Mutagenesis of barley malting quality QTLs with Ds transposons

Abstract

Various functional genomic tools are being used to identify and characterize genes in plants. The Activator/Dissociation (Ac/Ds) transposon-based approach offers great potential, especially in barley, due to its limited success of genetic transformation and its large genome size. The bias of the Ac/Ds system towards genic regions and its tendency toward localized transpositions can greatly enhance the discovery and tagging of genes linked to Ds. Barley is a key ingredient in malting and brewing industry; therefore, gene discovery in relation to malting has an industrial perspective. Malting quality in barley is a complex and quantitatively inherited trait. Two major quantitative trait loci (QTLs) affecting malting quality traits have been located on chromosome 4H. In this study, Ds was reactivated from parent transposants (TNP) lines, TNP-29 and TNP-79, where Ds was mapped in the vicinity of important malting QTLs. Reactivation of Ds was carried out both by conventional breeding and in vitro approaches. A threefold increase in reactivation frequency through the in vitro approach enabled the development of a new genomic resource for the dissection of malting QTL and gene discovery in barley. Identification of unique flanking sequences, using high-efficiency thermal asymmetric interlaced PCR and inverse PCR from these populations, has further emphasized the new location of Ds in the barley genome and provided new transposon mutants especially in β-GAL1, β-amylase-like gene and ABC transporter for functional genomic studies.

Comparative assessment of genetic diversity between wild and cultivated barley using gSSR and EST‐SSR markers

“…Genomic DNA was extracted using a modified phenol/chloroform method as described by Singh et al. . DNA quality and quantity was analysed using a Nanodrop spectrophotometer.…”

“…Prior to seeding, seeds of wild barley genotypes were vernalized at 4°C for 4 weeks. Genomic DNA was extracted using a modified phenol/chloroform method as described by Singh et al 2012. DNA quality and quantity was analysed using a Nanodrop spectrophotometer.…”

Induced Genetic Variation, TILLING and NGS-Based Cloning

“…Ds remobilisation has been obtained by two approaches, namely, by conventional crossing of the original Ds-carrying line with an AcTPase-expressing parent and by genetic transformation of the Ds-carrying line with a construct carrying the AcTPase gene. The latter approach unexpectedly showed a higher remobilisation rate, perhaps due to the alteration of the chromatin methylation pattern occurring during the in vitro cultivation phase (Singh et al 2012). Interestingly, Singh et al (2006) reported a very high insertion rate (86 %) in genic regions as observed in maize and rice, while Zhao et al (2006) observed some tendency for Ds elements to target low-copy, matrix-attachment regions.…”

Registration of the Barley Transposon‐Tagged Population II: Sixty‐One Lines Each Characterized for Ds Insertion Sites

“…This mutant has been useful for elucidating the control of cleistogamous flowering (Anwar et al, 2018). Other tagged barley lines have played a role in investigating the genes associated with root growth and malting quality (Kaur et al, 2013; Singh et al, 2012; Singh and Singh, 2017).…”