## 3 Results
### Efficient Introgression and Genetic Stability of Pod Shattering Resistance Allele  
Large-scale genotyping utilizing the KASP marker S12.68 revealed high-efficiency introgression of the PSR allele into the breeding lines (as presented in Table 3). Among \begin{table}
\begin{tabular}{c c c} \hline \hline
**Reagent** & **96-Well Plate Volume** & **384-Well Plate Volume** \\ \hline
2\(\times\) KASP V3 Master Mix (LGC) & 5 \(\mu\)L & 2.5 \(\mu\)L \\ \hline Genomic DNA Template & 2 \(\mu\)L & 1 \(\mu\)L \\ \hline Primer Master Mix & 0.14 \(\mu\)L & 0.07 \(\mu\)L \\ \hline ddH\({}_{2}\)O & 2.86 \(\mu\)L & 1.43 \(\mu\)L \\ \hline Total Volume & 10 \(\mu\)L & 5 \(\mu\)L \\ \hline \hline \end{tabular}
\end{table} 
Table 1: 96-well PCR plate or 384-well PCR plate system. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Step** & **Temperature** & **Time** & **Notes** \\ \hline
1 & 94 \({}^{\circ}\)C & 15 min & Initial Denaturation \\ \hline
2 & 94 \({}^{\circ}\)C & 20 s & Denaturation \\ \hline
3 & 61 \({}^{\circ}\)C & 60 s & Annealing/Extension (touchdown: \(-0.6\)\({}^{\circ}\)C per cycle) \\ \hline
4 & Go to Step 2 & — & Repeat for 9 cycles (total touchdown) \\ \hline
5 & 94 \({}^{\circ}\)C & 20 s & Denaturation \\ \hline
6 & 55 \({}^{\circ}\)C & 60 s & Annealing/Extension \\ \hline
7 & Go to Step 5 & — & Repeat for 26 cycles \\ \hline
8 & 37 \({}^{\circ}\)C & 60 s & Fluorescence Measurement \\ \hline \hline \end{tabular}
\end{table} 
Table 2: PCR Cycling conditions. the 634 test plants evaluated, the efficiency of PSR allele introgression reached an unprecedented level:  
Homozygous Resistant (HR): 73.34% (465 plants)  
Heterozygous (Het): 24.92% (158 plants)  
No Introgression (Susceptible): 0.16% (1 plant). This high-efficiency introgression represents a landmark achievement in the genetic improvement of complex traits in rapeseed, significantly surpassing the typical introgression efficiency (<40%) observed in conventional breeding programs. In-depth analysis of specific hybrid combinations (Table 1) revealed substantial variation: the KL01/NR24 population exhibited 100% homozygosity (53/53), whereas the NR20/KL01 population showed a high \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Material Source** & \begin{tabular}{c} **Homozygous** \\ **Susceptible** \\ \end{tabular} & \begin{tabular}{c} **Homozygous** \\ **Resistant** \\ \end{tabular} & \
\begin{tabular}{c} **Heterozygous** \\ **Detected** \\ \end{tabular} &
\begin{tabular}{c} **No Target Allele** \\ **Total** \\ \end{tabular} \\ \hline
0911 & 1 & & & & & 1 \\ KL01 & & 1 & & & & 1 \\ R10 & 1 & & & & & 1 \\ T8 & 1 & & & & & 1 \\ KL01/NR17 & & 23 & 2 & 3 & 28 \\ KL01/NR20 & & 22 & 2 & & 24 \\ KL01/NR21 & & 3 & 2 & & 5 \\ KL01/NR22 & & 26 & & & 26 \\ KL01/NR23 & & 24 & 3 & & 27 \\ KL01/NR24 & & 53 & & & 53 \\ KL01/NR25 & & 12 & 15 & & 27 \\ KL01/NR26 & & 26 & & 1 & 27 \\ KL01/NR27 & & 6 & & & 6 \\ NR17/KL01 & & 11 & 2 & & 13 \\ NR20/KL01 & & 1 & 8 & & 9 \\ NR21/KL01 & & 24 & & & 24 \\ NR22/KL01 & & 25 & & & 25 \\ NR23/KL01 & & 23 & 3 & & 26 \\ NR24/KL01 & & 26 & & 1 & 27 \\ NR25/KL01 & & 12 & 13 & & 25 \\ NR26/KL01 & & 15 & & & 15 \\ NR27/KL01 & & 26 & 1 & & 27 \\ W1/KL01 & & 11 & 15 & 1 & 27 \\ W2/KL01 & & 1 & 1 & & 1 \\ W3/KL01 & & 11 & 10 & 1 & 22 \\ W4/KL01 & & 11 & 5 & 2 & 18 \\ W5/KL01 & & 7 & 19 & & 26 \\ W6/KL01 & & 2 & & & 2 \\ W7/KL01 & & 12 & 14 & & 26 \\ W8/KL01 & & 26 & & 1 & 27 \\ W9/KL01 & & & 1 & & 1 \\ W10/KL01 & & 27 & & & 27 \\ Zao 5/KL01 & 1 & & 15 & & 16 \\ Yuyou 342/KL01 & & & 27 & & 27 \\ Total & 4 & 466 & 159 & 10 & 638 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Differences in introgression efficiency of cleavage angle resistance genes among different hybrid combinations. heterozyg rate of 88.9% (8/9). These results demonstrate that the efficiency of allele introgression is significantly influenced by the parental genetic background (Figure 1). Validation experiments using NR series materials further confirmed the stability of the resistance genotype.