Muhammad Amjad Nawaz

DNA molecular markers in plant breeding: current status and recent advancements in genomic selection and genome editing

ABSTRACT With the development of molecular marker technology in the 1980s, the fate of plant breeding has changed. Different types of molecular markers have been developed and advancement in sequencing technologies has geared crop improvement. To explore the knowledge about molecular markers, several reviews have been published in the last three decades; however, all these reviews were meant for researchers with advanced knowledge of molecular genetics. This review is intended to be a synopsis of recent developments in molecular markers and their applications in plant breeding and is devoted to early researchers with a little or no knowledge of molecular markers. The progress made in molecular plant breeding, genetics, genomic selection and genome editing has contributed to a more comprehensive understanding of molecular markers and provided deeper insights into the diversity available for crops and greatly complemented breeding stratagems. Genotyping-by-sequencing and association mapping based on next-generation sequencing technologies have facilitated the identification of novel genetic markers for complex and unstructured populations. Altogether, the history, the types of markers, their application in plant sciences and breeding, and some recent advancements in genomic selection and genome editing are discussed.

Genome-wide analysis of Family-1 UDP-glycosyltransferases in soybean confirms their abundance and varied expression during seed development.

Family-1 UDP-glycosyltransferases (EC 2.4.1.x; UGTs) are enzymes that glycosylate aglycones into glycoside-associated compounds with improved transport and water solubility. This glycosylation mechanism is vital to plant functions, such as regulation of hormonal homeostasis, growth and development, xenobiotic detoxification, stress response, and biosynthesis of secondary metabolites. Here, we report a genome-wide analysis of soybean that identified 149 putative UGTs based on 44 conserved plant secondary product glycosyl-transferase (PSPG) motif amino acid sequences. Phylogenetic analysis against 22 referenced UGTs from Arabidopsis and maize clustered the putative UGTs into 15 major groups (A-O); J, K, and N were not represented, but the UGTs were distributed across all chromosomes except chromosome 04. Leucine was the most abundant amino acid across all 149 UGT peptide sequences. Two conserved introns (C1 and C2) were detected in the most intron-containing UGTs. Publicly available microarray data on their maximum expression in the seed developmental stage were further confirmed using Affymetrix soybean IVT array and RNA sequencing data. The UGT expression models were designed, based on reads per kilobase of gene model per million mapped read (RPKM) values confirmed their maximally varied expression at globular and early maturation stages of seed development.

Environmental impacts of genetically modified plants: A review

Powerful scientific techniques have caused dramatic expansion of genetically modified crops leading to altered agricultural practices posing direct and indirect environmental implications. Despite the enhanced yield potential, risks and biosafety concerns associated with such GM crops are the fundamental issues to be addressed. An increasing interest can be noted among the researchers and policy makers in exploring unintended effects of transgenes associated with gene flow, flow of naked DNA, weediness and chemical toxicity. The current state of knowledge reveals that GM crops impart damaging impacts on the environment such as modification in crop pervasiveness or invasiveness, the emergence of herbicide and insecticide tolerance, transgene stacking and disturbed biodiversity, but these impacts require a more in-depth view and critical research so as to unveil further facts. Most of the reviewed scientific resources provide similar conclusions and currently there is an insufficient amount of data available and up until today, the consumption of GM plant products are safe for consumption to a greater extent with few exceptions. This paper updates the undesirable impacts of GM crops and their products on target and non-target species and attempts to shed light on the emerging challenges and threats associated with it. Underpinning research also realizes the influence of GM crops on a disturbance in biodiversity, development of resistance and evolution slightly resembles with the effects of non-GM cultivation. Future prospects are also discussed.

Genome and transcriptome-wide analyses of cellulose synthase gene superfamily in soybean.

The plant cellulose synthase gene superfamily belongs to the category of type-2 glycosyltransferases, and is involved in cellulose and hemicellulose biosynthesis. These enzymes are vital for maintaining cell-wall structural integrity throughout plant life. Here, we identified 78 putative cellulose synthases (CS) in the soybean genome. Phylogenetic analysis against 40 reference Arabidopsis CS genes clustered soybean CSs into seven major groups (CESA, CSL A, B, C, D, E and G), located on 19 chromosomes (except chromosome 18). Soybean CS expansion occurred in 66 duplication events. Additionally, we identified 95 simple sequence repeat makers related to 44 CSs. We next performed digital expression analysis using publically available datasets to understand potential CS functions in soybean. We found that CSs were highly expressed during soybean seed development, a pattern confirmed with an Affymatrix soybean IVT array and validated with RNA-seq profiles. Within CS groups, CESAs had higher relative expression than CSLs. Soybean CS models were designed based on maximum average RPKM values. Gene co-expression networks were developed to explore which CSs could work together in soybean. Finally, RT-PCR analysis confirmed the expression of 15 selected CSs during all four seed developmental stages.

Impact on environment, ecosystem, diversity and health from culturing and using GMOs as feed and food

Modern agriculture provides the potential for sustainable feeding of the worlds increasing population. Up to the present moment, genetically modified (GM) products have enabled increased yields and reduced pesticide usage. Nevertheless, GM products are controversial amongst policy makers, scientists and the consumers, regarding their possible environmental, ecological, and health risks. Scientific-and-political debates can even influence legislation and prospective risk assessment procedure. Currently, the scientifically-assessed direct hazardous impacts of GM food and feed on fauna and flora are conflicting; indeed, a review of literature available data provides some evidence of GM environmental and health risks. Although the consequences of gene flow and risks to biodiversity are debatable. Risks to the environment and ecosystems can exist, such as the evolution of weed herbicide resistance during GM cultivation. A matter of high importance is to provide precise knowledge and adequate current information to regulatory agencies, governments, policy makers, researchers, and commercial GMO-releasing companies to enable them to thoroughly investigate the possible risks.

Identification and expression profiling of a new β-amyrin synthase gene ( GmBAS3 ) from soybean

Cyclization of 2,3-oxidosqualene by different oxidosqualene cyclase (OSC) genes is responsible for sapogenin heterogeneity. The very first phase is the conversion of 2,3-oxidosqualene into β-amyrin by β-amyrin synthase (BAS) gene, a member of OSC family, in soy saponin biosynthesis pathway. This paper reports the identification of a new BAS gene (GmBAS3) and its expression pattern in soybean (Glycine max (L.) Merr.). GmBAS3 gene was identified by PCR/RACE method with an open reading frame of 2286 bp nucleotides encoding a 762 amino acid long protein devouring a characteristic QW motif repeated five times and DCTAE motif. GmBAS3 shared 96 and 92% homology with Glycyrrhiza uralensis BAS and Lotus japonicus putative BAS respectively. Expression of the gene was detected by RT-PCR in regard to seedlings age and tissue type. A spatio-temporal expression of GmBAS3 was found in 21-day-old seedlings in the hypocotyls, young leaves and mature leaves but not observed in stem and root tissues. No expression was perceived in 10-day-old seedling. This study also support the premise that β-amyrin synthesis hang on more than one type of BAS genes with there expression in different plant parts at different times.

Functional characterization of naturally occurring wild soybean mutant (sg-5) lacking astringent saponins using whole genome sequencing approach

Triterpenoid saponins are one of the most highly accumulated groups of functional components in soybean (Glycine max) and the oxidative reactions during their biosynthesis are required for their aglycone diversity. Natural mutants of soyasaponins in wild soybean (Glycine soja) are valuable resources for establishing the soyasaponin biosynthesis pathway and breeding new soybean varieties. In this study, we investigated the genetic mechanism behind the absence of group A saponins in a Korean wild soybean mutant, CWS5095. Whole genome sequencing (WGS) of CWS5095 identified four point mutations [Val6 → Asp, Ile231 → Thr, His294 → Gln, and Arg376 → Lys] in CYP72A69 (Glyma15g39090), which oxygenate the C-21 position of soyasapogenol B or other intermediates to produce soyasapogenol A, leading to group A saponin production. An in vitro enzyme activity assay of single-sited mutated clones indicated that the Arg376 > Lys mutation (a highly conserved mutation based on a nucleotide change from G → A at the 1,127th position) may lead to loss of gene function in the sg-5 mutant. A very high normalized expression value of 377 reads per kilo base per million (RPKM) of Glyma15g39090 in the hypocotyl axis at the early maturation seed-development stage confirmed their abundant presence in seed hypocotyls. A molecular dynamics analysis of the Arg376 > Lys mutation based on the CYP3A4 (a human CYP450) protein structure found that it was responsible for the increase in axis length toward the heme (active site), which is critically important for biological activity and ligand binding. Our results provide important information on how to eradicate bitter and astringent saponins in soybean by utilizing the reported mutation in Glyma15g39090, and its importance for seed hypocotyl development based on transcript abundance.

The tiered-evaluation of the effects of transgenic cry1c rice on Cyrtorhinus lividipennis , a main predator of Nilaparvata lugens

T1C-19, a newly developed transgenic cry1C rice line, expresses cry1C under the control of the maize ubiquitin promoter, and is highly resistant to lepidopteran pests of rice. Cyrtorhinus lividipennis is the major predator of the eggs and young nymphs of Nilaparvata lugens, which is the main non-target sap-sucking insect pest of Bt rice. C. lividipennis may be exposed to Cry1C protein, thus biosafety evaluations of transgenic cry1C rice on C. lividipennis should be conducted before the commercialization of T1C-19. In the current study, we tested the direct toxicity of elevated doses of Cry1C to C. lividipennis, effects of T1C-19 on the life-table parameters of C. lividipennis via preying planthoppers, and effects of T1C-19 on the population density and dynamics in rice fields. No detrimental effects on development, survival, female ratio and body weight of C. lividipennis were caused by direct exposure to elevated doses of the Cry1C protein or prey-mediated exposure to realistic doses of the protein. The population density and dynamics did not significantly differ between C. lividipennis in T1C-19 and non-transgenic rice fields. Thus, transgenic cry1C rice had no negative effects on C. lividipennis. This is the first report of the effects of transgenic cry1C rice on C. lividipennis.

Systems Identification and Characterization of Cell Wall Reassembly and Degradation Related Genes in Glycine max (L.) Merill, a Bioenergy Legume

Soybean is a promising biomass resource for generation of second-generation biofuels. Despite the utility of soybean cellulosic biomass and post-processing residues in biofuel generation, there is no comprehensive information available on cell wall loosening and degradation related gene families. In order to achieve enhanced lignocellulosic biomass with softened cell walls and reduced recalcitrance, it is important to identify genes involved in cell wall polymer loosening and degrading. Comprehensive genome-wide analysis of gene families involved in cell wall modifications is an efficient stratagem to find new candidate genes for soybean breeding for expanding biofuel industry. We report the identification of 505 genes distributed among 12 gene families related to cell wall loosening and degradation. 1262 tandem duplication events contributed towards expansion and diversification of studied gene families. We identified 687 Simple Sequence Repeat markers and 5 miRNA families distributed on 316 and 10 genes, respectively. Publically available microarray datasets were used to explore expression potential of identified genes in soybean plant developmental stages, 68 anatomical parts, abiotic and biotic stresses. Co-expression networks revealed transcriptional coordination of different gene families involved in cell wall loosening and degradation process.

RETRACTED ARTICLE: Beta-cyanoalanine synthase pathway as a homeostatic mechanism for cyanide detoxification as well as growth and development in higher plants

This review article has been retracted at the request of the Editors-in-Chief and the Reviews Editor. It has been using the published PhD thesis ‘‘Activity of the beta-cyanoalanine synthase pathway is associated with the response to abiotic stress by Arabidopsis thaliana’’ by Marylou Machingura as template without making reference to it. The original work can be consulted at http://opensiuc.lib. siu.edu/dissertations. The authors apologize to the Editors and readers as well as to the author of the original work. The online version of this article contains the full text of the retracted article as electronic supplementary material.