Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 72-78 http://cropscipublisher.com/index.php/tgg 74 simultaneously recognize three versions of TaGW2 in wheat (i.e. 6A, 6B, and 6D). We will use some computational tools to help with the design, which can reduce accidental damage to other genes (Wang et al., 2018). In order to knock out multiple versions of genes at the same time, multiple gRNAs can be strung together and connected with tRNA. In this way, a transcript can carry multiple gRNAs, which will be accurately cut in the cell, improving the overall editing efficiency (Xie et al., 2015). 3.2 Construction of editing vectors and system validation Usually, we will put the CRISPR/Cas9 system into a binary vector. This vector contains the Cas9 protein, which is driven by a strong promoter (such as the Ubi promoter in maize), and a gRNA module controlled by the wheat U6 promoter (Zhang et al., 2019). We use Agrobacterium to transfer this system into wheat cells, which is more stable than the gene gun and can reduce the number of transformations and gene silencing (Zhang et al., 2019). In transgenic wheat, we will check whether Cas9 and gRNA are expressed normally and confirm whether the target site is successfully edited. 3.3 Mutant identification and mutation type analysis We use PCR to amplify the target region of TaGW2 and then use sequencing to confirm whether mutations have occurred. In wheat, the most common mutation is "deletion", which sometimes exceeds 10 base pairs. Homozygous mutants or biallelic mutants can sometimes be obtained in the first generation (T0), and sometimes need to wait until the next few generations to appear (Zhang et al., 2019). The CRISPR/Cas9 system can continue to work in the next generation, sometimes generating new mutations in the offspring, which can bring more genetic changes (Wang et al., 2018). In order to ensure that this system does not cut randomly, we also perform off-target checks. Many studies have found that as long as the gRNA is well designed, off-target problems will hardly occur or cannot be detected at all. 4 Phenotypic Changes and Grain Weight in TaGW2 Mutants 4.1 Analysis of grain length, width, and thousand-kernel weight Studies have found that TaGW2 gene mutations can increase grain size and thousand-grain weight (TGW). Compared with the wild type, single-gene mutations can increase grain length by about 2%, width by 2%-3%, and TGW by 5%-7% (Simmonds et al., 2016; Wang et al., 2018; Bi et al., 2024). If two genes mutate together, the effect is more obvious, and TGW can be increased by 10%-12%. When all three genes mutate, the increase in TGW is the largest, reaching 16%-21% (Zhang et al., 2018). This increase is quite stable in different wheat varieties and environments. Moreover, the more mutations there are, the more obvious the increase in grain weight is. This shows that these genes have an additive effect. In particular, the two alleles TaGW2-6B and TaGW2-6A have a greater effect on grain width and weight (Yang et al., 2012). 4.2 Effects of gene editing on plant architecture and growth period Although we mainly want to improve grain traits by editing TaGW2, it sometimes affects other agronomic traits. Studies have found that some TaGW2 mutations are related to early heading and early maturity of wheat, and may also change plant height and ear structure (Jaiswal et al., 2015). However, most edited varieties have no major changes in the number of spikelets and grains. In other words, these mutations mainly make the grains themselves larger and do not affect the appearance of the whole plant much (Simmonds et al., 2016). Sometimes there is a small "trade-off": the grain weight increases, but the number of grains per ear may decrease slightly. But overall, the yield can still be maintained, or even higher (Zhai et al., 2017; Vicentin et al., 2024). 4.3 Correlation analysis between phenotype and genotype The number of copies of TaGW2 is negatively correlated with grain size and TGW. In other words, the more copies are mutated, the larger and heavier the grains are (Wang et al., 2018; Zhang et al., 2018). Genetic variation in the promoter region can also affect expression levels, which may be one of the reasons for the phenotypic differences between different varieties (Qin et al., 2014; Jaiswal et al., 2015). Now, we can use SNPs or haplotypes related to TaGW2 for molecular marker-assisted selection, so that we can more quickly select lines with larger grain weight and higher yield (Yang et al., 2012). Different combinations of homologous genes and the special reactions between varieties show that the relationship between genotype and phenotype is very critical. Understanding these relationships will help to better optimize breeding strategies (Bi et al., 2024).
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