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.

Dry direct-seeding of rice is rapidly increasing in China, but variable planting depth associated with machine sowing can lead to low seedling emergence rates. Phenotype analysis of 621 rice accessions showed that mesocotyl length (ML) was induced by deep soil covering and was important in deep-sowing tolerance in the field. Here, we performed and compared GWAS using three types of SNPs (non-synonymous SNP, non-synonymous SNPs and SNPs within promoters and 3 million randomly selected SNPs from the entire set of SNPs) and found that Non-Syn GWAS (GWAS using non-synonyomous SNP) decreased computation time and eliminated confounding by other loci relative to GWAS using randomly selected SNPs. Thirteen QTLs were finally detected, and two new major-effect genes, named OsML1 and OsML2, were identified by an integrated analysis. There were 2 and 7 non-synonymous SNPs in OsML1 and OsML2, respectively, from which 3 and 4 haplotypes were detected in cultivated rice. Combinations of superior haplotypes of OsML1 and OsML2 increased ML by up to 4 cm, representing high emergence rate (85%) in the field with 10 cm of soil cover. The studies provide key loci and naturally occurring alleles of ML that can be used in improving tolerance to dry direct-seeding.