Biology subjects

Isaac, P.

Publications and source records attributed to Isaac, P..

3 recordsLinked to original sources

bioRxiv2021

A novel approach to develop wheat chromosome-specific KASP markers for detecting Amblyopyrum muticum segments in doubled haploid introgression lines

Many wild relative species are being used in pre-breeding programmes to increase the genetic diversity of wheat. Genotyping tools such as single nucleotide polymorphism (SNP)-based arrays and molecular markers have been widely used to characterise wheat-wild relative introgression lines. However, due to the polyploid nature of the recipient wheat genome, it is difficult to develop SNP-based KASP markers that are codominant to track the introgressions from the wild species. Previous attempts to develop KASP markers have involved both exome- and PCR-amplicon-based sequencing of the wild species. But chromosome-specific KASPs assays have been hindered by homoeologous SNPs within the wheat genome. This study involved whole genome sequencing of the diploid wheat wild relative Amblyopyrum muticum and development of a SNP discovery pipeline that generated [~]38,000 SNPs in single-copy wheat genome sequences. New assays were designed to increase the density of Am. muticum polymorphic KASP markers. With a goal of one marker per 60 Mbp, 335 new KASP assays were validated as functional. Together with assays validated in previous studies, 498 well distributed chromosome-specific markers were used to recharacterize previously genotyped wheat-Am. muticum doubled haploid (DH) introgression lines. The chromosome specific nature of the KASP markers allowed clarification of which wheat chromosomes were involved with recombination events or substituted with Am. muticum chromosomes and the higher density of markers allowed detection of new small introgressions in these DH lines. Key MessageA novel methodology to generate chromosome-specific SNPs between wheat and its wild relative Amblyopyrum muticum and their use in the development of KASP markers to genotype wheat-Am. muticum introgression lines.

genetics↗

bioRxiv2020

Development of Wheat-Aegilops caudata Introgression Lines and Their Characterisation Using Genome-Specific KASP Markers

Aegilops caudata L. [syn. Ae. markgrafii (Greuter) Hammer], a diploid wild relative of wheat (2n = 2x = 14, CC), is an important source for new genetic variation for wheat improvement due to a variety of disease resistance factors along with tolerance for various abiotic stresses. Its practical utilisation in wheat improvement can be facilitated through the generation of genome-wide introgressions leading to a variety of different wheat-Ae. caudata recombinant lines. In this study, we report the generation of nine such wheat-Ae. caudata recombinant lines which were characterized using wheat genome-specific KASP (Kompetitive Allele Specific PCR) markers and multi-colour genomic in situ hybridization (mcGISH). Of these, six lines have stable homozygous introgressions from Ae. caudata and will be used for future trait analysis. Through a combination of molecular and cytological analysis of all the recombinant lines, we were able to physically map 182 KASP markers onto the seven Ae. caudata chromosomes, of which 155 were polymorphic specifically with only one wheat subgenome. Comparative analysis of the physical positions of these markers in the Ae. caudata and wheat genomes confirmed that the former had chromosomal rearrangements with respect to wheat, as previously reported. These wheat-Ae. caudata recombinant lines and KASP markers provide a useful genetic resource for wheat improvement with the latter having a wider impact as a tool for detection of introgressions from other Aegilops species into wheat.

genetics↗

bioRxiv2019

Rapid identification of homozygosity and site of wild relative introgressions in wheat through chromosome-specific KASP genotyping assays

For future food security it is important that wheat, one of the most widely consumed crops in the world, can survive the threat of abiotic and biotic stresses. New genetic variation is currently being introduced into wheat through introgressions from its wild relatives. For trait discovery, it is necessary that each introgression is homozygous and hence stable. Breeding programs rely on efficient genotyping platforms for marker-assisted selection (MAS). Recently, single nucleotide polymorphism (SNP) based markers have been made available on high-throughput Axiom(R) SNP genotyping arrays. However, these arrays are inflexible in their design and sample numbers, making their use unsuitable for long-term MAS. SNPs can potentially be converted into Kompetitive allele-specific PCR (KASP) assays which are comparatively cost-effective and efficient for low-density genotyping of introgression lines. However, due to the polyploid nature of wheat, KASP assays for homoeologous SNPs can have difficulty in distinguishing between heterozygous and homozygous hybrid lines in a backcross population. To identify co-dominant SNPs, that can differentiate between heterozygotes and homozygotes, we PCR-amplified and sequenced genomic DNA from potential single-copy regions of the wheat genome and compared them to orthologous copies from different wild relatives. A panel of 620 chromosome-specific KASP assays have been developed that allow rapid detection of wild relative segments and provide information on their homozygosity and site of introgression in the wheat genome. A set of 90 chromosome-nonspecific assays was also produced that can be used for genotyping introgression lines. These multipurpose KASP assays represent a powerful tool for wheat breeders worldwide.

plant biology↗