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

Abstract

TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN3) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M2 plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M3 progeny of 3,481 M2 individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M2 plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.

Barley ABI5 (Abscisic Acid INSENSITIVE 5) Is Involved in Abscisic Acid-Dependent Drought Response

“…In this study we focused on elucidating the role of HvABI5 in ABA signaling during barley response to drought stress. First, we have identified a hvabi5.d mutant using barley TILLING (Targeted Induced Local Lesions IN Genomes) platform created in our laboratory (Szurman-Zubrzycka et al, 2018). The identified TILLING mutant made it possible to initiate studies on the role of HvABI5 in regulation of ABA and drought responses in barley.…”

Identification of induced mutations in hexaploid wheat genome using exome capture assay

“…The majority of mutations were G/C to A/T transitions, while ~8% were transversions. Missense mutations (61% of the total) were largely found in coding regions while the remaining were silent (37.5%) and nonsense mutations (1.1%) [ 64 ].…”

TILLING in Cereal Crops for Allele Expansion and Mutation Detection by Using Modern Sequencing Technologies

“…The development of the TILLING method has increased the variety of plants with improved yield not only via reverse genetic approaches, but also by helping to improve targeted breeding. The application of TILLING to mutation breeding using radiation or chemical treatment has resulted in higher yielding rice, barley, and wheat (Table 2) [70][71][72]. Some of the key applications of TILLING to modify important traits of cereal crops are described below.…”

“…Through this screening, 382 mutations were identified, with a mutation density of one mutation per 477 kb. A total of 67 mutations were identified, of which 71% were missense and others were nonsense [71].…”