Breeding Tools Research Articles

Supplementation of Extender with Melatonin Improves the Motility, Mitochondrial Membrane Potential, and Fertilization Ability of Cryopreserved Brown-Marbled Grouper Sperm.

Sperm cryopreservation is a valuable tool for breeding, conservation, and genetic improvement in aquatic resources, while oxidative damage will cause a decline in sperm quality during this progress. Melatonin (MT), a natural antioxidant hormone, is used as an additive in sperm cryopreservation to reduce cellular damage from oxidative stress. Here, we aimed to investigate the effect of adding MT to the freezing medium in sperm cryopreservation of brown-marbled grouper (Epinephelus fuscoguttatus). Different concentrations of MT (0, 0.1, 0.25, and 0.5 mg/mL) were tested. We evaluated sperm motility, viability, apoptosis, mitochondrial membrane potential (MMP), and fertilization ability to assess the effects of MT supplementation. Our results demonstrated that the addition of MT to the extender improved the post-thaw motility, MMP, and fertilization ability of brown-marbled grouper sperm. The total motility, curvilinear velocity, straight linear velocity, and average path velocity in MT-treated groups (0.1 and 0.25 mg/mL) exhibited significantly higher values than that of the control group. A higher MMP (p < 0.05) was observed in the group treated with 0.25 mg/mL MT, suggesting that supplementation of MT in the extender might be able to protect mitochondrial membrane integrity effectively. Regarding fertilizing ability, 0.25 mg/mL MT yielded a significantly higher hatching rate than the control. An adverse effect was found with the concentration of MT up to 0.5 mg/mL, suggesting the possible toxicity of a high-dose addition. In this study, we optimized the sperm cryopreservation protocol of brown-marbled grouper, which might be valuable for sperm cryopreservation and sample commercialization of groupers and other fish.

Advancements in Enhancing Oil Quality in Rapeseed and Mustard: A Comprehensive Review

Rapeseed and mustard are important oilseed crops cultivated worldwide for their high oil content and versatile applications in food, feed, and industrial sectors. The quality of oil derived from these crops is influenced by various genetic, environmental, and agronomic factors. Breeding efforts aimed at improving oil quality traits in rapeseed and mustard have garnered significant attention in recent years due to their impact on market value, nutritional attributes, and industrial utility. In this review, we provide a comprehensive overview of breeding strategies and methodologies employed to enhance oil quality traits in rapeseed and mustard. We explore the genetic basis of oil quality traits, including fatty acid composition, erucic acid content, glucosinolate content, and tocopherol content, and discuss the importance of these traits for different end-uses. Furthermore, we highlight the role of molecular markers, genomics-assisted breeding, and biotechnological approaches in accelerating the breeding process and achieving targeted improvements in oil quality. The review also addresses the challenges and constraints associated with breeding for oil quality in rapeseed and mustard, including genotype-environment interactions, trait stability, and regulatory considerations. Additionally, we discuss emerging trends and future prospects in oil quality breeding, such as genome editing, metabolic engineering, and precision breeding, which offer novel avenues for achieving desired oil quality profiles while addressing sustainability and consumer preferences. Overall, this review underscores the significance of breeding for oil quality in rapeseed and mustard and provides insights into the latest advancements, challenges, and opportunities in this field. By integrating multidisciplinary approaches and harnessing the power of modern breeding tools and technologies, rapeseed and mustard breeders can continue to drive innovation and deliver oilseed crops with enhanced nutritional value, functional properties, and market competitiveness.

Developing breeding values for shedding using industry data from New Zealand sheep

ABSTRACT Changes in wool prices, labour costs and animal welfare have sparked interest in several new sheep traits. Shedding, the ability of moulting or seasonal wool loss in sheep, is one of them. Here, we aimed to develop breeding values for shedding using existing data from New Zealand flocks. We used breeder-collected shedding scores, 0 = ‘no shedding on any part of the body’ to 10 = ‘wool shed fully from all the body’, for Wiltshire and Wiltshire-composite lambs born between 2003 and 2022. After QC, 20,653 phenotypic records from 25,066 animals with pedigree information (393 sires and 7439 dams from 12 flocks) were included in the analyses. Genetic parameters and breeding values were obtained using a genetic additive model with pedigree data (BLUP model), assuming shedding scores were quantitative. The genetic model incorporated the following variables: date of birth, sex, birth and rearing rank, age of dam and contemporary group. We found a high heritability for lamb shedding, 0.54 (0.02), and estimated a low-to-moderate prediction accuracy of breeding values (ranging from 0.09 to 0.36) using forward-validation. Our results confirmed that shedding can be subject to selection and that pedigree-based breeding values for this trait can be a useful tool for breeders.

Novel electroporation-based genome editing of carnation plant tissues using RNPs targeting the anthocyanidin synthase gene.

A novel electroporation method for genome editing was performed using plant tissue samples by direct RNPs-introduction in carnation. Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a flower color mutant using the pink-flowered carnation 'Kane Ainou 1-go'. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of 'Kane Ainou 1-go' has not been previously isolated, we initially isolated the ANS gene from 'Kane Ainou 1-go' for characterization. Southern hybridization analysis confirmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efficiency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplified polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confirmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology.

Designing future peanut: the power of genomics-assisted breeding.

Integrating GAB methods with high-throughput phenotyping, genome editing, and speed breeding hold great potential in designing future smart peanut cultivars to meet market and food supply demands. Cultivated peanut (Arachis hypogaea L.), a legume crop greatly valued for its nourishing food, cooking oil, and fodder, is extensively grown worldwide. Despite decades of classical breeding efforts, the actual on-farm yield of peanut remains below its potential productivity due to the complicated interplay of genotype, environment, and management factors, as well as their intricate interactions. Integrating modern genomics tools into crop breeding is necessary to fast-track breeding efficiency and rapid progress. When combined with speed breeding methods, this integration can substantially accelerate the breeding process, leading to faster access of improved varieties to farmers. Availability of high-quality reference genomes for wild diploid progenitors and cultivated peanuts has accelerated the process of gene/quantitative locus discovery, developing markers and genotyping assays as well as a few molecular breeding products with improved resistance and oil quality. The use of new breeding tools, e.g., genomic selection, haplotype-based breeding, speed breeding, high-throughput phenotyping, and genome editing, is probable to boost genetic gains in peanut. Moreover, renewed attention to efficient selection and exploitation of targeted genetic resources is also needed to design high-quality and high-yielding peanut cultivars with main adaptation attributes. In this context, the combination of genomics-assisted breeding (GAB), genome editing, and speed breeding hold great potential in designing future improved peanut cultivars to meet market and food supply demands.

The Hessian fly resistance gene HvRHF1 is localized in an NBS-LRR gene cluster in barley.

Hessian fly (Mayetiola destructor Say) is a significant pest in cereal crops, causing substantial yield losses worldwide. While host resistance is the most efficient method for pest control, research on genetic characterization of Hessian fly resistance in barley (Hordeum vulgare L.) has been limited, and the underlying resistance mechanism remains largely unknown. In this study, we conducted fine mapping of a crucial Hessian fly resistance locus, known as HvRHF1, using a biparental population. Assisted with genetic markers and robust phenotyping assay, we pinpointed the HvRHF1 gene to an ~ 82kb region on chromosome 4H. Gene prediction and annotation revealed that the HvRHF1 locus comprises three complete NBS-LRR genes, which are characteristic of disease resistance genes. As a result, our study not only provides valuable resources for resistance in barley and genetic tools for breeding, but also identifies candidate genes that lay the foundation for cloning HvRHF1. This endeavor will significantly contribute to our understanding of the molecular mechanisms underlying cereal resistance to Hessian fly.

Improvement of economic and useful characters of wheat using RNA interference technology

Wheat is a strategic cereal crop of global importance and plays a leading role in the food supply of mankind. Despite the general trend to increase in its production, climatic changes leading to significant temperature changes, unpredictable precipitation or droughts and the appearance of new races of pathogens and pests significantly affect its yield. In order to prevent the negative impact of changes in climatic conditions on the productivity of this crop, it is necessary to develop innovative technologies for improving the resistance of wheat to environmental stresses. RNA interference (RNAi) represents a new potential tool for wheat breeding by introducing small non-coding RNA sequences with the ability to silence gene expression in a sequence-specific manner. A decrease in the expression of a certain gene determines the acquisition of a new characteristic through the elimination or accumulation of certain plant traits, which leads to biochemical or phenotypic changes that the original plants do not have. This literature review describes the progress achieved over the past decades in the application of RNAi to create wheat plants with improved economic and valuable traits. The main stages of the gene silencing mechanism mediated by short interfering RNA (siRNA) and microRNA (miRNA), features of their biogenesis, modes of action and distribution are presented. Examples of the use of various biotechnological approaches to wheat improvement using gene transformation, endogenous and exogenous double-stranded RNA molecules (dsRNA) are given. The possibility of using RNAi technology to increase the nutritional value and quality of grain, remove toxic compounds and allergens is highlighted. Considerable attention is paid to the practical results of various applications of RNAi to increase the resistance of wheat to biotic stress factors, in particular, viruses, bacteria, fungi, insect pests, and nematodes. Examples of the use of siRNA-mediated RNAi and the role of miRNA in improving wheat tolerance to abiotic stresses are summarized.

A Bayesian optimization R package for multitrait parental selection.

Selecting and mating parents in conventional phenotypic and genomic selection are crucial. Plant breeding programs aim to improve the economic value of crops, considering multiple traits simultaneously. When traits are negatively correlated and/or when there are missing records in some traits, selection becomes more complex. To address this problem, we propose a multitrait selection approach using the Multitrait Parental Selection (MPS) R package-an efficient tool for genetic improvement, precision breeding, and conservation genetics. The package employs Bayesian optimization algorithms and three loss functions (Kullback-Leibler, Energy Score, and Multivariate Asymmetric Loss) to identify parental candidates with desirable traits. The software's functionality includes three main functions-EvalMPS, FastMPS, and ApproxMPS-catering to different data availability scenarios. Through the presented application examples, the MPS R package proves effective in multitrait genomic selection, enabling breeders to make informed decisions and achieve strong performance across multiple traits.

Correlation and Path Analysis for Agronomic and Processing Quality Traits of Potato (&amp;lt;i&amp;gt;Solanum tuberosum&amp;lt;/i&amp;gt; L.) at Holetta, Central Ethiopia

Potato is the third most important food crop in terms of consumption in the world after rice and wheat. It is a nutrient-rich vegetable with just a small amount of fat and contains 16% carbohydrates, 2% proteins, 1% minerals, and 0.6% dietary fiber. The literature on path and correlation analysis and its application as a potato breeding tool is limited in comparison to its significance for processing purpose and the knowledge it adds for upcoming breeding work. The objective of this study was to determine the relation among tuber yield and processing quality traits of potato using correlation and path coefficient analysis. This experiment was conducted at Holetta Agricultural research Centre, Ethiopia during the main crop season of 2017. The experiment was laid out in randomized complete block design (RCBD) with three replications using 24 potato genotypes. Strong positive and significant correlation were found between total tuber yield and marketable tuber yield (r=0.98) at both genotypic and phenotypic levels. Stronger positive correlations were found between dry matter content and starch content (r= 1) and specific gravity (r=1). Path coefficient analysis of tuber yield and its components shows that dry matter content and marketable tuber yield exerted positive highest direct influence on total tuber yield. Specific gravity of tuber had high positive direct effect on dry matter content. So, to increase the performance of these traits for tuber yield and processing quality traits path analysis can be used. As a conclusion, most of the traits had positive correlations and direct effects with total tuber yield and dry matter content at phenotypic and genotypic levels. Therefore, those traits had practical importance in selection of potato genotypes for high total tuber yield and processing purpose.

Transient efflux inhibition improves plant regeneration by natural auxins.

Plant genome editing and propagation are important tools in crop breeding and production. Both rely heavily on the development of efficient in vitro plant regeneration systems. Two prominent regeneration systems that are widely employed in crop production are somatic embryogenesis (SE) and de novo shoot regeneration. In many of the protocols for SE or shoot regeneration, explants are treated with the synthetic auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D), since natural auxins, such as indole-3-acetic acid (IAA) or 4-chloroindole-3-acetic acid (4-Cl-IAA), are less effective or even fail to induce regeneration. Based on previous reports that 2,4-D, compared to endogenous auxins, is not effectively exported from plant cells, we investigated whether efflux inhibition of endogenous auxins could convert these auxins into efficient inducers of SE in Arabidopsis immature zygotic embryos (IZEs). We show that natural auxins and synthetic analogs thereof become efficient inducers of SE when their efflux is transiently inhibited by co-application of the auxin transport inhibitor naphthylphthalamic acid (NPA). Moreover, IZEs of auxin efflux mutants pin2 or abcb1 abcb19 show enhanced SE efficiency when treated with IAA or efflux-inhibited IAA, confirming that auxin efflux reduces the efficiency of Arabidopsis SE. Importantly, in contrast to the 2,4-D system, where only 50-60% of the embryos converted to seedlings, all SEs induced by transport-inhibited natural auxins converted to seedlings. Efflux-inhibited IAA, like 2,4-D, also efficiently induced SE from carrot suspension cells, whereas IAA alone could not, and efflux-inhibited 4-Cl-IAA significantly improved de novo shoot regeneration in Brassica napus. Our data provides new insights into the action of 2,4-D as an efficient inducer of plant regeneration but also shows that replacing this synthetic auxin for efflux-inhibited natural auxin significantly improves different types of plant regeneration, leading to a more synchronized and homogenous development of the regenerated plants.

Morpometric and molecular characterization of Surguli goat through CO1 gene in district Kohat

The present study was designed with the aim to study morphometric characterization as well as phylogeny and diversity of the local Surguli goat at their breeding tract district Kohat through mitochondrial DNA region, i.e., Cytochrome C Oxidase Subunit One (CO1) gene. Morphometric data and blood samples were collected from thirty (30) pure goats. Morphometric analysis showed that sex had significant effect (p < 0.05) on body weight, body length, hearth girth and horn length while no significant effect (p > 0.05) was observed for other characteristics. The results also indicated that age had significant effect (p < 0.05) on height at rump, ear length, horn length and tail length while no significant effect (p > 0.05) was observed for other characteristics. The phylogenetic analysis through CO1 nucleotide sequences within nucleotide range 1–767 showed nine polymorphic sites segregating into eight haplotypes. The mean intraspecific diversity and mean interspecific diversity were calculated as 0.23 and 2.36%, respectively. Phylogenetic tree revealed that Capra Ibex and native Surguli goat have common ancestors. The morphometric and molecular results obtained from the present study can be exploited as a selection tool for breeding and overall improvement.

Using spectral sensing in plant science

Digital cameras are widely used tools for plant monitoring in plant science today. Used to track plant growth or even visible symptoms, they are important tools for breeding and plant protection field trials. Nevertheless, its extension to measure the near infrared (NIR) region (700–1000 nm) includes great potential as plants show a higher light reflectance within this spectrum. Various applications have shown its use for disease detection, quantification, virus content estimation, and stress monitoring. As the next step is a comprehensive integration into agricultural routines, this study will show two use-cases with a high technological readiness level. One use-case shows a handheld multispectral sensor, which is used for manual measurements to detect and discriminate different virus types in sugar beet. In contrast, the second use-case shows a transfer to an UAV based disease quantification routine based on spectral imaging for Cercospora leaf spot. In addition, two prototypical workflows are shown for processing non-imaging and imaging spectral data in an agricultural setting. This study shows the state of the art in spectral sensing in the field for the two major sugar beet diseases – virus yellows and Cercospora leaf spot. Furthermore a future perspective for coming technological challenges regarding the integration of AI in sensors or robotic workflows is provided.

Multiple Foliar Fungal Disease Management in Tomatoes: A Comprehensive Approach

Foliar diseases are the significant production constraints in tomatoes. Among them, foliar fungal diseases in tomatoes, such as early blight (Alternaria linaria), Septoria leaf spot (Septoria lycopersici), and late blight (Phytophthora infestans), which is oomycetes, have higher economic significance. This paper will discuss the etiology, host range, distribution, symptoms, and disease cycle to help us understand the biology, followed by management approaches emphasizing the resistance breeding approach for these diseases. We provide an analytical review of crop improvement efforts, including conventional and molecular methods for improving these diseases’ resistance. We discuss the importance of modern breeding tools, including genomics, genetic transformation, and genome editing, to improve the resistance to these diseases in the future.

Development of a targeted genotyping platform for reproducible results within tetraploid and hexaploid blueberry

Blueberry (Vaccinium spp.) is one of the most economically important berry crops worldwide. Validation of genetic mapping studies is often hindered by asynchronous marker technology. The development of a standardized genotyping platform that targets a specific set of polymorphic loci can be a practical solution to unify the scientific and breeding community toward blueberry improvement. The objective of this study was to develop and evaluate a targeted genotyping platform for cultivated blueberries that is affordable, reproducible, and sufficiently high density to warrant large-scale adoption for genomic studies. The Flex-Seq platform was developed in a two-step procedure that resulted in 22,000 loci that yielded 194,365 single nucleotide polymorphisms when assessed in a diversity set of 192 samples including cultivated and other related wild Vaccinium species. Locus recovery averaged 89.4% in the cultivated polyploid blueberry (northern highbush [NHB], southern highbush [SHB], and rabbiteye [RE]) and on average 88.8% were polymorphic. While recovery of these loci was lower in the other Vaccinium species assayed, recovery remained high and ranged between 60.8% and 70.4% depending on the taxonomic distance to the cultivated blueberry targeted in this platform. NHB had the highest mean number of variants per locus at 9.7, followed by RE with 9.1, SHB with 8.5, and a range between 7.7 and 8.5 in other species. As expected, the total number of unique-in-state haplotypes exceeded the total number of variants in the domesticated blueberries. Phylogenetic analysis using a subset of the SNPs and haplotypes mostly conformed to known relationships. The platform also offers flexibility about the number of loci, depth of sequencing for accurate dosage calling, loci and haplotype reconstruction from increased fragment length. This genotyping platform will accelerate the development and improvement of blueberry cultivars through genomic-assisted breeding tools.

LbCas12a-nuclease-mediated tiling deletion for large-scale targeted editing of non-coding regions in rice

NTD method is the application of LbCas12a in the engineering of rice endogenous gene. Guided by libraries of crRNAs tiling on NCRs of SD1, 6 mutants with quantitative variations of rice height were generated and analyzed for the association between genotype and phenotype. Totally, NTD is a promising tool for molecular rice breeding.

Germination Assays for Comparable Seed Dormancy Depth and Distinction of Seed Color by Multiplex PCR in Wheat.

Seed dormancy is an important trait in cereal breeding, as it prevents preharvest sprouting (PHS). Although seed dormancy is a multifactorial trait, seed color has been demonstrated to be a major dormancy-related factor controlled by few genes. The R-1 gene is a seed color regulator that encodes a MYB-type transcription factor in wheat. A set of genetic markers designed against R-1 can provide a powerful tool for swift wheat breeding. Depth of seed dormancy varies not only among lines but also during seed development in each line. In this chapter, we describe how developmental seeds can be collected to perform germination tests, how seed color can be observed after NaOH staining, and how to genotype wheat R-1 genes using multiplex PCR.

Advanced Genetic Tools for Rice Breeding: CRISPR/Cas9 and Its Role in Yield Trait Improvement

Advanced Genetic Tools for Rice Breeding: CRISPR/Cas9 and Its Role in Yield Trait Improvement

Identification of Induced Copy Number Variation from Low Coverage Sequence Data.

Induced mutations have been an important tool for plant breeding and functional genomics for more than 80years. Novel mutations can be induced by treating seed or other plant cells with chemical mutagens or ionizing radiation. The majority of released mutant crop varieties were developed using ionizing radiation. This has been shown to create a variety of different DNA lesions including large (e.g., >=10,000bps) copy number variations (CNV). Detection of induced DNA lesions from whole genome sequence data is useful for choosing a mutagen dosage prior to committing resources to develop a large mutant population for forward or reverse-genetic screening. Here I provide a method for detecting large induced CNV from mutant plants that utilizes a new tool to streamline the process of obtaining read coverage directly from BAM files, comparing non-mutagenized controls and mutagenized samples, and plotting the results for visual evaluation. Example data is provided from low coverage sequence data from gamma-irradiated vegetatively propagated triploid banana.

Cas12a and MAD7, genome editing tools for breeding.

Food shortages due to population growth and climate change are expected to occur in the near future as a problem that urgently requires solutions. Conventional breeding techniques, notably crossbreeding and mutation breeding, are known for being inefficient and time-consuming in obtaining seeds and seedlings with desired traits. Thus, there is an urgent need for novel methods for efficient plant breeding. Breeding by genome editing is receiving substantial attention because it can efficiently modify the target gene to obtain desired traits compared with conventional methods. Among the programmable sequence-specific nucleases that have been developed for genome editing, CRISPR-Cas12a and CRISPR-MAD7 nucleases are becoming more broadly adopted for the application of genome editing in grains, vegetables and fruits. Additionally, ST8, an improved variant of MAD7, has been developed to enhance genome editing efficiency and has potential for application to breeding of crops.

A Review on High Throughput Phenotyping for Vegetable Crops

Abstract Conventional phenotyping approaches for vegetable crops such as Solanaceae, Bulb, and Root crops have contributed significantly to the development of numerous varieties. Despite this, traditional phenotyping procedures are insufficient because of the longer time required to produce a variety, poor genetic gain, environmental influences, and other externalities that impact phenotype-based selection. A novel recent approach of high throughput phenotyping (HTP) is regarded a potential tool for addressing the problems of traditional phenotyping. The advent of sensor, computer vision, automation, and sophisticated machine learning technologies sparked the creation of high-throughput phenotyping technology in the prior decade. HTP platforms are being used to conduct non-destructive evaluations of the whole plant system in a variety of crops. HTP provides precise measurements and suggests the collection of high-quality and accurate data, which is required for standardizing phenotyping for genetic dissection and genomic assisted breeding techniques such as genome-wide association studies (GWAS), linkage mapping, marker-assisted selection (MAS), and genomic selection (GS). The rest of this chapter examines the application of high-throughput phenotyping tools in genomic-assisted breeding for vegetable crops.