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Rice eating and cooking quality is largely determined by starch physicochemical properties. The diverse accessions in the USDA rice mini-core collection (URMC) facilitate extensive association analysis of starch physicochemical properties with molecular markers specific to starch biosynthesis related genes. To identify significant trait-marker associations that can be utilized in rice breeding programs for improved starch quality, we conducted two association analyses between 26 molecular markers derived from starch biosynthesis related genes and 18 parameters measured of starch physicochemical properties in two sets of the mini-core accessions successfully grown in two environments in China. Many significant trait-marker associations ( < 0.001) were detected in both association analyses. Five markers of gene, including the (CT) repeats, the G/T SNP of intron 1, the 23 bp sequence duplication (InDel) of exon 2, the A/C SNP of exon 6, and the C/T SNP of exon 10, were found to be primarily associated with starch traits related to apparent amylose content (AAC), and two markers targeting the 4,329-4,330 bp GC/TT SNPs and 4,198 bp G/A SNP of gene were mainly associated with traits related to gelatinization temperature (GT). Two new haplotypes were found in the mini-core collection based on the combinations of the 23 bp InDel and three SNPs (G/T of intron 1, A/C of exon 6, and C/T of exon 10) of gene. Furthermore, our analyses indicated that the (CT) polymorphisms of gene had a non-negligible effect on AAC related traits, as evidenced by significant variation in AAC related traits among rice accessions with the same SNPs but different (CT) repeats. As the five markers and the two markers showed consistent major effects on starch quality traits across studies, these markers should have priority for utilization in marker-assisted breeding.

[...]we have successfully mutated waxy gene to generate glutinous wheat with lower amylose content. PPO catalyses phenols oxidation into dark-coloured products, a feature often undesirable for wheat end-use products. [...]developing wheat cultivars with low PPO activity has always been an important goal in wheat breeding. [...]new allelic variations of the target genes (pinb, waxy, ppo and psy) were created in Fielder through Agrobacterium-delivered CRISPR/Cas9 system. Furthermore, many of the mutants had segregated out the CRISPR/Cas9 transgene. [...]we had successfully obtained new wheat germplasms with improved grain quality in hardness, starch composition and dough colour.

SH2 and BT2 are the large (SH2) and small (BT2) subunit of ADP‐glucose Pyrophosphorylase genes; GBSS, the WAXY gene encodes the enzyme GRANULE BOUND STARCH SYNTHASE; SS, the soluble starch synthase genes; SBE, starch branching enzyme genes; DBE, the debranching enzyme genes; Red crosses represent CRISPR/Cas9 targeted‐knockout activity. (b) The CRISPR/Cas9 construct to target the SH2 and WX genes in duplex. Bar, bialaphos resistance marker; E35S, 35S CaMV promoter; LB, T‐DNA left border; NLS, nuclear location signal sequence; Nos, Nos terminator; RB, T‐DNA right border; sgRNA‐SH2, single‐guide RNA targeting the SH2 gene; sgRNA‐WX, single‐guide RNA targeting the WX gene; Ubi, the promoter of the ubiquitin 1 gene; U6‐2, the maize endogenous U6 PolIII promoter. (c) and (d) The targeted mutations on the SH2 (c) and WX (d) genes. Counts, variant counts identified in 52 T1 events; Nucleotide sequence in red shows the mutations; PAM, proto‐spacer‐motif; Red dash in sequence, deletions; Blue sequence, the sgRNA location; Sequence underlined, the PAM sequence; sgRNA, single‐guide RNA; Var., variants of identified mutations; WT, the wild type. (e) and (f) The diagram of the genetic basis for SWC production (e) and the morphology of corn ears from genome edited lines (f).Texts in red indicate supersweet traits and genotypes while those in blue indicate waxy traits and genotypes. Acknowledgements This work was supported by grants from Beijing Municipal Science and Technology (Major Program D171100007717001), the Key Area Research and Development Program of Guangdong Province (2018B020202008), the China National Major Research and Development Program (2016YFD0101803), the National Science Foundation of China (No. 31771808) and the National Engineering Laboratory for Crop Molecular Breeding.

The genetic diversity and phenotypic variability of crop agronomic traits is valued by breeders for their benefits in crop breeding but are limited for most target traits. The rice Waxy (Wx) gene (LOC_Os06g04200) encodes granule‐bound starch synthase I (GBSSI), which determines the amylose content (AC) of endosperm by controlling amylose synthesis. The number of altered bases in each line (coloured in red) is indicated by the letter S followed by a number. (c) A structural model of Wxb constructed using the PROTEIN DATA BANK server; mutated residues contributing to the changes of AC are shown as spheres and are coloured (P124 in apricot, R125 in blue, R158 in red violet, G159 in white, V160 in green, D161 in red, T178 in orange and Y191 in purple). (d) Analysis of potential off‐target sites in the seven T1 edited lines. To determine the effect of these mutations on AC, we measured the apparent amylose contents (AACs) of grains from the seven mutant lines (Wxm5‐Wxm11), NIP (Wxb) and a 'soft rice' control Nangeng9108 (NG9108) (Wxmp) (Figure 1e).

Amylose biosynthesis is strictly associated with granule-bound starch synthase I (GBSSI) encoded by the Waxy gene. Mutagenesis of single bases in the Waxy gene, which induced by CRISPR/Cas9 genome editing, caused absence of intact GBSSI protein in grain of the edited line. The amylose and amylopectin contents of waxy mutants were zero and 31.73%, while those in the wild type were 33.50% and 39.00%, respectively. The absence of GBSSI protein led to increase in soluble sugar content to 37.30% compared with only 10.0% in the wild type. Sucrose and β-glucan, were 39.16% and 35.40% higher in waxy mutants than in the wild type, respectively. Transcriptome analysis identified differences between the wild type and waxy mutants that could partly explain the reduction in amylose and amylopectin contents and the increase in soluble sugar, sucrose and β-glucan contents. This waxy flour, which showed lower final viscosity and setback, and higher breakdown, could provide more option for food processing.Key messageThe new zero amylose barley was obtained via the CRISPR/Cas9 genome editing, showing higher soluble sugar content and lower final viscosity and setback.

Background Starch, a major component of wheat ( Triticum aestivum L.) grain, plays a crucial role in determining processing quality. Granule-bound starch synthase I (GBSSI), the enzyme primarily responsible for elongating α-1,4-glucan chains into linear amylose molecules, is a key determinant of starch quality. In this study, a mutant population of the wheat cultivar SM126, a high-quality variety form Sichuan, China, was generated using ethyl methanesulfonate (EMS) mutagenesis. This research investigates the effects of GBSSI inactivation on starch structure and functionality. Results A waxy mutant (Wx-Abd) was identified by screening an M4 seed library with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of grain endosperm flour. DNA sequencing revealed a single nucleotide polymorphism (SNP) in the fourth exon, causing a premature stop codon and inactivation of the Wx-Abd allele. In previous work, the Wx-abD mutant was identified in the M2 generation, and crossing the M2-31 line with the M4-6165 line produced four distinct Wx protein subunits in the SM126 background. Comparisons between the Wx-abd line and the wild-type SM126 (Wx-AbD) showed significant differences in starch properties. The Wx-abd line exhibited reduced Wx gene expression, a distinct surface depression on starch granules, and a higher proportion of B-type starch granules. Notably, it exhibited significantly lower amylose content (7.02%) compared to SM126 (22.32%), along with a reduction in total starch content. Additionally, the Wx-abd line showed a higher gelatinization temperature. Conclusion Inactivation of GBSSI in the Wx-abd line resulted in altered starch structure, particularly a decrease in amylose content and changes in granule morphology. These findings suggest that the Wx-abd line represents a valuable genetic resource for wheat breeding programs focused on improving starch quality for food production, with its high agronomic performance making it suitable for further breeding applications.

The waxy gene mutation causes waxy maize grain to have a sticky quality. China has numerous waxy maize landraces and is thought to be the place of origin of waxy maize. The most abundant waxy maize resources in China are located in the Yunnan province and its surrounding areas. We collected 57 waxy maize landraces from Yunnan province and cloned and sequenced the waxy gene from its fourth to eighth exon. Two new waxy gene mutations, named wx-Cin4 and wx-124 , were identified. The wx-Cin4 mutation is a 466-bp retrotransposon inserted into exon six. The wx-124 mutation is a 116-bp miniature inverted-repeat transposable element inserted into exon seven. This is the first time a 124 -type mutation has been found in a maize waxy gene. The discovery of the two specific waxy mutations from landraces collected in Yunnan province provides new evidence supporting the hypothesis that China is the origin area for waxy maize.

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.

Summary It is well known that the overall quality of japonica/geng rice is superior to that of indica/xian rice varieties. However, the molecular mechanisms underlying the quality disparities between these two subspecies of rice are still largely unknown. In this study, we have pinpointed a gene homologous to SLR1, termed LCG1, exhibiting significant expression during early caryopsis development and playing a specific role in regulating rice chalkiness and taste by affecting the accumulation of grain storage components, starch granule structure and chain length distribution of amylopectin. LCG1 physically interacts with OsBP5 and indirectly influences the expression of the amylose synthesis gene Waxy (Wx) by hindering the transcriptional activity of the OsBP5/OsEBP89 complex. Notably, sequence variations in the promoter region of LCG1 result in enhanced transcription in japonica rice accessions. This leads to elevated LCG1 expression in CSSL‐LCG1Nip, thereby enhancing rice quality. Our research elucidates the molecular mechanism underlying the impact of the LCG1‐OsBP5/OsEBP89‐Wx regulatory pathway on rice chalkiness and taste quality, offering new genetic resources for improving the indica rice quality.

The cooking and eating quality of rice grains is a major focus from a consumer’s perspective and is mainly determined by the apparent amylose content (AAC) of the starch. Waxy rice, a type of rice with an AAC of less than 2%, is an important goal for the breeding of high-quality rice. In recent years, the cloning of the Waxy ( Wx) gene has revealed the molecular mechanism of the formation of waxy traits in rice. However, there have been limited studies on the physicochemical properties, such as gelatinization temperature, rapid viscosity analyzer profile, and amylopectin fine structure of wx mutants. In the current study, a rapid and highly efficient strategy was developed through the CRISPR/Cas9 gene-editing system for generating wx mutants in the background of five different rice varieties. The wx mutation significantly reduced the AAC and starch viscosity but did not affect the major agronomic traits (such as plant height, panicle number per plant, grain number per panicle, and seed-setting frequency). Incorporation of the wx mutation into varieties with low initial AAC levels resulted in further reduction in AAC, but without significantly affecting the original, desirable gelatinization traits and amylopectin structure types, suggesting that parents with low initial AAC should be preferred in breeding programs.