Molecular Plant Breeding 2024, Vol.15, No.3, 144-154 http://genbreedpublisher.com/index.php/mpb 150 blister rust, exhibited a strong association with disease resistance. This association was validated through PCR-based genotyping, demonstrating the potential of SNP markers in identifying resistant individuals and expediting forest restoration efforts (Table 1) (Wright et al., 2022). Table 1 Fit of observed (O) diploid genotype frequencies to expected (E) Hardy–Weinberg proportions in a random sample of putatively susceptible, Cr1r trees and in a selected sample of resistant, Cr1R treesa (Adopted from Wright et al., 2022) Genotype Random sample Selected sample O E O E A/A 75 76.0 16 24.8 A/B 12 12.0 0 4.7 A/C 15 12.9 64 41.8 B/B 1 0.5 1 0.2 B/C 0 1.0 7 3.9 C/C 0 0.6 5 17.6 Total 103 103.0NS 93 93.0* Note: NS, not significant (P < 0.45); *P < 9.5 × 10–9; A haplotype (restriction sites 1, 2, and 6): 81 and 359 bp; B haplotype (1, 2, and 6 plus 3 and 4): 81 30 86 and 243 bp; and C haplotype (1, 2, and 6 plus 5): 81 187 and 172 bp Wright et al. (2022) compares observed (O) and expected (E) diploid genotype frequencies under Hardy-Weinberg equilibrium in a random sample of putatively susceptible Cr1r trees and a selected sample of resistant Cr1R trees. In the random sample, the observed frequencies closely match the expected ones, indicating no significant deviation from Hardy-Weinberg proportions (P < 0.45). However, in the selected sample of resistant trees, significant deviations are observed (P < 9.5 × 10–9), particularly with the A/C and B/C genotypes showing substantial differences between observed and expected frequencies. This suggests strong selection pressure for resistance traits. The haplotypes are defined by restriction sites, highlighting genetic variations influencing disease resistance. This research underscores the importance of specific haplotypes in breeding programs aimed at enhancing disease resistance in Cr1R trees. In another study, the genomic architecture of quantitative disease resistance in sugar pine was investigated using quantitative trait loci mapping and genome-wide association studies. The study identified 453 SNPs involved in various biological functions, including disease resistance. These findings suggest that newly reported genes may have partial resistance or epistatic effects on qualitative disease resistance genes, providing a deeper understanding of the complex genomic architecture of disease resistance in long-generation trees (Weiss et al., 2020). Furthermore, the application of the CRISPR/Cas9 system in developing disease-resistant cultivars has been systematically reviewed, highlighting its versatility and effectiveness in editing genes and noncoding sequences. This study underscores the potential of CRISPR/Cas9 in promoting tolerance to multiple abiotic and biotic stresses, thereby improving the overall health and resilience of pine species (Nascimento et al., 2023). 8 Challenges and Future Perspectives 8.1 Technical challenges in pine genome editing Genome editing in pine species presents several technical challenges. One significant issue is the complexity of the pine genome, which is large and highly repetitive, making precise editing difficult (Liu et al., 2019). Additionally, the long generation time of pine trees complicates the process of validating edits and observing phenotypic changes (Weiss et al., 2020). The efficiency of current genome editing tools, such as CRISPR/Cas9, is also a concern, as these tools need to be optimized for the unique characteristics of pine genomes (Mushtaq et al., 2019). Furthermore, the delivery of genome editing components into pine cells is challenging due to the thick cell walls and the recalcitrant nature of pine tissues to transformation (Mishra et al., 2021).
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