Biology subjects

Prusty, M. R.

Publications and source records attributed to Prusty, M. R..

4 recordsLinked to original sources

bioRxiv2024

From Pigments to Precision: Exploring Genetic Transformation and Genome Editing in Wheat and Barley

Genetic engineering of wheat is complex due to its large genome size, the presence of numerous genes with high sequence similarities, and a multitude of repetitive elements. In addition, genetic transformation of wheat has been difficult, mainly due to poor regeneration in tissue cultures. Recent advances in plant biotechnology, particularly the use of the regenerative genes GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF), have provided new tools for wheat transformation and regeneration. Another transformative tool is the RUBY system, that involves genetic engineering of three betalain biosynthesis genes, providing a noninvasive, visually detectable red pigment. In this study, we used the GRF4-GIF1 chimera along with the RUBY system to advance transformation and gene editing in wheat and barley. The GRF4-GIF1 chimera significantly aided wheat regeneration; however, it had an opposite effect in barley, where it inhibited the regeneration process. Therefore, we primarily generated RUBY transgenic barley lines using constructs that did not include the GRF4-GIF1 chimera. Additionally, we used the RUBY cassette for fast assessment of gene editing by knockingout the first betalain biosynthetic gene in RUBY-positive transgenic wheat plants, resulting in a change of leaf color from red to green. The edited RUBY wheat lines lost more than just the red color. They also lost betalain-related traits, such as being less likely to get leaf rust (Puccinia triticina) and salt stress. Importantly, the loss of RUBY did not affect plant viability, making it a useful tool for genome editing and a viable alternative to destructive methods.

genomics↗

bioRxiv2021

Plasmotype condition nuclear pleiotropic effects on clock and fitness in barley

In plants, the role of chloroplasts and mitochondria (plasmotype) in controlling circadian clock plasticity and overall plant robustness has not been elucidated. In this study, we investigated the rhythmicity of chlorophyll fluorescence (Chl F) clock output, and fitness in the field at optimal and elevated temperatures, in three different barley populations. First, we examined a reciprocal DH population between two wild barley (Hordeum vulgare ssp. spontaneum), in which we identified two pleiotropic QTLs (frp2.1 and amp7.1) that modulate clock and fitness including conditioning of these effects by plasmotype diversity. In the second population, a complete diallel consisting of 11 genotypes (reciprocal hybrids differing in plasmotype), we observed a gradual reduction in plasmotype, ranging from 26% and 15% for Chl F and clock measurements to 5.3% and 3.7% for growth and reproductive traits, respectively. The third population studied was a collection of cytolines in which nine different wild plasmotypes replaced the cultivated Noga (H. vulgare) plasmotype. Here, the order and magnitude of the effects of the plasmotypes differed from what we observed in the diallel population, with the greatest effect of plasmotype diversity observed for clock period and amplitude. Comparison of the chloroplast sequences suggests several candidate genes in the plastid-encoded RNA polymerase (PEP) complex that may be responsible for the observed plasmotype effects. Overall, our results unravel previously unknown cytonuclear epistatic interactions that controls clock performance while also having pleiotropic effects on a plant field characteristics.

plant biology↗

bioRxiv2020

Loss of Photosynthetic Rhythm Thermal Plasticity Under Domestication and RepurposingDrivers of Circadian Clock (DOC) Loci for Adaptive Breeding in Barley

Circadian clock rhythms are critical to control physiological and development traits, allowing, plants to adapt to changing environments. Here we show that the circadian rhythms of cultivated barley (Hordeum vulgare) have slowed and amplitude increased under domestication by comparing with its wild ancestor (H. spontaneum). Moreover, we show a significant loss of thermal plasticity during barley evolution for the period and more extensively for amplitude. Our genetic analysis indicates that wild allele at epistatic loci, which mutually condition clock variation and its thermal plasticity in interspecific crosses, are absent in a contemporary barley breeding panel. These epistatic interactions include conditioned effects of Drivers of Circadian (DOC) clock loci on chromosome 3 and 5, which mediate amplitude decrease and period lengthening, respectively, under domestication. Notably, two significant loci, DOC3.1 and DOC5.1, which are not associated with clock diversity in cultivated breeding material, do show pleiotropic effects on flowering time and grain yield at multiple experimental sites across the U.S. in a temperature-dependent manner. We suggest that transition from winter growth of wild barley (H. spontaneum) to spring growth of modern cultivars included the loss and repurposing of circadian clock regulators to yield adaptation by mechanisms yet to be clarified. Significance statementCircadian clock rhythms are crucial factors affecting crop adaptation to changing environments. If faced with increased temperature plants could respond with temperature compensation adaptation and maintain clock rhythms, or they can change period and/or amplitude to adapt. We used a combination of approaches: high-throughput clock analysis under optimal and elevated heat conditions, genome-wide association study (GWAS) with cultivated and wild diversity panels to identify changes under domestication and quantitative trait loci (QTL) that control the clock and its responses, and QTL-environment association for testing environmentally-conditioned effects of these QTLongrain yield and flowering timingacross US. Our findings provide insights into changes of circadian rhythms under domestication and genetic tools for plant breeders to develop better-adapted cultivars to changing environments.

genetics↗

bioRxiv2020

RECAS9: Recombining wild species introgression via mitotic gene editing in barley

Genetic loci underlying variation in traits with agronomic importance or genetic risk factors in human diseases have been identified by linkage analysis and genome-wide association studies. However, narrowing down the mapping to the individual causal genes and variations within these is much more challenging, and so is the ability to break linkage drag between beneficial and unfavourable loci in crop breeding. We developed RECAS9 as a transgene-free approach for precisely targeting recombination events by delivering CRISPR/Cas9 ribonucleotide protein (RNP) complex into heterozygous mitotic cells for the barley (Hordeum vulgare) Heat3.1 locus. A wild species (H. spontaneum) introgression in this region carries the agronomical unfavourable tough rachis phenotype (non-brittle) allele linked with a circadian clock accelerating QTL near GIGANTEA gene. We delivered RNP, which was targeted between two single nucleotide polymorphism (SNPs), to mitotic calli cells by particle bombardment. We estimated recombination events by next generation sequencing (NGS) and droplet digital PCR (ddPCR). While NGS analysis grieved from confounding effects of PCR recombination, ddPCR analysis allowed us to associate RNP treatment on heterozygous individuals with significant increase of homologous directed repair (HDR) between cultivated and wild alleles, with recombination rate ranging between zero to 57%. These results show for the first time in plants a directed and transgene free mitotic recombination driven by Cas9 RNP, and provide a starting point for precise breeding and fine scale mapping of beneficial alleles from crop wild relatives.

genetics↗