Dataset Information

Improvement of marker-based predictability of Apparent Amylose Content in japonica rice through GBSSI allele mining.

ABSTRACT: BACKGROUND:Apparent Amylose Content (AAC), regulated by the Waxy gene, represents the key determinant of rice cooking properties. In occidental countries high AAC rice represents the most requested market class but the availability of molecular markers allowing specific selection of high AAC varieties is limited. RESULTS:In this study, the effectiveness of available molecular markers in predicting AAC was evaluated in a collection of 127 rice accessions (125 japonica ssp. and 2 indica ssp.) characterized by AAC values from glutinous to 26%. The analyses highlighted the presence of several different allelic patterns identifiable by a few molecular markers, and two of them, i.e., the SNPs at intron1 and exon 6, were able to explain a maximum of 79.5% of AAC variation. However, the available molecular markers haplotypes did not provide tools for predicting accessions with AAC higher than 24.5%. To identify additional polymorphisms, the re-sequencing of the Waxy gene and 1kbp of the putative upstream regulatory region was performed in 21 genotypes representing all the AAC classes identified. Several previously un-characterized SNPs were identified and four of them were used to develop dCAPS markers. CONCLUSIONS:The addition of the SNPs newly identified slightly increased the AAC explained variation and allowed the identification of a haplotype almost unequivocally associated to AAC higher than 24.5%. Haplotypes at the waxy locus were also associated to grain length and length/width (L/W) ratio. In particular, the SNP at the first intron, which identifies the Wxa and Wxb alleles, was associated with differences in the width of the grain, the L/W ratio and the length of the kernel, most likely as a result of human selection.

SUBMITTER: Biselli C

PROVIDER: S-EPMC3904453 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Improvement of marker-based predictability of Apparent Amylose Content in japonica rice through GBSSI allele mining.

Biselli Chiara C Cavalluzzo Daniela D Perrini Rosaria R Gianinetti Alberto A Bagnaresi Paolo P Urso Simona S Orasen Gabriele G Desiderio Francesca F Lupotto Elisabetta E Cattivelli Luigi L Valè Giampiero G

Rice (New York, N.Y.) 20140102 1

<h4>Background</h4>Apparent Amylose Content (AAC), regulated by the Waxy gene, represents the key determinant of rice cooking properties. In occidental countries high AAC rice represents the most requested market class but the availability of molecular markers allowing specific selection of high AAC varieties is limited.<h4>Results</h4>In this study, the effectiveness of available molecular markers in predicting AAC was evaluated in a collection of 127 rice accessions (125 japonica ssp. and 2 in ...[more]

PMID: 24383761

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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.

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.

Dataset Information

Improvement of marker-based predictability of Apparent Amylose Content in japonica rice through GBSSI allele mining.

Publications

Improvement of marker-based predictability of Apparent Amylose Content in japonica rice through GBSSI allele mining.

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