Dataset Information

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

ABSTRACT: 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).

SUBMITTER: Liu Y

PROVIDER: S-EPMC6977447 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Liu Yi Y Zhang Anning A Wang Feiming F Wang Jiahong J Bi Junguo J Kong Deyan D Zhang Fenyun F Luo Lijun L Liu Guolan G Yu Xinqiao X

Breeding science 20191102 4

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) meth ...[more]

PMID: 31988636

Similar Datasets

Project description:BackgroundThe rice Waxy (Wx) gene plays a major role in seed amylose synthesis and consequently controls grain amylose content. Wx gene expression is highly regulated at the post-transcriptional level. In particular, the GT/TT polymorphism at the 5'splicing site of its 1st intron greatly affects this intron's splicing efficiency and defines two predominant Wx alleles, Wxa and Wxb. Wxa rice often harbours intermediate to high amylose contents, whereas Wxb rice exhibits low to intermediate amylose contents. By deleting the Wx 1st intron using CRISPR/Cas9 technology, we generate a completely novel Wx allele and further investigate how intron removal affects Wx gene expression and rice grain amylose content.ResultsCRISPR/Cas9-mediated targeted deletion of the Wx 1st intron was performed on 4 rice inbred lines: KY131 (Wxb), X32 (Wxb), X35 (Wxa) and X55 (Wxlv). Deletion of the 1st intron occurred in 8.6-11.8% of the primary transformants of these 4 inbred lines. Compared to wild-type plants, amylose content was significantly increased from 13.0% to approximately 24.0% in KY131 and X32 mutant lines, which both carried the Wxb allele. However, no significant difference in amylose content was observed between wild-type plants and X35 and X55 mutant lines, which carried the Wxa and Wxlv alleles, respectively. Wx gene expression analysis of wild-type plants and mutants yielded results that were highly consistent with amylose content results. KY131 and X32 mutants accumulated increased levels of steady mRNA transcripts compared with wild-type plants, whereas steady mRNA levels were not altered in X35 and X55 mutants compared with wild-type plants. Grain quality, including appearance quality and eating and cooking quality, which are tightly associated with amylose content, was also assessed in wild-type and mutant plants, and data were presented and analysed.ConclusionsThis study presents a novel and rapid strategy to increase amylose content in inbred rice carrying a Wxb allele. Our data strongly suggest that the 1st intron of the Wx gene regulates Wx gene expression mainly at the post-transcriptional level in rice. This finding is in contrast to a previous hypothesis suggesting that it influences Wx gene transcription. In addition, removal of the first intron generates a completely novel Wx allele. Further studies on this new Wx allele will provide invaluable insights into the regulation of Wx gene expression, which will help researchers engineer new Wx alleles to facilitate the breeding of rice cultivars with better eating and cooking quality.

Project description:Apparent amylose content (AAC) is one of the most important parameters in rice quality evaluation. In this study, four rice reference materials used to test rice AAC were developed. The AAC of rice reference materials were measured by a spectrophotometric method with a defatting procedure, calibrated from potato amylose and waxy rice amylopectin at the absorption wavelengths of 620 and 720 nm. Homogeneity test (n = 20) was judged by F-test based on the mean squares of among and within bottles, and short- and long-term stability monitoring was performed by T-test to check if there was significant degradation at the delivery temperature of under 40 °C (14 days) and at 0-4 °C storage condition (18 months), respectively. After joint evaluation by ten laboratories, Dixion and Cochran statistical analyses were presented. The expanded uncertainties were calculated based on the uncertainty of homogeneity, short- and long-term stability, and inter-laboratory validation containing factor k = 2. It found that the four reference materials were homogenous and stable, and had the AAC (g/100 g, k = 2) of 2.96 ± 1.01, 10.68 ± 0.66, 17.18 ± 1.04, and 16.09 ± 1.29, respectively, at 620 nm, and 1.46 ± 0.49, 10.44 ± 0.56, 16.82 ± 0.75, and 24.33 ± 0.52, respectively, at 720 nm. It was indicated that 720 nm was more suitable for the determination of rice AAC with lower uncertainties. The determinations of the AAC of 11 rice varieties were carried out by two methods, the method without defatting and with calibration from the four rice reference materials and the method with a defatting procedure and calibrating from potato amylose and waxy rice amylopectin. It confirmed that the undefatted rice reference materials could achieve satisfactory results to test the rice samples with the AAC ranging from 1 to 25 g/100 g. It would greatly reduce the time cost and improve testing efficiency and applicability, and provide technical support for the high-quality development of the rice industry.

Dataset Information

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

Publications

Development and validation of a PCR-based functional marker system for identifying the low amylose content-associated gene <i>Wx</i> <i><sup>hp</sup></i> in rice.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets