CRISPR/Cas-Based Diagnostics in Agricultural Applications.

Pests and disease-causing pathogens frequently impede agricultural production. An early and efficient diagnostic tool is crucial for effective disease management. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein (Cas) have recently been harnessed to develop diagnostic tools. The CRISPR/Cas system, composed of the Cas endonuclease and guide RNA, enables precise identification and cleavage of the target nucleic acids. The inherent sensitivity, high specificity, and rapid assay time of the CRISPR/Cas system make it an effective alternative for diagnosing plant pathogens and identifying genetically modified crops. Furthermore, its potential for multiplexing and suitability for point-of-care testing at the field level provide advantages over traditional diagnostic systems such as RT-PCR, LAMP, and NGS. In this review, we discuss the recent developments in CRISPR/Cas based diagnostics and their implications in various agricultural applications. We have also emphasized the major challenges with possible solutions and provided insights into future perspectives and potential applications of the CRISPR/Cas system in agriculture.

Genome-wide investigation of SnRK2 gene family in two jute species: Corchorus olitorius and Corchorus capsularis.

BACKGROUND: Sucrose non-fermenting-1 (SNF1)-related protein kinase 2 (SnRK2), a plant-specific serine/threonine kinase family, is associated with metabolic responses, including abscisic acid signaling under biotic and abiotic stresses. So far, no information on a genome-wide investigation and stress-mediated expression profiling of jute SnRK2 is available. Recent whole-genome sequencing of two Corchorus species prompted to identify and characterize this SnRK2 gene family. RESULT: We identified seven SnRK2 genes of each of Corchorus olitorius (Co) and C. capsularis (Cc) genomes, with similar physico-molecular properties and sub-group patterns of other models and related crops. In both species, the SnRK2 gene family showed an evolutionarily distinct trend. Highly variable C-terminal and conserved N-terminal regions were observed. Co- and CcSnRK2.3, Co- and CcSnRk2.5, Co- and CcSnRk2.7, and Co- and CcSnRK2.8 were upregulated in response to drought and salinity stresses. In waterlogging conditions, Co- and CcSnRk2.6 and Co- and CcSnRK2.8 showed higher activity when exposed to hypoxic conditions. Expression analysis in different plant parts showed that SnRK2.5 in both Corchorus species is highly expressed in fiber cells providing evidence of the role of fiber formation. CONCLUSION: This is the first comprehensive study of SnRK2 genes in both Corchorus species. All seven genes identified in this study showed an almost similar pattern of gene structures and molecular properties. Gene expression patterns of these genes varied depending on the plant parts and in response to abiotic stresses.

Evaluation of In-Field Inoculation Methods for Characterization of Macadamia Germplasm to Husk Spot Incidence and Severity.

Husk spot, a fungal disease of macadamia pericarps (Pseudocercospora macadamiae), induces premature abscission in several major commercial cultivars. Breeding for resistance to husk spot is a priority of the Australian macadamia industry. Due to the large tree size of macadamia and high numbers of progeny in breeding populations, inoculating for resistance screening is laborious and time consuming. Previously utilized methods included direct applications of P. macadamiae suspensions and the hanging of bags of diseased husks above developing fruit in tree canopies. In this study, both methods were modified to allow for efficient application in large-scale breeding populations, and their efficacy was evaluated. Two quantities of diseased husk per bag, 'large' (75 g) and 'small' (30 g), and two concentrations of sprayed P. macadamiae suspensions, 'stock' (5 × 105 propagules/ml) and 'dilute' (2.5 × 105 propagules/ml), were tested across two fruiting seasons. Treatments were compared against a control (sterile water) in commercial cultivars A38 and A4. Husk spot incidence and severity produced by small bags were significantly affected by season. A significant season effect was less common for other treatments. All four treatments infected over 50% of target fruit in each season, but the highest husk spot incidence across both seasons (≥85%) was produced from large bags. Overall, the large bags were the most reliable method for infection of target fruit. Results also demonstrate the importance of considering the effect of season when selecting husk spot inoculation methods.

Precision genome editing in plants using gene targeting and prime editing: existing and emerging strategies.

Precise modification of plant genomes, such as seamless insertion, deletion, or replacement of DNA sequences at a predefined site, is a challenging task. Gene targeting (GT) and prime editing are currently the best approaches for this purpose. However, these techniques are inefficient in plants, which limits their applications for crop breeding programs. Recently, substantial developments have been made to improve the efficiency of these techniques in plants. Several strategies, such as RNA donor templating, chemically modified donor DNA template, and tandem-repeat homology-directed repair, are aimed at improving GT. Additionally, improved prime editing gRNA design, use of engineered reverse transcriptase enzymes, and splitting prime editing components have improved the efficacy of prime editing in plants. These emerging strategies and existing technologies are reviewed along with various perspectives on their future improvement and the development of robust precision genome editing technologies for plants.

The genome of the endangered Macadamia jansenii displays little diversity but represents an important genetic resource for plant breeding.

Macadamia, a recently domesticated expanding nut crop in the tropical and subtropical regions of the world, is one of the most economically important genera in the diverse and widely adapted Proteaceae family. All four species of Macadamia are rare in the wild with the most recently discovered, M. jansenii, being endangered. The M. jansenii genome has been used as a model for testing sequencing methods using a wide range of long read sequencing techniques. Here, we report a chromosome level genome assembly, generated using a combination of Pacific Biosciences sequencing and Hi-C, comprising 14 pseudo-molecules, with a N50 of 52 Mb and a total genome assembly size of 758 Mb of which 56% is repetitive. Completeness assessment revealed that the assembly covered -97.1% of the conserved single copy genes. Annotation predicted 31,591 protein coding genes and allowed the characterization of genes encoding biosynthesis of cyanogenic glycosides, fatty acid metabolism, and anti-microbial proteins. Re-sequencing of seven other genotypes confirmed low diversity and low heterozygosity within this endangered species. Important morphological characteristics of this species such as small tree size and high kernel recovery suggest that M. jansenii is an important source of these commercial traits for breeding. As a member of a small group of families that are sister to the core eudicots, this high-quality genome also provides a key resource for evolutionary and comparative genomics studies.

Genomic Prediction for Whole Weight, Body Shape, Meat Yield, and Color Traits in the Portuguese Oyster Crassostrea angulata.

Genetic improvement for quality traits, especially color and meat yield, has been limited in aquaculture because the assessment of these traits requires that the animals be slaughtered first. Genotyping technologies do, however, provide an opportunity to improve the selection efficiency for these traits. The main purpose of this study is to assess the potential for using genomic information to improve meat yield (soft tissue weight and condition index), body shape (cup and fan ratios), color (shell and mantle), and whole weight traits at harvest in the Portuguese oyster, Crassostrea angulata. The study consisted of 647 oysters: 188 oysters from 57 full-sib families from the first generation and 459 oysters from 33 full-sib families from the second generation. The number per family ranged from two to eight oysters for the first and 12-15 oysters for the second generation. After quality control, a set of 13,048 markers were analyzed to estimate the genetic parameters (heritability and genetic correlation) and predictive accuracy of the genomic selection for these traits. The multi-locus mixed model analysis indicated high estimates of heritability for meat yield traits: 0.43 for soft tissue weight and 0.77 for condition index. The estimated genomic heritabilities were 0.45 for whole weight, 0.24 for cup ratio, and 0.33 for fan ratio and ranged from 0.14 to 0.54 for color traits. The genetic correlations among whole weight, meat yield, and body shape traits were favorably positive, suggesting that the selection for whole weight would have beneficial effects on meat yield and body shape traits. Of paramount importance is the fact that the genomic prediction showed moderate to high accuracy for the traits studied (0.38-0.92). Therefore, there are good prospects to improve whole weight, meat yield, body shape, and color traits using genomic information. A multi-trait selection program using the genomic information can boost the genetic gain and minimize inbreeding in the long-term for this population.

Genomic selection and genetic gain for nut yield in an Australian macadamia breeding population.

BACKGROUND: Improving yield prediction and selection efficiency is critical for tree breeding. This is vital for macadamia trees with the time from crossing to production of new cultivars being almost a quarter of a century. Genomic selection (GS) is a useful tool in plant breeding, particularly with perennial trees, contributing to an increased rate of genetic gain and reducing the length of the breeding cycle. We investigated the potential of using GS methods to increase genetic gain and accelerate selection efficiency in the Australian macadamia breeding program with comparison to traditional breeding methods. This study evaluated the prediction accuracy of GS in a macadamia breeding population of 295 full-sib progeny from 32 families (29 parents, reciprocals combined), along with a subset of parents. Historical yield data for tree ages 5 to 8 years were used in the study, along with a set of 4113 SNP markers. The traits of focus were average nut yield from tree ages 5 to 8 years and yield stability, measured as the standard deviation of yield over these 4 years. GBLUP GS models were used to obtain genomic estimated breeding values for each genotype, with a five-fold cross-validation method and two techniques: prediction across related populations and prediction across unrelated populations. RESULTS: Narrow-sense heritability of yield and yield stability was low (h2 = 0.30 and 0.04, respectively). Prediction accuracy for yield was 0.57 for predictions across related populations and 0.14 when predicted across unrelated populations. Accuracy of prediction of yield stability was high (r = 0.79) for predictions across related populations. Predicted genetic gain of yield using GS in related populations was 474 g/year, more than double that of traditional breeding methods (226 g/year), due to the halving of generation length from 8 to 4 years. CONCLUSIONS: The results of this study indicate that the incorporation of GS for yield into the Australian macadamia breeding program may accelerate genetic gain due to reduction in generation length, though the cost of genotyping appears to be a constraint at present.

Discovery of Single Nucleotide Polymorphisms for Resistance to Abnormal Vertical Growth in Macadamia.

Abnormal vertical growth (AVG) syndrome is a serious threat to the Australian macadamia industry as it decreases the yield of nuts by as much as 70% per annum. A lack of information on the cause of AVG has hindered the development of an effective disease management strategy. Discovery of genetic markers associated with disease resistance can be used as tool for rapid selection of elite cultivars, hence helps in efficient disease management. Differences in field susceptibility of macadamia cultivars provide an opportunity for discovery of genetic markers that are associated with host resistance. REML mixed model analysis was performed to estimate the AVG rating of 51 cultivars from multiple origins using phenotypic data from 359 trees planted in four sites. Most of the Hawaiian cultivars were found as susceptible, while selections from the Australian macadamia industry breeding program were predominantly resistant. All the cultivars were genotyped for 13,221 DArTseq-based single nucleotide polymorphism (SNP) markers. A bulked sample analysis was performed using 20 genotypes each at the extremes of AVG phenotypic ratings. Ten SNP markers were predicted to be associated with AVG resistance and two arbitrarily selected SNP markers were validated using PCR and Sanger sequencing. Our findings suggest that AVG resistance in the commercial cultivars may be derived from the genomic introgression of Macadamia tetraphylla through interspecific hybridization. The results may support marker-assisted selection for macadamia germplasm with AVG resistance.

Genetic Structure of Wild Germplasm of Macadamia: Species Assignment, Diversity and Phylogeographic Relationships.

Macadamia is an Australian native rainforest tree that has been domesticated and traded internationally for its premium nuts. Common cultivars rely upon a limited gene pool that has exploited only two of the four species. Introducing a more diverse germplasm will broaden the genetic base for future crop improvement and better adaptation for changing environments. This study investigated the genetic structure of 302 accessions of wild germplasm using 2872 SNP and 8415 silicoDArT markers. Structure analysis and principal coordinate analysis (PCoA) assigned the 302 accessions into four distinct groups: (i) Macadamia integrifolia, (ii) M. tetraphylla, and (iii) M. jansenii and M. ternifolia, and (iv) admixtures or hybrids. Assignment of the four species matched well with previous characterisations, except for one M. integrifolia and four M. tetraphylla accessions. Using SNP markers, 94 previously unidentified accessions were assigned into the four distinct groups. Finally, 287 accessions were identified as pure examples of one of the four species and 15 as hybrids of M. integrifolia and M. tetraphylla. The admixed accessions showed the highest genetic diversity followed by M. integrifolia, while M. ternifolia and M. jansenii accessions were the least diverse. Mantel test analysis showed a significant correlation between genetic and geographic distance for M. integrifolia (r = 0.51, p = 0.05) and a positive but not significant correlation for M. tetraphylla (r = 0.45, p = 0.06). This study provides a population genetics overview of macadamia germplasm as a background for a conservation strategy and provides directions for future macadamia breeding.

Genome-wide association studies for yield component traits in a macadamia breeding population.

BACKGROUND: Breeding for new macadamia cultivars with high nut yield is expensive in terms of time, labour and cost. Most trees set nuts after four to five years, and candidate varieties for breeding are evaluated for at least eight years for various traits. Genome-wide association studies (GWAS) are promising methods to reduce evaluation and selection cycles by identifying genetic markers linked with key traits, potentially enabling early selection through marker-assisted selection. This study used 295 progeny from 32 full-sib families and 29 parents (18 phenotyped) which were planted across four sites, with each tree genotyped for 4113 SNPs. ASReml-R was used to perform association analyses with linear mixed models including a genomic relationship matrix to account for population structure. Traits investigated were: nut weight (NW), kernel weight (KW), kernel recovery (KR), percentage of whole kernels (WK), tree trunk circumference (TC), percentage of racemes that survived from flowering through to nut set, and number of nuts per raceme. RESULTS: Seven SNPs were significantly associated with NW (at a genome-wide false discovery rate of < 0.05), and four with WK. Multiple regression, as well as mapping of markers to genome assembly scaffolds suggested that some SNPs were detecting the same QTL. There were 44 significant SNPs identified for TC although multiple regression suggested detection of 16 separate QTLs. CONCLUSIONS: These findings have important implications for macadamia breeding, and highlight the difficulties of heterozygous populations with rapid LD decay. By coupling validated marker-trait associations detected through GWAS with MAS, genetic gain could be increased by reducing the selection time for economically important nut characteristics. Genomic selection may be a more appropriate method to predict complex traits like tree size and yield.

Scion control of miRNA abundance and tree maturity in grafted avocado.

BACKGROUND: Grafting is the common propagation method for avocado and primarily benefits orchard production by reducing the time to tree productivity. It also allows use of scions and rootstocks specifically selected for improved productivity and commercial acceptance. Rootstocks in avocado may be propagated from mature tree cuttings ('mature'), or from seed ('juvenile'). While the use of mature scion material hastens early bearing/maturity and economic return, the molecular factors involved in the role of the scion and/or rootstock in early bearing/reduced juvenility of the grafted tree are still unknown. RESULTS: Here, we utilized juvenility and flowering associated miRNAs; miR156 and miR172 and their putative target genes to screen pre-graft and post-graft material in different combinations from avocado. The abundance of mature miR156, miR172 and the miR156 target gene SPL4, showed a strong correlation to the maturity of the scion and rootstock material in avocado. Graft transmissibility of miR156 and miR172 has been explored in annual plants. Here, we show that the scion may be responsible for grafted tree maturity involving these factors, while the rootstock maturity does not significantly influence miRNA abundance in the scion. We also demonstrate that the presence of leaves on cutting rootstocks supports graft success and contributes towards intergraft signalling involving the carbohydrate-marker TPS1. CONCLUSION: Here, we suggest that the scion largely controls the molecular 'maturity' of grafted avocado trees, however, leaves on the rootstock not only promote graft success, but can influence miRNA and mRNA abundance in the scion. This constitutes the first study on scion and rootstock contribution towards grafted tree maturity using the miR156-SPL4-miR172 regulatory module as a marker for juvenility and reproductive competence.

Ultra-high-throughput DArTseq-based silicoDArT and SNP markers for genomic studies in macadamia.

Macadamia (Macadamia integrifolia, M. tetraphylla and hybrids) is an Australian native nut crop and has a significant economic value in the food industries worldwide. Long juvenility along with traditional breeding strategies impede quick genetic improvement of this crop. The existing cultivars constitute only second to fourth generation of the wild germplasm in the rainforest. The utilisation of molecular markers for genomic selection and genome-wide association studies may accelerate genetic gains. Identification of a robust, reproducible, and cost-effective marker system is instrumental in increasing the efficiency of genomic studies. This study is the first to report the potential of two ultra-high-throughput diversity array technology (DArT) markers (silicoDArT and SNP) in macadamia. Both markers were used to identify the genetic diversity and population structure in 80 macadamia cultivars. Parentage analysis of 25 scions in a rootstock trial was conducted to confirm plant identity where recorded identities did not corroborate with phenotypic field observations. A total of 22,280 silicoDArT and 7,332 SNP markers were reported, of which 11,526 silicoDArT and 3,956 SNP markers were used for analyses after screening with quality control parameters including >95% call rate, >95% reproducibility, and >0.05 one ratio. The average polymorphic information content (PIC) values of silicoDArT and SNP markers were 0.29 and 0.21, respectively. Genetic variance among the cultivars ranged from 0.003 to 0.738 in silicoDArT and 0.004 to 0.412 in SNP markers. Four distinct population groups were identified from SNP data analysis. Most of the accessions used in this study were descended from two or more populations. Cluster analysis clearly separated genotypes of distinct origins, such as the Hawaii Agricultural Experiment Station and Hidden Valley Plantation accessions. Two wild accessions of Macadamia jansenii and M. ternifolia were found to be distantly related to the cultivars. Wild germplasm individuals and their hybrids with cv. '660' formed separate clusters, suggesting that crossing between wild and cultivated genepools can extend genetic diversity. DArTseq-based SNP markers were successfully utilized to confirm the genetic identity of 25 scions in a rootstock trial. Our study suggests that DArT platforms are a robust system for the facilitation of genomic studies with regard to macadamia.