Regulation of Amylose Content by Single Mutations at an Active Site in the Wx-B1 Gene in a Tetraploid Wheat Mutant.

The granule-bound starch synthase I (GBSSI) encoded by the waxy gene is responsible for amylose synthesis in the endosperm of wheat grains. In the present study, a novel Wx-B1 null mutant line, M3-415, was identified from an ethyl methanesulfonate-mutagenized population of Chinese tetraploid wheat landrace Jianyangailanmai (LM47). The gene sequence indicated that the mutated Wx-B1 encoded a complete protein; this protein was incompatible with the protein profile obtained using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed the lack of Wx-B1 protein in the mutant line. The prediction of the protein structure showed an amino acid substitution (G470D) at the edge of the ADPG binding pocket, which might affect the binding of Wx-B1 to starch granules. Site-directed mutagenesis was further performed to artificially change the amino acid at the sequence position 469 from alanine (A) to threonine (T) (A469T) downstream of the mutated site in M3-415. Our results indicated that a single amino acid mutation in Wx-B1 reduces its activity by impairing its starch-binding capacity. The present study is the first to report the novel mechanism underlying Wx-1 deletion in wheat; moreover, it provided new insights into the inactivation of the waxy gene and revealed that fine regulation of wheat amylose content is possible by modifying the GBSSI activity.

A single base change at exon of Wx-A1 caused gene inactivation and starch properties modified in a wheat EMS mutant line.

BACKGROUND: Wheat is an essential source of starch. The GBSS or waxy genes are responsible for synthesizing amylose in cereals. The present study identified a novel Wx-A1 null mutant line from an ethyl methanesulfonate (EMS)-mutagenized population of common wheat cv. SM126 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel analyses. RESULTS: The alignment of the Wx-A1 gene sequences from the mutant and parental SM126 lines showed only one single nucleotide polymorphism causing the appearance of a premature stop codon and Wx-A1 inactivation. The lack of Wx-A1 protein resulted in decreased amylose, total starch and resistant starch. The starch morphology assessment revealed that starch from mutant seeds was more wrinkled, increasing its susceptibility to digestion. Regarding the starch thermodynamic properties, the gelatinization temperature was remarkably reduced in the mutant compared to parental line SM126. The digestibility of native, gelatinized, and retrograded starches was analyzed for mutant M4-627 and the parental SM126 line. In the M4-627 line, rapidly digestible starch contents were increased, whereas resistant starch was decreased in the three types of starch. CONCLUSION: Waxy protein is essential for starch synthesis. The thermodynamic characteristics were decreased in the Wx-A1 mutant line. The digestibility properties of starch were also affected. Therefore, the partial waxy mutant M3-627 might play a significant role in food improvement. Furthermore, it might also be used to produce high-quality noodles. © 2021 Society of Chemical Industry.

Influence of single-nucleotide polymorphisms in the gene encoding granule-bound starch synthase I on amylose content in Vietnamese rice cultivars.

Amylose content is one of the most important factors influencing the physical and chemical properties of starch in rice. Analysis of 352 Vietnamese rice cultivars revealed a wide range of variation in apparent amylose content and the expression level of granule-bound starch synthase. On the basis of single-nucleotide polymorphisms (SNP) at the splicing donor site of the first intron and in the coding region of the granule-bound starch synthase I gene, Waxy gene, alleles can be classified into seven groups that reflect differences in apparent amylose content. The very low and low apparent amylose content levels were tightly associated with a G to T in the first intron whereas intermediate and high amylose was associated with a T genotype at SNP in exon 10. The correlation between the combination of T genotype at SNP in the first intron, C in exon 6, or C in exon 10 was predominant among low amylose rice varieties. Our analysis confirmed the existence of Wx (op) allele in Vietnamese rice germplasm. The results of this study suggest that the low amylose properties of Vietnamese local rice germplasm are attributable to spontaneous mutations at exons, and not at the splicing donor site.

The transformation of the photo-thermo sensitive genic male-sterile line 261S of rice via an expression vector containing the anti-Waxy gene.

Transgenic photo-thermo sensitive genic male sterility Oryza sativa L. cv. "261S" plants with the anti-Waxy gene were successfully obtained using an Agrobacterium tumefaciens-mediated co-transformation method. Marker-free homozygous transgenic lines with the anti-Waxy gene were obtained. The setting seed rates of the transgenic plants via self-pollination or via crossing with the restorer line WX99075 rice and the 1000-grain weight of the transgenic plants and the F2 hybrid seeds obtained by crossing the transgenic or non-transgenic plants with the restorer line WX99075 rice, and the number of panicles of the transgenic plants and yields of the F2 hybrid rice, were analysed. Quality indexes of the transgenic plants and of the F2 hybrid seeds were analysed. Our researches results indicate that hybrid female and hybrid descendant edibility could be improved via the introduction of the anti-Waxy gene, but the grain yields of the reserve seeds via self-pollination of the transgenic photo-thermo sensitive genic sterile lines and of the hybrid rice were not affected.

Generating waxy rice starch with target type of amylopectin fine structure and gelatinization temperature by waxy gene editing.

Waxy rice, which lacks amylose, is an important variant in rice, and its starches have been widely used. New waxy rice varieties generated via the CRISPR/Cas9 gene-editing system is described. Herein, four waxy rice starches with different physicochemical properties were successfully obtained by the CRISPR/Cas9 editing Waxy (Wx) gene. The results showed that the amylose content (AC) of wx mutant starches ranged from 0.26 to 1.78 %, and CZBwx1 starches had the best gel consistency and highest water solubility among all wx mutants. Mutations of Wxb allele produced more short-chains (degree of chain polymerization (DP) 6-11), and less medium- and long-chains (DP12-70) than that of Wxa, while the AC of Wxa allele mutants was higher than the mutations of Wxb allele. The gelatinization temperature (GT) of wxa mutant starches was higher than that of wxb mutant starches. Moreover, we found that the GT and amylopectin fine structure type of waxy rice starch did not change after Wx gene editing. Based on these findings, it is possible to produce waxy rice starch with different physicochemical properties, containing target GT and chain length distributions of amylopectin.

Simple and rapid molecular techniques for identification of amylose levels in rice varieties.

The polymorphisms of Waxy (Wx) microsatellite and G-T single-nucleotide polymorphism (SNP) in the Wx gene region were analyzed using simplified techniques in fifteen rice varieties. A rapid and reliable electrophoresis method, MetaPhor agarose gel electrophoresis (MAGE), was effectively employed as an alternative to polyacrylamide gel electrophoresis (PAGE) for separating Wx microsatellite alleles. The amplified products containing the Wx microsatellite ranged from 100 to 130 bp in length. Five Wx microsatellite alleles, namely (CT)(10), (CT)(11), (CT)(16), (CT)(17), and (CT)(18) were identified. Of these, (CT)(11) and (CT)(17) were the predominant classes among the tested varieties. All varieties with an apparent amylose content higher than 24% were associated with the shorter repeat alleles; (CT)(10) and (CT)(11), while varieties with 24% or less amylose were associated with the longer repeat alleles. All varieties with intermediate and high amylose content had the sequence AGGTATA at the 5'-leader intron splice site, while varieties with low amylose content had the sequence AGTTATA. The G-T polymorphism was further verified by the PCR-AccI cleaved amplified polymorphic sequence (CAPS) method, in which only genotypes containing the AGGTATA sequence were cleaved by AccI. Hence, varieties with desirable amylose levels can be developed rapidly using the Wx microsatellite and G-T SNP, along with MAGE.

A straightforward plant prime editing system enabled highly efficient precise editing of rice Waxy gene.

CRISPR Cas9-mediated genome editing is highly efficient at targeted site-specific gene knock-out through NHEJ (Non-Homology End Joining), but ineffective for specific DNA integration through HDR (Homology Directed Repair) for precise gene editing. Base editors can make limited base substitutions but only within restricted small windows of the protospacer. Prime editing has been applied in plants with various degrees of success. However, several questions such as low and inconsistent editing efficiencies across different target sites need to be addressed. We compared two prime editing approaches PE3 and PE2 at two neighboring target sites within rice Waxy gene to partially address those questions. A straightforward PE2 plant prime editing system retrofitted from a regular CRISPR-Cas9 editing system can deliver highly efficient up to 66.7% precise gene editing. Various forms of precise editing including base substitutions, small deletions and insertions can be accurately achieved. The secondary structure variations of different pegRNAs may be the primary reason for inconsistent editing across different target sites and should be the optimization focus to further improve plant prime editing.

Pan-genome analysis of three main Chinese chestnut varieties.

Chinese chestnut (Castanea mollissima Blume) is one of the earliest domesticated plants of high nutritional and ecological value, yet mechanisms of C. mollissima underlying its growth and development are poorly understood. Although individual chestnut species differ greatly, the molecular basis of the formation of their characteristic traits remains unknown. Though the draft genomes of chestnut have been previously released, the pan-genome of different variety needs to be studied. We report the genome sequence of three cultivated varieties of chestnut herein, namely Hei-Shan-Zhai-7 (H7, drought-resistant variety), Yan-Hong (YH, easy-pruning variety), and Yan-Shan-Zao-Sheng (ZS, early-maturing variety), to expedite convenience and efficiency in its genetics-based breeding. We obtained three chromosome-level chestnut genome assemblies through a combination of Oxford Nanopore technology, Illumina HiSeq X, and Hi-C mapping. The final genome assemblies are 671.99 Mb (YH), 790.99 Mb (ZS), and 678.90 Mb (H7), across 12 chromosomes, with scaffold N50 sizes of 50.50 Mb (YH), 65.05 Mb (ZS), and 52.16 Mb (H7). Through the identification of homologous genes and the cluster analysis of gene families, we found that H7, YH and ZS had 159, 131, and 91 unique gene families, respectively, and there were 13,248 single-copy direct homologous genes in the three chestnut varieties. For the convenience of research, the chestnut genome database was constructed. Based on the results of gene family identification, the presence/absence variations (PAVs) information of the three sample genes was calculated, and a total of 2,364, 2,232, and 1,475 unique genes were identified in H7, YH and ZS, respectively. Our results suggest that the GBSS II-b gene family underwent expansion in chestnut (relative to nearest source species). Overall, we developed high-quality and well-annotated genome sequences of three C. mollissima varieties, which will facilitate clarifying the molecular mechanisms underlying important traits, and shortening the breeding process.

Assessment of the Characteristics of Waxy Rice Mutants Generated by CRISPR/Cas9.

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.

Unearthed New Indel in the Waxy Gene in Glutinous Rice Landraces from Thailand.

<b>Background and Objective:</b> Glutinous rice landraces have played important role in northeastern and northern region of Thailand and the Lao People's Democratic Republic (PDR) by ethnic groups in this area, both as staple food and ritual in rice culture since ancient times. At a present number of these rice, cultivars have decreased and disappeared from villages without considerations and/or explore DNA variation. This study compared target DNA sequences of <i>waxy</i> genes of a collection of glutinous rice landraces both upland and lowland rice. <b>Materials and Methods:</b> A collection of 50 glutinous rice landraces was explored DNA variation in the <i>Waxy</i> gene by re-sequencing DNA of the two segments (i.e., promoter, exon1 and intron1) of a gene. <b>Results:</b> New InDel of two deletion G at position 11 (GG-) and C at position 15 (CC-) were observed in DNA sequences of the promoter region and 5'untranslalted region of both upland and lowland rice accessions. Further, the (CT)₁₇ and (CT)₁₈ were two alleles in these glutinous rice landraces from northern and northeastern Thailand. All glutinous rice landraces exhibited a characteristic of low amylose content or glutinous rice: T Single Nucleotide Polymorphisms (SNP) (AGTTATA) in the first intron splice site. <b>Conclusion:</b> New InDel in DNA sequences of the promoter region of the <i>waxy</i> gene in glutinous rice landraces was first reported. This implies that it may reflect the status of genetic background in glutinous rice landraces in Thailand.

Association Analysis of Markers Derived from Starch Biosynthesis Related Genes with Starch Physicochemical Properties in the USDA Rice Mini-Core Collection.

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 (P < 0.001) were detected in both association analyses. Five markers of Waxy gene, including the (CT)n 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 SSIIa 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 Waxy gene. Furthermore, our analyses indicated that the (CT)n polymorphisms of Waxy 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 Waxy SNPs but different (CT) n repeats. As the five Waxy markers and the two SSIIa markers showed consistent major effects on starch quality traits across studies, these markers should have priority for utilization in marker-assisted breeding.

Resistant starch: Variation among high amylose rice varieties and its relationship with apparent amylose content, pasting properties and cooking methods.

Resistant starch (RS), which is not hydrolyzed in the small intestine, has proposed health benefits. We evaluated 40 high amylose rice varieties for RS content in cooked rice and a 1.9-fold difference was found. Some varieties had more than two-fold greater RS content than a US long-grain intermediate-amylose rice. The high amylose varieties were grouped into four classes according to paste viscosity and gelatinization temperature based on genetic variants of the Waxy and Starch Synthase IIa genes, respectively. RS content was not different between the four paste viscosity-gelatinization temperature classes. Multiple linear regression analysis showed that apparent amylose content and pasting temperature were strong predictors of RS within each class. Two cooking methods, fixed water-to-rice ratio/time and in excess-water/minimum-cook-time, were compared using six rice varieties that were extremes in RS in each of the genetic variant classes, no difference in RS content due to cooking method was observed.

Dosage effects of Waxy gene on the structures and properties of corn starch.

The objective of this study was to understand dosage effects of the Waxy gene on the structures of amylose and amylopectin and on the properties of corn starch. Reciprocal crossing of isogenic normal and waxy corn lines was conducted to develop hybrids with different dosages (0, 1, 2, 3) of Waxy gene in the endosperm. The amylose content of starch and proportions of branch chains of DP 17-30 and extra-long branch chains (DP>100) of amylopectin were positively correlated with the Waxy-gene dosage. Proportions of short (DP<17) and long branch-chains (DP 30-80), however, were negatively correlated with the Waxy-gene dosage. The gelatinization conclusion-temperature and temperature-range of the starch were negatively correlated with the Waxy-gene dosage, indicating that amylose facilitated dissociation of the surrounding crystalline regions. These results helped us understand the function of granule-bound starch synthase I in the biosynthesis of amylose and amylopectin and impacts of Waxy-gene dosages on the properties of corn starch.