# Development and Application of an SNP Marker for High-Throughput Detection and Utilization of the badh2 Gene in Rice Breeding

## TL;DR

This study develops efficient genetic tools to detect and utilize the badh2 gene in rice breeding, helping to improve aromatic rice varieties.

## Contribution

The development of a multiplex-ready PCR assay and a KASP marker for high-throughput badh2 genotyping in rice.

## Key findings

- Eight japonica aromatic lines had a 7 bp deletion in badh2-E2, while 12 indica lines had an 8 bp deletion in badh2-E7.
- A KASP marker enabled successful screening of an elite aromatic rice line with superior agronomic traits.
- The PCR assay showed high accuracy in genotyping 200 rice plants, with only one potential heterozygous case.

## Abstract

Background: As a key rice breeding resource, aromatic rice is widely cultivated in agriculture due to its unique aroma. Badh2 mutations cause function loss, enabling rice’s characteristic aroma. Methods: In this study, we analyzed several badh2 mutation types across 8 japonica and 16 indica aromatic rice lines. Based on the 7 bp deletion in badh2-E2 identified in japonica aromatic lines, we developed a multiplex-ready PCR assay for badh2 genotyping. Additionally, leveraging the deletion mutation in badh2-E7 from the indica aromatic line Yexiang, we designed a KASP marker. Results: All 8 japonica aromatic lines carried a 7 bp deletion in badh2-E2, while 12 indica aromatic lines harbored an 8 bp deletion in badh2-E7, and 4 additional indica aromatic lines exhibited an 8 bp deletion in badh2-E2. The multiplex-ready PCR assay was used to screen 200 individual plants from the aromatic rice line Jia 58: 199 plants showed the expected results, while the remaining 1 exhibited two fluorescent signal peaks—suggesting that it may be a heterozygous individual. Using the KASP marker, we performed genotyping analysis on F7 progeny individuals derived from the cross between Yexiang (aromatic line) and Yuenongsimiao (non-aromatic line). Combined with phenotypic observations, we successfully screened out an elite aromatic line named Zhexiangzhenhe, which not only possesses aroma but also maintains superior agronomic traits. Conclusions: The multiplex-ready PCR assay and KASP markers facilitate high-throughput genotyping in large-scale breeding populations, providing breeders with a rapid and efficient selection tool to accelerate aromatic trait improvement in rice.