radish (<i>Raphanus sativus</i> L. <i>var. sativus</i>) using two-step TILLING

Abstract

Radish (Raphanus sativus L. var. sativus), a widely cultivated root vegetable crop, possesses a large sink organ (the root), implying that photosynthetic activity in radish can be enhanced by altering both the source and sink capacity of the plant. However, since radish is a self-incompatible plant, improved mutation-breeding strategies are needed for this crop. TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful method used for reverse genetics. In this study, we developed a new TILLING strategy involving a two-step mutant selection process for mutagenized radish plants: the first selection is performed to identify a BC1M1 line, that is, progenies of M1 plants crossed with wild-type, and the second step is performed to identify BC1M1 individuals with mutations. We focused on Rubisco as a target, since Rubisco is the most abundant plant protein and a key photosynthetic enzyme. We found that the radish genome contains six RBCS genes and one pseudogene encoding small Rubisco subunits. We screened 955 EMS-induced BC1M1 lines using our newly developed TILLING strategy and obtained six mutant lines for the six RsRBCS genes, encoding proteins with four different types of amino acid substitutions. Finally, we selected a homozygous mutant and subjected it to physiological measurements.

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 ).…”