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.

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.

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.

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.

Genome-Wide Association Study on Total Starch, Amylose and Amylopectin in Barley Grain Reveals Novel Putative Alleles.

The content and composition of starch in cereal grains are closely related to yield. Few studies have been done on the identification of the genes or loci associated with these traits in barley. This study was conducted to identify the genes or loci controlling starch traits in barley grains, including total starch (TS), amylose (AC) and amylopectin (AP) contents. A large genotypic variation was found in all examined starch traits. GWAS analysis detected 13, 2, 10 QTLs for TS, AC and AP, respectively, and 5 of them were commonly shared by AP and TS content. qTS-3.1, qAC-6.2 and qAP-5.1 may explain the largest variation of TS, AC and AP, respectively. Four putative candidate genes, i.e., HORVU6Hr1G087920, HORVU5Hr1G011230, HORVU5Hr1G011270 and HORVU5Hr1G011280, showed the high expression in the developing barley grains when starch accumulates rapidly. The examined 100 barley accessions could be divided into two groups based on the polymorphism of the marker S5H_29297679, with 93 accessions having allele GG and seven accessions having AA. Moreover, significantly positive correlation was found between the number of favorable alleles of the identified QTLs and TS, AC, AP content. In conclusion, the identified loci or genes in this study could be useful for genetic improvement of grains starch in barley.

A null allele of granule bound starch synthase (Wx-B1) may be one of the major genes controlling chapatti softness.

Chapatti (unleavened flatbread) is a staple food in northern India and neighboring countries but the genetics behind its processing quality are poorly understood. To understand the genes determining chapatti quality, differentially expressed genes were selected from microarray data of contrasting chapatti cultivars. From the gene and trait association studies, a null allele of granule bound starch synthase (GBSS; Wx-B1) was found to be associated with low amylose content and good chapatti quality. For validation, near-isogenic lines (NILs) of this allele were created by marker assisted backcross (MAB) breeding. Background screening indicated 88.2 to 96.7% background recovery in 16 selected BC3F5 NILs. Processing quality and sensory evaluation of selected NILs indicated improvement in chapatti making quality. Traits that showed improvement were mouthfeel, tearing strength and softness indicating that the Wx-B1 may be one of the major genes controlling chapatti softness.

Starch properties of high and low amylose proso millet (Panicum miliaceum L.) genotypes are differentially affected by varying salt and pH.

Variation in salt content and in pH are common in starch-based foods and can affect starch properties and final product texture. Fifteen accessions of proso millet starch with diverse amylose content were selected to investigate single factor and interaction effects of pH and NaCl on thermal, pasting, and textural properties. Pasting properties and gelatinization temperatures were markedly altered by salt addition. Changes in pH only had substantial effects on ΔH, but other properties were generally stable under different pH conditions. From two-way ANOVA, interactive effects of salt and pH were found to affect ΔH. The response of starch of different genotypes in terms of thermal and pasting properties differed under the same pH and salinity conditions. The reason is likely that ions in the starch-water system performed the roles of both reducing water activity and building of hydrogen bonds, which will have opposite effects on starch gelatinization.

Morphology, structure, properties and applications of starch ghost: A review.

Starch ghost, an insoluble structure of gelatinized starch, plays an important role in the applications of starch. In this review, we summarized the preparation, morphology, structure, properties and applications of starch ghost. The preparation steps of starch ghost include gelatinization, purification and preservation, and many factors influence the yield of starch ghost. The morphology and content of starch ghost can be influenced by many factors like starch resource and amylose content. Ghosts from non-waxy starches are composed of amylopectin with long branch-chains and amylose. These molecules cross-link to each other to reinforce the structure, and tend to form B-type double helix in ghosts from high-amylose starches. Some surface proteins that bind tightly to starch granules are also present in starch ghost. Protein and lipid are thought to have limited effects on the structural stability, but they make a big difference in the morphology of starch ghost. Starch ghost shows a different resistance to amylase among various starches, but it can be further digested under the high shear force. The mechanical, enzymatic hydrolysis and electrochemical properties of starch ghost make it widely used as emulsifier, stabilizer, thickener and starch-based films or gels in food and non-food processing industries.

Genetic diversity analysis of specialty glutinous and low-amylose rice (Oryza sativa L.) landraces of Assam based on Wx locus and microsatellite diversity.

The sticky rice of Assam is traditionally classified as bora (glutinous) and chokuwa (semi-glutinous) based on their stickiness after cooking. The Waxy (Wx) gene encodes for granule-bound starch synthase (GBSS) that controls the synthesis of amylose, which is a key determinant of rice end-use quality attributes. In this report, we analysed the level of variation in grain quality traits in a collection of bora and chokuwa cultivars, and examined the nucleotide diversity at the Wx locus of selected rice accessions to identify the possible cause of low-amylose in these rice cultivar groups. The Wx gene sequencing from 24 bora and chokuwa cultivars revealed several nucleotide variations that can explain the variation in the amylose phenotypes. The nucleotide polymorphisms in the downstream intron regions were similar to those reported in Bangladeshi Beruin cultivars. Among the Wx polymorphisms, the CTn microsatellite in exon 1 and G/T SNP in intron 1 (G/T-Int1) should be considered for marker assisted breeding involving bora cultivars. The Wx gene tree, classified the bora accessions possessing the G/T-Int1 SNP as japonicas. However, cluster analysis using microsatellite markers classified the bora and chokuwa cultivars as indica, and intermediate of indica-aus. The findings of this study supplemented our understanding on the evolution of the Wx gene under human selection. The results will assist plant breeders to effectively improve the bora and chokuwa landraces.