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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 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.

[...]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.

Waxy corn possessing high amylopectin is widely employed as an industrial product. Traditional corn contains ~ 70–75% amylopectin, whereas waxy corn with the mutant waxy1 ( wx1 ) gene possesses ~ 95–100% amylopectin. Marker-assisted breeding can greatly hasten the transfer of the wx1 allele into normal corn. However, the available gene-based marker(s) for wx1 are not always polymorphic between recipient and donor parents, thereby causing a considerable delay in the molecular breeding program. Here, a 4800 bp sequence of the wx1 gene was analyzed among seven wild-type and seven mutant inbreds employing 16 overlapping primers. Three polymorphisms viz., 4 bp InDel (at position 2406 bp) in intron-7 and two SNPs (C to A at position 3325 bp in exon-10 and G to T at position 4310 bp in exon-13) differentiated the dominant ( Wx1 ) and recessive ( wx1 ) allele. Three breeder-friendly PCR markers (WxDel4, SNP3325_CT1, and SNP4310_GT2) specific to InDel and SNPs were developed. WxDel4 amplified 94 bp among mutant-type inbreds, while 90 bp was amplified among wild-type inbreds. SNP3325_CT1 and SNP4310_GT2 revealed the presence-absence polymorphisms with an amplification of 185 bp and 189 bp of amplicon, respectively. These newly developed markers showed 1:1 segregation in both BC 1 F 1 and BC 2 F 1 generations, while 1:2:1 segregation was observed in BC 2 F 2 . The recessive homozygotes ( wx1wx1 ) of BC 2 F 2 identified by the markers possessed significantly higher amylopectin (97.7%) compared to the original inbreds ( Wx1Wx1: 72.7% amylopectin). This is the first report of novel wx1 gene-based markers. The information generated here would help in accelerating the development of waxy maize hybrids.

Waxy gene mutations cause the stickiness of maize grains. China is rich in waxy maize landraces and is considered to be the origin of waxy maize. At present, the Waxy alleles found in Chinese waxy maize include wx - D7 , wx - D10 , wx - Cin4 , and wx - 124 , of which wx - Cin4 and wx - 124 are characterized as transposon insertion mutations. The Yunnan area has the most abundant Chinese waxy maize; however, there are still a large number of waxy maize landraces with unknown Waxy alleles. In this study, 20 waxy maize landraces from Yunnan Province of China were used as research materials for waxy gene analysis, and a new Waxy allele was detected. Molecularly, this allele is characterized by an ~ 5.4-Kb retrotransposon Reina inserted in the tenth intron of the Waxy gene, and we named this allele wx - Reina . Reina is a member of the long terminal repeat (LTR) retrotransposon family. This Reina transposon has the same LTR sequence at both ends, so this mutation is a newly formed mutation. Through reverse transcription PCR (RT-PCR) analysis, we found that Reina insertion results in the deletion of exons 10 and 11 in the transcript of wx - Reina, so its original gene function is lost. The discovery of wx - Reina further enriches the knowledge of types of waxy alleles in Chinese waxy maize. Combined with our previous studies, we believe that transposon activity is one of the main driving forces for the formation of Waxy gene alleles in Chinese waxy maize.

Background In the context of climate change, maize is facing unprecedented heat stress (HS) threats during grain filling. Understanding how HS affects yield is the key to reducing the impact of climate change on maize production. Suyunuo5 (SYN5) and Yunuo7 (YN7) were used as materials, and four temperature gradients of 28℃ (day)/20℃ (night; T0, control), 32 °C/24°C (T1, mild HS), 36 °C/28°C (T2, moderate HS), and 40 °C/32°C (T3, severe HS) were set up during grain filling to explore the physiological mechanism of different degrees HS affecting photosynthetic characteristics of leaves in this study. Results Results showed that HS accelerated the degradation of chlorophyll, disturbed the metabolism of reactive oxygen species, reduced the activity of antioxidant enzymes, and caused leaf damage. Heat stress induced the down-regulation of photosynthesis-related genes, which results in the decrease of enzymatic activities involved in photosynthesis, thereby inhibiting photosynthesis and reducing yield. Integrated analysis showed that the degree of the negative influence of three HS types during grain filling on leaves and yield was T3 > T2 > T1. The increase in HS disturbed leaf physiological activities and grain filling. Meanwhile, this study observed that the YN7 was more heat tolerance than SYN5 and thus it was recommended to use YN7 in waxy maize planting areas with frequent high temperatures. Conclusions Heat stress during grain filling caused premature senescence of the leaves by inhibiting the ability of leaves to photosynthesize and accelerating the oxidative damage of cells, thereby affecting the waxy maize yield. Our study helped to simulate the productivity of waxy maize under high temperatures and provided assistance for a stable yield of waxy maize under future climate warming.

Background Kernel development and starch formation are the primary determinants of maize yield and quality, which are considerably influenced by drought stress. To clarify the response of maize kernel to drought stress, we established well-watered (WW) and water-stressed (WS) conditions at 1–30 days after pollination (dap) on waxy maize ( Zea mays L. sinensis Kulesh ). Results Kernel development, starch accumulation, and activities of starch biosynthetic enzymes were significantly reduced by drought stress. The morphology of starch granules changed, whereas the grain filling rate was accelerated. A comparative proteomics approach was applied to analyze the proteome change in kernels under two treatments at 10 dap and 25 dap. Under the WS conditions, 487 and 465 differentially accumulated proteins (DAPs) were identified at 10 dap and 25 dap, respectively. Drought induced the downregulation of proteins involved in the oxidation–reduction process and oxidoreductase, peroxidase, catalase, glutamine synthetase, abscisic acid stress ripening 1, and lipoxygenase, which might be an important reason for the effect of drought stress on kernel development. Notably, several proteins involved in waxy maize endosperm and starch biosynthesis were upregulated at early-kernel stage under WS conditions, which might have accelerated endosperm development and starch synthesis. Additionally, 17 and 11 common DAPs were sustained in the upregulated and downregulated DAP groups, respectively, at 10 dap and 25 dap. Among these 28 proteins, four maize homologs (i.e., A0A1D6H543, B4FTP0, B6SLJ0, and A0A1D6H5J5) were considered as candidate proteins that affected kernel development and drought stress response by comparing with the rice genome. Conclusions The proteomic changes caused by drought were highly correlated with kernel development and starch accumulation, which were closely related to the final yield and quality of waxy maize. Our results provided a foundation for the enhanced understanding of kernel development and starch formation in response to drought stress in waxy maize.

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).

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.

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.