Understanding the amylose biosynthesis and regulation mechanisms in Tartary buckwheat by the endosperm transcriptome.

Starch serves as a crucial energy source for both plants and humans, predominantly synthesized and stored in endosperms, tubers, rhizomes, and cotyledons. Given the significant role of amylose in determining the quality of starchy crops, optimizing its content has become a key objective in current crop breeding efforts. Tartary buckwheat, a dicotyledonous plant, notably accumulates high levels of amylose in its endosperm, surpassing common cereals like rice and maize. However, the mechanisms underlying amylose accumulation, distribution, and regulation in Tartary buckwheat remain unclear. Here, amylose content was determined across various tissues and organs of Tartary buckwheat, identifying with the endosperm as the primary site for its biosynthesis and accumulation. RNA sequencing analysis of endosperms from different developmental stages identified 35 genes potentially involved in starch biosynthesis, with 13 genes showing high endosperm-specific expression, suggesting crucial roles in starch biosynthesis. Additionally, the transcription factor FtNF-YB2, which was specifically highly expressed in the endosperm, was discovered to enhance amylose synthesis. Moreover, promoters with potential endosperm-specific activity were identified, advancing our understanding of amylose regulation. Additionally, this study also demonstrates that brassinosteroids (BR) positively influence amylose biosynthesis in Tartary buckwheat endosperm. These findings provide essential insights into the mechanisms of understanding amylose biosynthesis, accumulation and regulation in Tartary buckwheat, offering significant implications for future breeding strategies.

QTL Mapping and Candidate Gene Analysis for Starch-Related Traits in Tartary Buckwheat (Fagopyrum tataricum (L.) Gaertn).

Starch is the main component that determines the yield and quality of Tartary buckwheat. As a quantitative trait, using quantitative trait locus (QTL) mapping to excavate genes associated with starch-related traits is crucial for understanding the genetic mechanisms involved in starch synthesis and molecular breeding of Tartary buckwheat varieties with high-quality starch. Employing a recombinant inbred line population as research material, this study used QTL mapping to investigate the amylose, amylopectin, and total starch contents across four distinct environments. The results identified a total of 20 QTLs spanning six chromosomes, which explained 4.07% to 14.41% of the phenotypic variation. One major QTL cluster containing three stable QTLs governing both amylose and amylopectin content, qClu-4-1, was identified and located in the physical interval of 39.85-43.34 Mbp on chromosome Ft4. Within this cluster, we predicted 239 candidate genes and analyzed their SNP/InDel mutations, expression patterns, and enriched KEGG pathways. Ultimately, five key candidate genes, namely FtPinG0004897100.01, FtPinG0002636200.01, FtPinG0009329200.01, FtPinG0007371600.01, and FtPinG0005109900.01, were highlighted, which are potentially involved in starch synthesis and regulation, paving the way for further investigative studies. This study, for the first time, utilized QTL mapping to detect major QTLs controlling amylose, amylopectin, and total starch contents in Tartary buckwheat. The QTLs and candidate genes would provide valuable insights into the genetic mechanisms underlying starch synthesis and improving starch-related traits of Tartary buckwheat.

Multiomics of a rice population identifies genes and genomic regions that bestow low glycemic index and high protein content.

To counter the rising incidence of diabetes and to meet the daily protein needs, we created low glycemic index (GI) rice varieties with protein content (PC) surpassing 14%. In the development of recombinant inbred lines using Samba Mahsuri and IR36 amylose extender (IR36ae) as parental lines, we identified quantitative trait loci and genes associated with low GI, high amylose content (AC), and high PC. By integrating genetic techniques with classification models, this comprehensive approach identified candidate genes on chromosome 2 (qGI2.1/qAC2.1 spanning the region from 18.62 Mb to 19.95 Mb), exerting influence on low GI and high amylose. Notably, the phenotypic variant with high value was associated with the recessive allele of the starch branching enzyme 2b (sbeIIb). The genome-edited sbeIIb line confirmed low GI phenotype in milled rice grains. Further, combinations of alleles created by the highly significant SNPs from the targeted associations and epistatically interacting genes showed ultralow GI phenotypes with high amylose and high protein. Metabolomics analysis of rice with varying AC, PC, and GI revealed that the superior lines of high AC and PC, and low GI were preferentially enriched in glycolytic and amino acid metabolisms, whereas the inferior lines of low AC and PC and high GI were enriched with fatty acid metabolism. The high amylose high protein recombinant inbred line (HAHP_101) was enriched in essential amino acids like lysine. Such lines may be highly relevant for food product development to address diabetes and malnutrition.

Combining genome-wide association study and linkage mapping in the genetic dissection of amylose content in maize (Zea mays L.).

KEY MESSAGE: Integrated linkage and association analysis revealed genetic basis across multiple environments. The genes Zm00001d003102 and Zm00001d015905 were further verified to influence amylose content using gene-based association study. Maize kernel amylose is an important source of human food and industrial raw material. However, the genetic basis underlying maize amylose content is still obscure. Herein, we used an intermated B73 × Mo17 (IBM) Syn10 doubled haploid population composed of 222 lines and a germplasm set including 305 inbred lines to uncover the genetic control for amylose content under four environments. Linkage mapping detected 16 unique QTL, among which four were individually repeatedly identified across multiple environments. Genome-wide association study revealed 17 significant (P = 2.24E-06) single-nucleotide polymorphisms, of which two (SYN19568 and PZE-105090500) were located in the intervals of the mapped QTL (qAC2 and qAC5-3), respectively. According to the two population co-localized loci, 20 genes were confirmed as the candidate genes for amylose content. Gene-based association analysis indicated that the variants in Zm00001d003102 (Beta-16-galactosyltransferase GALT29A) and Zm00001d015905 (Sugar transporter 4a) affected amylose content across multi-environment. Tissue expression analysis showed that the two genes were specifically highly expressed in the ear and stem, respectively, suggesting that they might participate in sugar transport from source to sink organs. Our study provides valuable genetic information for breeding maize varieties with high amylose.

The Waxy Gene Has Pleiotropic Effects on Hot Water-Soluble and -Insoluble Amylose Contents in Rice (Oryza sativa) Grains.

Rice (Oryza sativa) is a cereal crop with a starchy endosperm. Starch is composed of amylose and amylopectin. Amylose content (AC) is the principal determinant of rice quality, but varieties with similar ACs can still vary substantially in their quality. In this study, we analyzed the total AC (TAC) and its constituent fractions, the hot water-soluble amylose content (SAC) and hot water-insoluble amylose content (IAC), in two sets of related chromosome segment substitution lines of rice with a common genetic background grown in two years. We searched for quantitative trait loci (QTLs) associated with SAC, IAC, and TAC and identified one common QTL (qSAC-6, qIAC-6, and qTAC-6) on chromosome 6. Map-based cloning revealed that the gene underlying the trait associated with this common QTL is Waxy (Wx). An analysis of the colors of soluble and insoluble starch-iodine complexes and their λmax values (wavelengths at the positions of their peak absorbance values) as well as gel permeation chromatography revealed that Wx is responsible for the biosynthesis of amylose, comprising a large proportion of the soluble fractions of the SAC. Wx is also involved in the biosynthesis of long chains of amylopectin, comprising the hot water-insoluble fractions of the IAC. These findings highlight the pleiotropic effects of Wx on the SAC and IAC. This pleiotropy indicates that these traits have a positive genetic correlation. Therefore, further studies of rice quality should use rice varieties with the same Wx genotype to eliminate the pleiotropic effects of this gene, allowing the independent relationship between the SAC or IAC and rice quality to be elucidated through a multiple correlation analysis. These findings are applicable to other valuable cereal crops as well.

Molecular insights on the origin and development of waxy genotypes in major crop plants.

Starch is a significant ingredient of the seed endosperm with commercial importance in food and industry. Crop varieties with glutinous (waxy) grain characteristics, i.e. starch with high amylopectin and low amylose, hold longstanding cultural importance in some world regions and unique properties for industrial manufacture. The waxy character in many crop species is regulated by a single gene known as GBSSI (or waxy), which encodes the enzyme Granule Bound Starch Synthase1 with null or reduced activity. Several allelic variants of the waxy gene that contribute to varying levels of amylose content have been reported in different crop plants. Phylogenetic analysis of protein sequences and the genomic DNA encoding GBSSI of major cereals and recently sequenced millets and pseudo-cereals have shown that GBSSI orthologs form distinct clusters, each representing a separate crop lineage. With the rapidly increasing demand for waxy starch in food and non-food applications, conventional crop breeding techniques and modern crop improvement technologies such as gene silencing and genome editing have been deployed to develop new waxy crop cultivars. The advances in research on waxy alleles across different crops have unveiled new possibilities for modifying the synthesis of amylose and amylopectin starch, leading to the potential creation of customized crops in the future. This article presents molecular lines of evidence on the emergence of waxy genes in various crops, including their genesis and evolution, molecular structure, comparative analysis and breeding innovations.

Population Structure and Genetic Diversity of Shanlan Landrace Rice for GWAS of Cooking and Eating Quality Traits.

The Shanlan landrace rice in Hainan Province, China, is a unique upland rice germplasm that holds significant value as a genetic resource for rice breeding. However, its genetic diversity and its usefulness in rice breeding have not been fully explored. In this study, a total of eighty-four Shanlan rice, three typical japonica rice cultivars, and three typical indica rice cultivars were subjected to resequencing of their genomes. As a result, 11.2 million high-quality single nucleotide polymorphisms (SNPs) and 1.6 million insertion/deletions (InDels) were detected. Population structure analysis showed all the rice accessions could be divided into three main groups, i.e., Geng/japonica 1 (GJ1), GJ2, and Xian/indica (XI). However, the GJ1 group only had seven accessions including three typical japonica cultivars, indicating that most Shanlan landrace rice are different from the modern japonica rice. Principal component analysis (PCA) showed that the first three principal components explained 60.7% of the genetic variation. Wide genetic diversity in starch physicochemical parameters, such as apparent amylose content (AAC), pasting viscosity, texture properties, thermal properties, and retrogradation representing the cooking and eating quality was also revealed among all accessions. The genome-wide association study (GWAS) for these traits was conducted and identified 32 marker trait associations in the entire population. Notably, the well-known gene Waxy (Wx) was identified for AAC, breakdown viscosity, and gumminess of the gel texture, and SSIIa was identified for percentage of retrogradation and peak gelatinization temperature. Upon further analysis of nucleotide diversity in Wx, six different alleles, wx, Wxa, Wxb, Wxin, Wxla/mw, and Wxlv in Shanlan landrace rice were identified, indicating rich gene resources in Shanlan rice for quality rice breeding. These findings are expected to contribute to the development of new rice with premium quality.

Grain quality characteristics analysis and application on breeding of Yuenongsimiao, a high-yielding and disease-resistant rice variety.

Rice quality is one of the main targets of rice breeding and is a complex trait that involves grain appearance, milling, cooking, eating and nutritional quality. For many years, rice breeding has contended with imbalances in rice yield, quality, and disease and lodging resistance. Here, the milling and appearance quality, cooking quality, starch rapid viscosity analyzer (RVA) profile, and nutritional quality of grains of Yuenongsimiao (YNSM), an indica rice variety with high yield, high quality and disease resistance, were determined. YNSM had excellent appearance and quality, with low amylose contents and high gel consistency, and these characteristics exhibited significant correlations with the RVA profile such as hot paste viscosity, cool paste viscosity, setback viscosity, and consistency. Moreover, 5 genes related to length-to-width ratio (LWR) as well as the Wx gene were used to detect the main quality genotype of YNSM. The results showed that YNSM is a semilong-grain rice with a relatively high brown rice rate, milled rice rate and head rice yield and low chalkiness. The results indicated that the LWR and food quality of YNSM might be related to gs3, gw7 and Wxb. This study also reports the quality characteristics of hybrid rice developed using YNSM as a restorer line. The quality characteristics and the genotype for grain quality determined through gene analysis in YNSM may facilitate the breeding of new rice varieties that achieve a balance of grain yield, resistance and quality.

Development of Soft Rice Lines by Regulating Amylose Content via Editing the 5'UTR of the Wx Gene.

The type of soft rice with low amylose content (AC) is more and more favored by consumers for its better eating and cooking quality, as people's quality of life continuously improves in China. The Wx gene regulates the AC of rice grains, thus affecting the degree of softness of the rice. Mei Meng B (MMB), Tian Kang B (TKB), and DR462 are three indica rice maintained lines with good morphological characters, but also with undesirably high AC. Therefore, CRISPR/Cas9 technology was used to edit the Wx gene of these lines to create a batch of soft rice breeding materials. New gene-edited lines MMB-10-2, TKB-21-12, and DR462-9-9, derived from the above parental lines, respectively, were selected in the T2 generations, with an AC of 17.2%, 16.8%, and 17.8%, and gel consistency (GC) of 78.6 mm, 77.4 mm, and 79.6 mm, respectively. The rapid viscosity analysis (RVA) spectrum showed that the three edited lines had a better eating quality as compared to the corresponding wild type, and showing new characteristics, different from the high-quality soft rice popular in the market. There was no significant difference in the main agronomic traits in the three edited lines compared to the corresponding wild types. Moreover, the chalkiness of DR462-9-9 was reduced, resulting in an improved appearance of its polished rice. The present study created soft rice germplasms for breeding improved quality hybrid rice, without changing the excellent traits of their corresponding wild type varieties.

Allelic Diversification of the Wx and ALK Loci in Indica Restorer Lines and Their Utilisation in Hybrid Rice Breeding in China over the Last 50 Years.

Hybrid rice technology has been used for more than 50 years, and eating and cooking quality (ECQ) has been a major focus throughout this period. Waxy (Wx) and alkaline denaturation (ALK) genes have received attention owing to their pivotal roles in determining rice characteristics. However, despite significant effort, the ECQ of restorer lines (RLs) has changed very little. By contrast, obvious changes have been seen in inbred rice varieties (IRVs), and the ECQ of IRVs is influenced by Wx, which reduces the proportion of Wxa and increases the proportion of Wxb, leading to a decrease in amylose content (AC) and an increase in ECQ. Meanwhile, ALK is not selected in the same way. We investigated Wx alleles and AC values of sterile lines of female parents with the main mating combinations in widely used areas. The results show that almost all sterile lines were Wxa-type with a high AC, which may explain the low ECQ of hybrid rice. Analysis of hybrid rice varieties and RLs in the last 5 years revealed serious homogenisation among hybrid rice varieties.

Post-genomics revolution in the design of premium quality rice in a high-yielding background to meet consumer demands in the 21st century.

The eating and cooking quality (ECQ) of rice is critical for determining its economic value in the marketplace and promoting consumer acceptance. It has therefore been of paramount importance in rice breeding programs. Here, we highlight advances in genetic studies of ECQ and discuss prospects for further enhancement of ECQ in rice. Innovations in gene- and genome-editing techniques have enabled improvements in rice ECQ. Significant genes and quantitative trait loci (QTLs) have been shown to regulate starch composition, thereby affecting amylose content and thermal and pasting properties. A limited number of genes/QTLs have been identified for other ECQ properties such as protein content and aroma. Marker-assisted breeding has identified rare alleles in diverse genetic resources that are associated with superior ECQ properties. The post-genomics-driven information summarized in this review is relevant for augmenting current breeding strategies to meet consumer preferences and growing population demands.

Unraveling novel and rare mutations for alpha-amylase and key transcription factors in EMS-induced wheat mutants for amylose by TILLING.

BACKGROUND: TILLING (Targeting Induced Local Lesions in Genomes) is a reverse-genetic strategy that is used to locate an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and cost-effective detection of induced point mutations in populations of chemically mutagenized individuals. Grain amylose content is the major constraints for its nutritional quality and have drawn research interest. Identification of allelic variations in genes involved in starch biosynthesis in wheat endosperm is pre-requisite to amenable for nutritional quality improvement. METHODS AND RESULTS: In this study, 44 EMS-induced (M4 generation) mutant lines having variation for amylose content were used for TILLING sequencing. Overall 2098.08 kb of the sequence was analyzed, and the average mutation density was 1/65.56 kb. In analysis, at the high depth score a total of 32 variations were identified including three natural variations, 76% transitions, 10% transversions, and 14% InDels respectively. The substitutions led to intronic variants, UTRs and up-downstream gene variants in Alpha-amylase, TabZIP77.1, TabZIP1 and Myb respectively. In the Myb transcription factor two missense mutations recorded namely Myb_7B c.680G > A and c.1358 T > C led to p.Gly227Asp and p.Met453Thr and c.1390G > A one substitution in Myb_7D led to p.Val464Ile. CONCLUSION: The identified missense substitutions were predicted to affect the protein function; hence they may have a probable role in context to the amylose content in mutants. The mutations ascertained in the current study will help in gene discovery in wheat and identified mutants can be used as genetic resources to improve nutritional quality of wheat.

Rice starch multi-level structure and functional relationships.

Starch from 15 different rice genotypes with amylose content (AC) ranging 1.5%-30.6% were investigated for relationships between structures and properties. For parameters related to the granular level, the most important relationships were found for AC, average chain lengths (ACL) of the amylopectin (AP) fb1 chains having a length of DP 13-24, crystallinity, and the thickness of the crystalline (dc) and the amorphous lamellae (da) of the starch granule. AC and dc were negatively correlated with the peak gelatinization temperature (Tp), thermal enthalpy (ΔH), and peak viscosity (PV), but positively correlated with swelling power. ACLfb1 and da, as compared to AC and dc, had the opposite effects on these parameters, demonstrating important roles of specific molecular and lamellar structures on the starch granular stability. For the gelatinized systems, increasing ACLfb1 decreased retrogradation, while AC increased retrogradation by increasing the resistant starch (RS) content, storage modulus (G'), and setback (SB).

Effects of post-silking low temperature on the physicochemical properties of waxy maize starch.

Low temperature (LT) at late growth stages is an important abiotic stress that affects the grain end-use quality of summer maize. In the present work, two experiments were conducted to study the effects of LT on the structural and functional properties of starches using two waxy maize hybrids ('Suyunuo5' and 'Yunuo7'). In field trial, the plants were sown on July 1 (normal sowing date) and August 1 (late sowing date). In pot trial, the plants were sown on July 1, grown at natural environment till silking, and suffered two post-silking temperatures (normal temperature and LT were set as 28/20 and 23/15 °C, respectively). The result showed that the starch was composed of more small granules with oval polytope when sown late (August 1) or subjected to LT post-silking. The LT-stressed starch presented high proportion of short amylopectin chains and low relative crystallinity (RC). LT reduced the pasting viscosity, gelatinization enthalpy, and gelatinization temperatures but increased the retrogradation tendency. In conclusion, the low pasting viscosity and high retrogradation tendency under LT condition were caused by the decreased granule size, amylopectin chain length, and RC.

Potassium Fertilization Stimulates Sucrose-to-Starch Conversion and Root Formation in Sweet Potato (Ipomoea batatas (L.) Lam.).

A field experiment was established to study sweet potato growth, starch dynamic accumulation, key enzymes and gene transcription in the sucrose-to-starch conversion and their relationships under six K2O rates using Ningzishu 1 (sensitive to low-K) and Xushu 32 (tolerant to low-K). The results indicated that K application significantly improved the biomass accumulation of plant and storage root, although treatments at high levels of K, i.e., 300-375 kg K2O ha-1, significantly decreased plant biomass and storage root yield. Compared with the no-K treatment, K application enhanced the biomass accumulation of plant and storage root by 3-47% and 13-45%, respectively, through promoting the biomass accumulation rate. Additionally, K application also enhanced the photosynthetic capacity of sweet potato. In this study, low stomatal conductance and net photosynthetic rate (Pn) accompanied with decreased intercellular CO2 concentration were observed in the no-K treatment at 35 DAT, indicating that Pn was reduced mainly due to stomatal limitation; at 55 DAT, reduced Pn in the no-K treatment was caused by non-stomatal factors. Compared with the no-K treatment, the content of sucrose, amylose and amylopectin decreased by 9-34%, 9-23% and 6-19%, respectively, but starch accumulation increased by 11-21% under K supply. The activities of sucrose synthetase (SuSy), adenosine-diphosphate-glucose pyrophosphorylase (AGPase), starch synthase (SSS) and the transcription of Susy, AGP, SSS34 and SSS67 were enhanced by K application and had positive relationships with starch accumulation. Therefore, K application promoted starch accumulation and storage root yield through regulating the activities and genes transcription of SuSy, AGPase and SSS in the sucrose-to-starch conversion.

Variability in waxy (Wx) allele, in-vitro starch digestibility, glycemic response and textural behaviour of popular Northern Himalayan rice varieties.

Eight commonly cultivated and consumed rice varieties of Northern Himalayan regions and a popular high amylose rice variety were characterized at Wx locus and evaluated for resistant starch (RS), in-vitro starch digestibility, predicted glycemic index (pGI), glycemic load (GL) and textural parameters. Cytosine and thymine repeats (CT)n at waxy locus (Wx) showed high association with apparent amylose content (AAC). Both pGI and GL varied substantially within the selected varieties. The pGI was relatively lower in high and intermediate amylose Indica varieties compared to low amylose Japonica ones. However, Koshikari despite being a low amylose variety showed relatively lower pGI and GL, due to its higher RS, dietary fiber, protein and fat content. It was thus presumed that in addition to AAC, RS and other grain components also affect the glycemic response. Inherent resistance to enzymatic hydrolysis was also found to be higher in firm textured and less sticky rice varieties. The genotypes-Lalat, Basmati-1509 and Koshikari, in view of their low to moderate pGI and relatively higher RS content, can be explored in future breeding programmes to develop rice varieties whose consumption will help to prevent hyper/hypo glycemic responses in Northern Himalayan regions, where daily staple diet is rice.

Genome-wide analysis of RING-type E3 ligase family identifies potential candidates regulating high amylose starch biosynthesis in wheat (Triticum aestivum L.).

In ubiquitin-mediated post-translational modifications, RING finger families are emerged as important E3 ligases in regulating biological processes. Amylose and amylopectin are two major constituents of starch in wheat seed endosperm. Studies have been found the beneficial effects of high amylose or resistant starch on health. The ubiquitin-mediated post-translational regulation of key enzymes for amylose/amylopectin biosynthesis (GBSSI and SBEII) is still unknown. In this study, the genome-wide analysis identified 1272 RING domains in 1255 proteins in wheat, which is not reported earlier. The identified RING domains classified into four groups-RING-H2, RING-HC, RING-v, RING-G, based on the amino acid residues (Cys, His) at metal ligand positions and the number of residues between them with the predominance of RING-H2 type. A total of 1238 RING protein genes were found to be distributed across all 21 wheat chromosomes. Among them, 1080 RING protein genes were identified to show whole genome/segmental duplication within the hexaploid wheat genome. In silico expression analysis using transcriptome data revealed 698 RING protein genes, having a possible role in seed development. Based on differential gene expression and correlation analysis of 36 RING protein genes in diverse (high and low) amylose mutants and parent, 10 potential RING protein genes found to be involved in high amylose biosynthesis and significantly associated with two starch biosynthesis genes; GBSSI and SBEIIa. Characterization of mutant lines using next-generation sequencing method identified unique mutations in 698 RING protein genes. This study signifies the putative role of RING-type E3 ligases in amylose biosynthesis and this information will be helpful for further functional validation and its role in other biological processes in wheat.

Posttranslational Modification of Waxy to Genetically Improve Starch Quality in Rice Grain.

The waxy (Wx) gene, encoding the granule-bound starch synthase (GBSS), is responsible for amylose biosynthesis and plays a crucial role in defining eating and cooking quality. The waxy locus controls both the non-waxy and waxy rice phenotypes. Rice starch can be altered into various forms by either reducing or increasing the amylose content, depending on consumer preference and region. Low-amylose rice is preferred by consumers because of its softness and sticky appearance. A better way of improving crops other than downregulation and overexpression of a gene or genes may be achieved through the posttranslational modification of sites or regulatory enzymes that regulate them because of their significance. The impact of posttranslational GBSSI modifications on extra-long unit chains (ELCs) remains largely unknown. Numerous studies have been reported on different crops, such as wheat, maize, and barley, but the rice starch granule proteome remains largely unknown. There is a need to improve the yield of low-amylose rice by employing posttranslational modification of Wx, since the market demand is increasing every day in order to meet the market demand for low-amylose rice in the regional area that prefers low-amylose rice, particularly in China. In this review, we have conducted an in-depth review of waxy rice, starch properties, starch biosynthesis, and posttranslational modification of waxy protein to genetically improve starch quality in rice grains.

Novel Wx alleles generated by base editing for improvement of rice grain quality.

Amylose content (AC), which is regulated by the Waxy (Wx) gene, is a major indicator of eating and cooking quality (ECQ) in rice (Oryza sativa). Thus far, only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein. Here, nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors. The nucleotide substitutions led to changes in 15 amino acid residues of Wx, and a series of novel Wx alleles with ACs of 0.3%-29.43% (wild type with AC of 19.87%) were obtained. Importantly, the waxyabe2 allele showed a "soft rice" AC, improved ECQ, favorable appearance, and no undesirable agronomic traits. The transgenes were removed from the waxyabe2 progeny, generating a promising breeding material for improving rice grain quality.

Molecular characterization of five novel Wx-A1 alleles in common wheat including one silent allele by transposon insertion.

Wheat starch is composed of two glucose polymers, amylose and amylopectin. Although several starch synthases are responsible for its synthesis, only the waxy protein is associated with the amylose synthesis. The waxy protein composition of 45 Spanish common wheat landraces from Andalusia (southern Spain) was evaluated. Within these materials, five novel alleles for the Wx-A1 gene were detected. Four of them showed functional proteins (Wx-A1p, Wx-A1q, Wx-A1r and Wx-A1s), although some amino acid changes were found in the mature protein sequence. However, one of them (Wx-A1t) exhibited loss of the Wx-A1 protein, and its base sequence contained one large insert (1,073 bp) in the tenth exon, that interrupted the ORF of the Wx-A1 gene. This insert exhibited the characteristics of a Class II transposon of the Mutator superfamily, which had not been described previously, and has been named Baetica. The conservation of such inserts could be related to their low effect on vital properties of the plants, as occurs with most of the genes associated with technological quality. In conclusion, the evaluation of old wheat landraces showed that, in addition to their use as alternative crops, these materials could be a useful source of interesting genes in wheat quality improvement.