Development and validation of a PCR-based functional marker system for identifying the low amylose content-associated gene Wx(hp) in rice

Low amylose content (AC) is a desirable trait for rice (Oryza sativa L.) cooking quality and is selected in soft rice breeding. The Wx(hp) allele was derived from a Yunnan rice landrace in China, Haopi, with low AC. To efficiently and rapidly utilize the low amylose content-associated gene Wx(hp) in rice molecular breeding programs, we developed a tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method, according to the single-nucleotide variation of the Wx(hp). Four Wx(hp)-specific primers were used to perform PCR assays using genomic DNA extracted from several rice varieties. Based on the band pattern of the amplified products after electrophoresis, this method can accurately distinguish three Wx(hp)-related genotypes (i.e., Wx(hp) homozygotes, Wx(hp) heterozygotes, and wild-type), and the genotypes completely correspond to the appearance of mature endosperm. This method represents a novel approach that is both inexpensive and highly efficient and can be widely used for genotyping Wx(hp) alleles in rice germplasm collections and may aid breeding programs with marker-assisted selection (MAS).