Waxy Gene Research Articles

Evolutionary insights into the Andean genus Nicandra (Solanoideae, Solanaceae)

Nicandra (Solanaceae) is a small Andean genus, historically considered monospecific, with N. physalodes as its only species. This species is native to the South American Andes but naturalized in many countries worldwide. Recently, N. john-tyleriana and N. yacheriana were described using morphological evidence, which are endemic to Peru. Sequence data from four DNA markers, the plastidial intergenic spacers trnL-trnF and ndhF-rpl32, and the nuclear ITS region and GBSSI (waxy) gene, were used to test the identity of the nominal species using genomic evidence, to reconstruct the genus phylogenetic history through Maximum Likelihood and Bayesian Inference, and to estimate divergence times within the genus. The three Nicandra species formed distinct partitions and monophyletic lineages. Nicandra physalodes, originated in the late Miocene (ca. 8 My), was resolved as the first splitting branch, sister to the siblings N. john-tyleriana and N. yacheriana, which were originated at the end of the Miocene (ca. 5 My). The crown ages for the three species were estimated from the early Pliocene (N. physalodes) to the mid-Pleistocene (N. john-tyleriana and N. yacheriana), which is consistent with hypotheses of Neotropical species diversification. Open questions remain about the biogeography of the genus.

New highlights on Old World giant Phragmites (Poaceae) using leaf and floral bract microscopic characters

The genus Phragmites includes taxa that are very difficult to distinguish macroscopically, and even genetically. To better discriminate these taxa, microscopic traits, such as the epidermis of floral bracts and leaves, and leaf anatomy, were investigated. In the Old World, four distinct giant reeds species (> 3 m) were delineated: two tropical species, P. karka and P. mauritianus, and two Mediterranean species, P. altissimus and P. frutescens. Previously included within P. australis sensu lato, the rehabilitation of P. altissimus and P. frutescens is supported by relevant distinctive characteristics. Conversely, the small reeds (< 3 m) from temperate to cold regions form a distinct group. Although this group is still difficult to subdivide with epidermis traits, three taxa can be separated using leaf anatomy: both the Afro-European and the Australian-E. Asian P. australis sensu stricto, and P. japonicus. Using iodine green solution, we link our morphological analyses with previous genetic clustering, the green coloration of floral bracts and margin leaf teeth matches with the presence of nuclear waxy gene bands in P. frutescens and P. mauritianus (100 bp), and P. altissimus (200 bp), while P. karka, P. australis subsp. australis and P. japonicus have no coloration nor waxy band. While leaf anatomy features seem to be correlated with eco-climatic conditions, the epidermal traits delimited other clades. For leaf epidermis P. karka, P. mauritianus, and P. frutescens present the more ancestral characters, whereas P. australis, P. japonicus, and P. altissimus possess derived traits. Lastly, floral bract epidermis traits separate two divergent evolutionary lineages from the basal P. karka.

HvGBSSI mutation at the splicing receptor site affected RNA splicing and decreased amylose content in barley.

Granule-bound starch synthase I (HvGBSSI) is encoded by the barley waxy (Wx-1) gene and is the sole enzyme in the synthesis of amylose. Here, a Wx-1 mutant was identified from an ethyl methane sulfonate (EMS)-mutagenized barley population. There were two single-base mutations G1086A and A2424G in Wx-1 in the mutant (M2-1105). The G1086A mutation is located at the 3′ splicing receptor (AG) site of the fourth intron, resulting in an abnormal RNA splicing. The A2424G mutation was a synonymous mutation in the ninth intron. The pre-mRNA of Wx-1 was incorrectly spliced and transcribed into two abnormal transcripts. The type I transcript had a 6 bp deletion in the 5′ of fifth exon, leading to a translated HvGBSSI protein lacking two amino acids with a decreased starch-binding capacity. In the type II transcript, the fourth intron was incorrectly cleaved and retained, resulting in the premature termination of the barley Wx-1 gene. The mutations in the Wx-1 decreased the enzymatic activity of the HvGBSSI enzyme and resulted in a decreased level in amylose content. This work sheds light on a new Wx-1 gene inaction mechanism.

Waxy is an important factor for grain fissure resistance and head rice yield as revealed by a genome-wide association study.

Head rice yield (HRY) is an essential quality trait, and is sensitive to environmental stresses during the grain-filling, harvest, and postharvest stages. It is therefore important for rice production and global food security to select for superior HRY traits; however, the molecular basis of this trait remains unknown. Using diverse rice germplasm material, we performed a genome-wide association study of grain fissure resistance (GFR), the phenotype most associated with HRY, and found that the granule-bound starch synthase I gene Waxy is an important gene controlling GFR. Analysis of near-isogenic lines demonstrated that genetic variations in Waxy conferred different levels of tolerance to fissuring in grains. The null allele wx resulted in the highest GFR, while alleles that increased amylose synthesis reduced GFR. Increases in amylose content led to increases in the ratio of the widths of the amorphous layer to the semi-crystalline layer of the starch granules, and also to increased occurrence of chalkiness. The layer structure determined GFR by affecting the degree of swelling of granules in response to moisture, and chalkiness acted as an accelerator of moisture infiltration to rapidly increase the number of swelling granules. Our study reveals the molecular basis of GFR and HRY, thus opening the door for further understanding of the molecular networks of GFR and HRY.

Molecular screening of the landraces from Turkey and modern bread wheat (Triticum aestivum L.) cultivars for HMW-GS, wbm, waxy genes and Lr34 gene

Molecular screening of the landraces from Turkey and modern bread wheat (Triticum aestivum L.) cultivars for HMW-GS, wbm, waxy genes and Lr34 gene

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.

Phylogeny, character evolution and biogeography of the genus Sclerophylax (Solanaceae)

AbstractThe genus Sclerophylax (Atropina clade, Solanaceae) has 14 species recognized to date, all except two endemic to Argentina. To clarify its phylogenetic relationships, molecular sequence data from the trnL‐trnF and the rpl32F‐trnL intergenic spacer regions, the waxy gene and the ITS region were employed in maximum likelihood and Bayesian inference analyses. Divergence times were estimated for the combined dataset using secondary calibrations. Ancestral distribution areas were reconstructed by Bayesian binary analyses and seven morphological traits (one continuous, six discrete) were traced. Sclerophylax is monophyletic and its sister clade is Lycium + Nolana. Sclerophylax is divided into S. sect. Caducifructus (1 sp.) and the monophyletic sect. Sclerophylax (11 spp.). Sclerophylax sect. Sclerophylax includes five well‐supported clades that are also supported by morphological synapomorphies; circumscription of some species remains uncertain, probably as a result of rapid diversifications, but four lineages are recognized by a combination of morphological traits. The ancestor of Sclerophylax was an annual, procumbent/decumbent plant with rhomboidal leaves and sessile, persistent, 2–3‐seeded fruits. The most variable traits were leaf shape and life form, while flower size was shown to be highly informative in diagnosing clades within Sclerophylax. The area of origin for Sclerophylax was the Prepuna biogeographical province, from which it has spread in recent geological times. A total of 12 species are recognized in Sclerophylax, with an updated synonymy, lectotypifications, and re‐circumscriptions provided.

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.

Assessment of Marker-trait Associations for Amylose Content, Gelatinization Temperature and Yield in Sri Lankan Rice Varieties

Rice provides a balanced diet, and regular rice consumption helps maintain a healthy life and prevents many common illnesses. Amylose content and gelatinization temperature are considered major physicochemical factors which significantly affect the nutrition and cooking quality of rice. The Waxy (Wx) gene controls the synthesis of amylose in the endosperm and the ALK gene controls the gelatinization temperature of rice. Both genes are present on chromosome 6. Improving the rice traits is crucially essential to the cultivators and the consumers to overcome the increasing demand for high-quality and nutritionally rich rice. Many studies have been successfully conducted to improve the agronomical traits of rice. However, the concern for improving nutrition and cooking quality-related traits of rice is less common among the breeders. Therefore, considering the current trends, introducing consumer-friendly novel varieties that can fulfill the nutritional requirements is essential to face the increasing domestic demand for high-quality rice. In the present study, we used frequently grown 54 local rice varieties and carried out a marker-based genetic analysis for three traits: amylose content, gelatinization temperature, and yield. Then statistical analysis was carried out using quantitative data of different agronomical and physicochemical traits to identify the correlation and potential to improve such essential traits. The level of amylose content is significantly associated with the gelatinization temperature indicating that AC and GT have a positive correlation. The DNA markers SSIIa and Seq1-2 markers showed monomorphic patterns indicating the narrow genetic basis of the rice varieties tested. This study revealed that existing high-yielding rice varieties could be successfully employed to increase the amylose content while decreasing the level of gelatinization temperature through breeding strategies to make nutritionally rich consumer-friendly novel varieties.

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.

Development of the PARMS Markers of the Waxy Gene and Utilization in Discriminating Wild Accessions, and Cultivated Rice (Oryza sativa L.) with Different Eating and Cooking Quality

Amylose content (AC) is the major indicator of rice eating and cooking quality (ECQ). Its synthesis in rice endosperm is mainly regulated by the protein, granule-bound starch synthase 1, which is encoded by the waxy gene (Os06g0133000, LOC_Os06g04200). The diversity of AC is largely attributable to the allelic variation at the Wx locus and the development of effective and accurate functional molecular markers to target rice variant alleles is crucial in the breeding strategy. In the present study, we developed six pairs of penta-primer amplification refractory mutation system (PARMS) markers to distinguish between Wxlv, Wxa, Wxin, Wxb, Wxmp, and Wxop,hp. These markers were successfully used to screen the genotype of large assets of genetic resources including 98 wild accessions, 55 cultivars, and 22 parental lines. Our results showed that Wxb in a low AC type was predominant in Guangxi cultivated rice as a result of cultural preference, while Wxlv in the wild accessions. Moreover, our findings surprisingly revealed the presence of Wxb in wild accession, which is a new outcome that may contribute to understanding the origin, selection and domestication processes of rice. These functional markers could be effectively used in marker-assisted breeding to improve selection efficiency of cultivars with desired AC in the early generation.

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.

Du13 encodes a C2 H2 zinc-finger protein that regulates Wxb pre-mRNA splicing and microRNA biogenesis in rice endosperm.

SummaryAmylose content is a crucial physicochemical property responsible for the eating and cooking quality of rice (Oryza sativa L.) grain and is mainly controlled by the Waxy (Wx) gene. Previous studies have identified several Dull genes that modulate the expression of the Wxb allele in japonica rice by affecting the splicing efficiency of the Wxb pre‐mRNA. Here, we uncover dual roles for a novel Dull gene in pre‐mRNA splicing and microRNA processing. We isolated the dull mutant, du13, with a dull endosperm and low amylose content. Map‐based cloning showed that Du13 encodes a C2H2 zinc‐finger protein. Du13 coordinates with the nuclear cap‐binding complex to regulate the splicing of Wxb transcripts in rice endosperm. Moreover, Du13 also regulates alternative splicing of other protein‐coding transcripts and affects the biogenesis of a subset of microRNAs. Our results reveal an evolutionarily conserved link between pre‐mRNA splicing and microRNA biogenesis in rice endosperm. Our findings also provide new insights into the functions of Dull genes in rice and expand our knowledge of microRNA biogenesis in monocots.

Analysis of starch structure and functional properties of tetraploid wheat (Triticum turgidum L.) with differing waxy protein composition.

An increased demand for food has mirrored the increasing global population. Obesity and diabetes are two disorders induced by poor eating choices. Consequently, there is an urgent need to develop modified foods that can ameliorate such illnesses. The objective of this study was to explore the effect of Waxy genes on the structural and functional properties of starch, with the aim of improving food quality. Wild-type tetraploid wheat was compared with three mutants with different Waxy gene combinations. The proportion of B-type granules was higher in the mutants than in the wild-type (Wx-AB), and there were significant changes in the starch granule size, number, and phenotype in the Wx free mutant (Wx-ab). The lowest branch chain length was observed in Wx-ab, whereas Wx-AB had the highest branch chain length of DP ≥ 37. Wx-ab had the highest degree of crystallinity. The crystallinity trend followed the order Wx-ab>Wx-Ab>Wx-aB>Wx-AB. The amount of slowly digestible starch (SDS) was higher in native, gelatinized, and retrograded starch in the mutant. The amount of retrograded starch was closer to gelatinized starch than to native starch. Waxy proteins make a substantial contribution to starch structure. A lack of waxy proteins reduced the unit chains markedly compared with the control. Waxy proteins significantly affected the smaller and longer chains of starch. The lines with differing waxy composition had different effects on food digestion. The Wx-AB in native starch and Wx-Ab in gelatinized starch can control obesity and diabetes by slow-digesting carbohydrates and high resistance to digestion. © 2022 Society of Chemical Industry.

Authentication of carnaroli rice by HRM analysis targeting nucleotide polymorphisms in the Alk and Waxy genes

Carnaroli is a high quality and priced variety, being considered as one of the finest Italian rice varieties due to its sensorial and rheological properties and, thus being a potential adulteration target. The present work aimed at exploiting polymorphisms in the Alk (A/G and GC/TT in exon 8) and Waxy ((CT)n and G/T in intron 1) genes by HRM analysis to differentiate Carnaroli rice from closely related varieties. The HRM method targeting the Alk gene did not allow gathering the Carnaroli subgroup genotypes in the same cluster. The HRM approach targeting Waxy gene successfully discriminated the varieties sold as Carnaroli from all the others with high level of confidence (>98%), which corroborated sequencing data. Its applicability to commercial rice samples was successful. Therefore, the proposed new HRM method can be considered a simple, specific, high-throughput and cost-effective tool for the authentication of Carnaroli rice, contributing to valorise such premium variety.

Type‐2 Diabetes and Identification of Major Genetic Determinants of Glycemic Index in Rice – A Review

AbstractRice consumers of different parts of the world always face the risk of diabetes and related health complications. Dietary intervention is the best adapted measure to maintain blood glucose levels and manage the diseases in rice consuming populations. The glycemic index of rice varieties is negatively correlated to the amylose content. Research studies have proven that the consumption of rice varieties with low glycemic index has beneficial effects in controlling diabetes. The genetic basis for the diversity observed for amylose content in different rice varieties is well elucidated. Waxy gene (Wx) is known to encode Granule–Bound Starch Synthase I (GBSS I) enzyme, which determines the amylose content in rice. Different allelic variations of Wx gene exist, which can be correlated to varying amylose content in rice. The PCR‐based markers for detecting single nucleotide polymorphism mutation at Wx locus provides a cost‐effective alternative to genotype rice varieties. This review outlines the anti‐diabetic properties of rice and strategies to identify rice varieties with a low glycemic index at the molecular level. The molecular basis of the glycemic index allows breeders to successfully identify rice germplasms with low glycemic index and engineer rice varieties with high grain yield and low glycemic index.

Waxy allele diversity in waxy maize landraces of Yunnan Province, China

Waxy maize is one of the main fresh-eating maize types, and a mutation of the waxy gene causes the waxy character of maize grains. China is rich in waxy maize landraces, and Yunnan and its surrounding areas, are the place of origin and genetic diversity center of Chinese waxy maize. The six known waxy alleles of Chinese waxy maize are wx-D7, wx-D10, wx-Cin4, wx-124, wx-Reina, and wx-Xuanwei. The mutation sites of these alleles all occur in the coding region of the waxy gene, however, the mechanism by which the waxy characteristic is caused by the mutation in the regulatory region has only been reported rarely in maize. In this study, 405 waxy maize landraces from Yunnan were used as materials to identify the insertion and deletion of a large sequence fragment in the upstream ∼3.5 kb regulatory region of the waxy gene by molecular marker detection. Three different waxy alleles were identifed in this study: wx-PIF/Harbinger, wx-hAT and wxElote2. These three types of mutations all represented transposons inserted into the regulatory region of the waxy gene. Wx-PIF/Harbinger was a 304-bp MITE class transposon insertion belonging to the PIF/Harbinger family, while wx-hAT was a 560-bp MITE class transposon insertion belonging to the hAT family, and wx-Elote2 was a 6 560-bp LTR-like transposon insertion. In this study, the alleles were identifed for more than 70% of the waxy maize landraces in Yunnan, which provids a basis for the utilization of these waxy maize landraces.

The Physicochemical Properties of Starch Are Affected by Wxlv in Indica Rice.

Amylose largely determines rice grain quality profiles. The process of rice amylose biosynthesis is mainly driven by the waxy (Wx) gene, which also affects the diversity of amylose content. The present study assessed the grain quality profiles, starch fine structure, and crystallinity characteristics of the near-isogenic lines Q11(Wxlv), NIL(Wxa), and NIL(Wxb) in the indica rice Q11 background containing different Wx alleles. Q11(Wxlv) rice contained a relatively higher amylose level but very soft gel consistency and low starch viscosity, compared with rice lines carrying Wxa and Wxb. In addition, starch fine structure analysis revealed a remarkable decrease in the relative area ratio of the short amylopectin fraction but an increased amylose fraction in Q11(Wxlv) rice. Chain length distribution analysis showed that Q11(Wxlv) rice contained less amylopectin short chains but more intermediate chains, which decreased the crystallinity and lamellar peak intensity, compared with those of NIL(Wxa) and NIL(Wxb) rice. Additionally, the starches in developing grains showed different accumulation profiles among the three rice lines. Moreover, significant differences in starch gelatinization and retrogradation characteristics were observed between near-isogenic lines, which were caused by variation in starch fine structure. These findings revealed the effects of Wxlv on rice grain quality and the fine structure of starch in indica rice.

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

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. 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. 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.

The Low Copy Nuclear Gene Region, Granule Bound Starch Synthase (GBSS1), as a Novel Mini-DNA Barcode for the Identification of Different Sage (Salvia) Species.

Morphological similarity within species makes the identification and authentication of Salvia species challenging, especially in dietary supplements that contain processed root or leaf powder of different sage species. In the present study, the species discriminatory power of 2 potential DNA barcode regions from the nuclear genome was evaluated in 7 medicinally important Salvia species from the family Lamiaceae. The nuclear internal transcribed spacer 2 and the exon 9 - 14 region of low copy nuclear gene WAXY coding for granule-bound starch synthase 1 were tested for their species discrimination ability using distance, phylogenetic, and BLAST-based methods. A novel 2-step PCR method with 2 different annealing temperatures was developed to achieve maximum amplification from genomic DNA. The granule-bound starch synthase 1 region showed higher amplification and sequencing success rates, higher interspecific distances, and a perfect barcode gap for the tested species compared to the nuclear internal transcribed spacer 2. Hence, these novel mini-barcodes generated from low copy nuclear gene regions (granule-bound starch synthase) that were proven to be effective barcodes for identifying 7 Salvia species have potential for identification and authentication of other Salvia species.