Genome-Wide Development and Validation of Cost-Effective KASP Marker Assays for Genetic Dissection of Heat Stress Tolerance in Maize

Abstract

Maize is the third most important cereal crop worldwide. However, its production is vulnerable to heat stress, which is expected to become more and more severe in coming years. Germplasm resilient to heat stress has been identified, but its underlying genetic basis remains poorly understood. Genomic mapping technologies can fill the void, provided robust markers are available to tease apart the genotype-phenotype relationship. In the present investigation, we used data from an RNA-seq experiment to identify single nucleotide polymorphisms (SNPs) between two contrasting lines, LM11 and CML25, sensitive and tolerant to heat stress, respectively. The libraries for RNA-seq were made following heat stress treatment from three separate tissues/organs, comprising the top leaf, ovule, and pollen, all of which are highly vulnerable to damage by heat stress. The single nucleotide variants (SNVs) calling used STAR mapper and GATK caller pipelines in a combined approach to identify highly accurate SNPs between the two lines. A total of 554,423, 410,698, and 596,868 SNVs were discovered between LM11 and CML25 after comparing the transcript sequence reads from the leaf, pollen, and ovule libraries, respectively. Hundreds of these SNPs were then selected to develop into genome-wide Kompetitive Allele-Specific PCR (KASP) markers, which were validated to be robust with a successful SNP conversion rate of 71%. Subsequently, these KASP markers were used to effectively genotype an F2 mapping population derived from a cross of LM11 and CML25. Being highly cost-effective, these KASP markers provide a reliable molecular marker toolkit to not only facilitate the genetic dissection of the trait of heat stress tolerance but also to accelerate the breeding of heat-resilient maize by marker-assisted selection (MAS).

Development and validation of diagnostic SNP markers for quality control genotyping in a collection of four rice (Oryza) species

“…In this study, KASP markers were successfully developed for 158 of 224 DArTseq-based SNPs sites submitted for conversion (Supplementary Table S1 ) . This constitutes a design success rate of 71%, similar to what has been reported previously in other crops 47 . Failure to successfully convert some of the markers can possibly be due to nearby polymorphisms in the sequences of the accessions included in this panel or the presence of paralogous sequences.…”

Genetics of Germination and Seedling Traits under Drought Stress in a MAGIC Population of Maize

“…Zm00001d037273 was associated with the CL/RL ratio and implicated in heat stress response ( HSR ). Jagtap et al [ 83 ] detected Zm00001d007215 in hybrids tolerant to water stress during the maize kernel-filling stage in a previous study. This is a differentially abundant proteins ( DAP ) gene involved in the response to drought stress [ 84 ].…”

Development of GBTS and KASP Panels for Genetic Diversity, Population Structure, and Fingerprinting of a Large Collection of Broccoli (Brassica oleracea L. var. italica) in China

“…For the fingerprinting of the broccoli lines, few KASP markers will be more cost effective and flexible. KASP assay has been widely used by plant breeders for genetic diversity study, genetic mapping, and genetic purity test in several major crops including pigeon pea (Saxena et al, 2012), chickpea (Hiremath et al, 2012), maize (Jagtap et al, 2020), cotton (Islam et al, 2015), tomato (Devran et al, 2016), peanut (Zhao et al, 2017), wheat (Rasheed et al, 2016), and rice (Pariasca-Tanaka et al, 2015;Cheon et al, 2018;Steele et al, 2018;Yang G. et al, 2019). In the present study, out of 28,220 filtered SNPs, 13,621 SNPs with PIC values between 0.2 and 0.5 were retained for KASP assay design.…”

“…However, only 8,768 (64.4%) SNPs could be designed as KASP markers successfully. This low conversion rate may have occurred probably due to the presence of duplicate loci, paralogous sequence, or incorrect primer design (Semagn et al, 2014;Steele et al, 2018;Jagtap et al, 2020). Optimizing the PCR conditions may improve the rate of successful KASP assay design.…”