Waxy Alleles Research Articles

Determination of amylose content of traditional rice varieties of Kerala using PCR-based molecular markers

The present study uses molecular approach to assess the amount of amylose in collected rice genotypes using PCR based markers. The polymorphism of Waxy allele which encodes the major enzyme catalyzing amylose synthesis was analyzed in the collected rice samples to determine the amylose content in the samples. All the rice varieties under study expressed Wxa allele corresponding to intermediate to high amylose content. This study proves that simple PCR technique can be used to detect Single nucleotide polymorphism mutation at Wx locus and provides a cost-effective alternative to predict amylose content in rice.

The Breeding of Waxy Sorghum Using Traditional Three-Line Method and Marker-Assisted Selection

Sorghum (Sorghum bicolor) exhibits drought resistance and environmental adaptability, making it a crucial cereal crop for semi-arid regions. It has a wide range of uses, including as food, feed, brooms, alcohol production, and bioethanol. In particular, Taiwan imports nearly 50,000 tons of sorghum annually, primarily for the production of sorghum liquor. However, the ideal raw material for high-quality sorghum liquor is waxy sorghum, and not all sorghum varieties imported or promoted in Taiwan are of this waxy type. Consequently, there is a shortage of sufficient waxy sorghum raw materials to meet the demands of the Taiwan market. The occurrence of waxy sorghum (wx) is caused by the mutation of granule-bound starch synthase I (GBBS I), and there are currently several known types of mutants, carrying different wxa, wxb, and wxc waxy alleles. Among them, wxc is a novel mutation type, and in native sorghum in Taiwan, individuals with the waxy allele wxc have been found. The three-line method is a commonly used breeding strategy, which simplifies the process of emasculation to obtain hybrid F1 offspring. In this study, imported sorghum variety Liangnuo No.1 (with male sterility), native glutinous sorghum variety SB6 from Taiwan (carrying the wxc waxy allele), and sorghum reference genome variety BTx623 were used as research materials. The goal was to use the three-line method to produce waxy sorghums, including the male sterile line (A-line), male sterile maintenance line (B-line), and fertility-restoring line (R-line). The breeding results showed that by using backcross breeding, molecular-assisted selection, and traditional field selection methods, high-quality three-line materials (A-, B-, R-lines, named CNA1, CNB1 CNR1, respectively) and F1 hybrid (CNH1) with favorable agronomic traits and yield quality were successfully obtained.

Segregation of molecular markers associated with Bph3 gene in BC5F2 population derived from Ciherang rice variety as the recipient parent

Brown planthopper (BPH), an important insect pest of rice, can cause severe damage and significant yield loss. An efficient and environmentally friendly method to control this pest is by planting resistant varieties. The brown planthopper resistance gene Bph3 from Rathu Heenati variety is closely linked to the Waxy gene that regulates amylose synthesis, the primary determinant of rice eating and cooking quality. The purposes of this study were to analyze the segregation ratio of SSR markers associated with Bph3 gene and to identify individual plants carrying homozygous allele for Bph3 gene in a BC5F2 population derived from a backcross that used Ciherang as the recurrent recipient parent. Analyses using RM589, RM586, and RM588 markers linked to Bph3 gene and RM190 marker linked to the Waxy gene indicated that the ratio of genotype segregation for each marker related to Bph3 gene did not deviate from the expected ratio with the χ 2 value was 0.956, 0.587, and 1.467 for RM589, RM586, and RM588, respectively (χ2 table = 5.991, α=0,05). All individual plants had a homozygous allele from Ciherang for Waxy gene based on the RM190 marker. As many as 25 individual plants which have homozygous Bph3 allele from Rathu Heenati and homozygous Waxy allele from Ciherang can be further developed as promising BPH-resistant rice varieties with eating and cooking quality similar to Ciherang.

Structure characteristics of mutation sites in two waxy alleles from Yunnan waxy maize (Zea mays L. var. certaina Kulesh) landraces.

A large number of waxy maize landraces are distributed in Yunnan and surrounding areas, and abundant waxy alleles of different types are distributed in these landraces. The identification of waxy alleles is helpful to the protection and utilization of these waxy landraces. This study introduced structure characteristics of waxy genes from two specific landraces of Yunnan, Zinuoyumi and Myanmar Four-Row Wax. Zinuoyumi has two waxy alleles wx-Cin4 and wx-Cin4-2; Myanmar Four-Row Wax has three waxy alleles wx-D10, wx-Reina and wx-D11. The wx-Cin4-2 and wx-D11 are two types of waxy alleles first reported in this study. The wx-Cin4-2 has two mutation sites, deletion of 30 bp in exon 10, insertion of a 1,267 bp non-long terminal repeat (non-LTR) retrotransposon Cin4 in intron 10, and 13 bp extra sequence were found at 5' end of the Cin4; the mutation site of wx-D11 is a 1,082 bp deletion from exons 11 to 14 of the waxy gene and is replaced with a 72 bp filler sequence. This study enriched the type of waxy allele from Yunnan waxy maize landraces and further discussed the molecular basis for the formation of mutation sites of wx-Cin4-2 and wx-D11.

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.

Combined effects of SSII-2RNAi and different Wx alleles on rice grain transparency and physicochemical properties

Near-isogenic lines Nip(Wxb/SSII-2), Nip(Wxb/ss2-2), Nip(Wxmw/SSII-2), Nip(Wxmw/ss2-2), Nip(Wxmp/SSII-2) and Nip(Wxmp/ss2-2) in the Nipponbare (Nip) background containing the SSII-2RNAi cassette combined with different Waxy (Wx) alleles were investigated in terms of rice grain transparency and quality profiles. Rice lines carrying the SSII-2RNAi cassette displayed downregulation of SSII-2, SSII-3 and Wx genes. Introduction of the SSII-2RNAi cassette decreased apparent amylose content (AAC) in all transgenic lines, but grain transparency differed between low AAC rice lines. Grains from Nip(Wxb/SSII-2) and Nip(Wxb/ss2-2) were transparent, while those of rice were increasingly translucent with decreasing moisture due to cavities within starch granules. Rice grain transparency was positively correlated with grain moisture and AAC, but negatively correlated with cavity area within starch granules. Starch fine structure analysis revealed a marked increase in short amylopectin chains with DP 6–12, but a decrease in intermediate chains with DP 13–24, resulting in decreased gelatinisation temperature. Starch crystalline structure analysis showed that the transgenic rice starches have lower crystallinity and lamellar repeat distance than controls due to differences in starch fine structure. The results highlight the molecular basis underpinning rice grain transparency, and provide strategies for improving rice grain transparency.

Identification of waxy genotype in sorghum genetic resources using waxy gene-based markers and iodine staining methods

Sorghum is widely recognized as a versatile crop with significant value in the production of food and energy. Sorghum with waxy trait (low amylose) is more palatable to the food industries and more appealing to consumers than nonwaxy variety. However, waxy sorghum has not yet been introduced to Indonesia, making it difficult to use sorghum to support the government of Indonesia's initiative to diversify the country's main foods. This study aimed at identify the waxy traits in a set of sorghum germplasm collected in Indonesia using waxy gene-based markers and iodine staining screening approaches. A total of 48 (27 introduced and 21 local) sorghum accessions collected in the IAARD gene bank along with 4 national sorghum varieties, which were subjected to both molecular analysis and iodine staining assay. The presence of waxy allele in the sorghum gene bank collection was identified using DNA markers associated with two waxy alleles (wxa and wxc). Iodine staining assay performed in the present study was applied to verify the waxy phenotype in sorghum grain and waxy genotypes in sorghum pollen. Based on molecular analysis and pollen staining, five local sorghum accessions were classified as heterowaxy sorghum. These accessions showed reddish-brown color in their endosperm after treated by iodine staining which confirmed them as waxy phenotype. These findings would be beneficial for further improvement of grain quality in sorghum breeding programs

Fine-tuning grain amylose contents by genome editing of Waxy cis-regulatory region in rice.

Rice grain amylose contents (ACs) is a key quantitative trait influencing eating and cooking quality. Regulating the expression level of Waxy, a key gene controlling ACs, and in turn fine-tuning the grain ACs, is an ideal approach to improve grain quality of rice varieties. Based on CRISPR/Cas9 genome editing technology, we designed eight targets in the cis-regulatory region of Wxa background, screened phenotypic changes of the transgenic lines and generated eight novel Waxy alleles with altered grain ACs. Among the eight alleles, we found that a 407-bp non-homologous substitution (NHS) in the 5'UTR-intron caused by genome editing regulated Waxy expression and decreased grain ACs by 2.9%. Moreover, embedding the 407-bp NHS into the cis-regulatory region of Wxb allele can also affect gene activity. Our work suggested the effect of 5'UTR-intron on Waxy gene expression regulation, and provided a potentially useful allele in breeding that can finely adjust rice grain ACs.

Genome-Wide Association Study (GWAS) Reveals an SNP Associated with Waxy Trait and Development of a Functional Marker for Predicting Waxy Maize (Zea mays L. var. ceratina)

Waxy maize (Zea mays L. var. ceratina) is a special type of maize characterized by a sticky texture when cooked, due to high amylopectin content in the endosperm. Waxy maize is popular in China and Southeast Asia for fresh consumption. Breeding strategies have been used to improve the quality of waxy maize, including hybrid breeding by crossing super sweet maize and waxy maize. However, the lack of a marker has limited the efficiency of breeding for the waxy trait, especially because the waxy allele is recessive. In this study, we conducted a genome-wide association study (GWAS) in an association panel consisting of 213 inbred lines and recombinant inbred lines (RILs) of field maize and waxy maize to identify loci associated with the waxy kernel phenotype. The genotypic data were 155,768 SNPs derived from the high-density 600 K maize genotyping array for single-nucleotide polymorphisms (SNPs). The GWAS results identified the qWx9 locus on chromosome 9 (25.06–25.18 Mb) associated with the trait. Based on the most significantly associated SNP (AX-90613979, −log10(P) = 6.8)), which was located on Wx1, a MassArray marker was developed and validated in a panel of 139 maize lines containing waxy maize and sweet maize with different amylose content. The newly developed marker had a significant association with amylose content (R2 value of 0.81, p < 0.001) and clearly distinguished between waxy maize and sweet maize lines that had different amylose content. This marker will be useful for maize breeding programs for the waxy trait, as well as for breeding programs for hybrid maize combining the sweetness and waxy traits. The gene-based SNP markers could aid breeders by eliminating the costs and time required to perform lengthy field trials and help to accelerate sweet maize and waxy maize breeding programs.

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.

English

Wheat breeding programs are increasingly using molecular tools to improve efficiency and speed of developing productive cultivars. Molecular markers such as Kompetitive Allele Specific PCR (KASP) have now become a principal approach to study genetic relationship among various wheat species. The objective of our research study was to identify and distinguished the wheat parents and their hybrid for genes controlling major agronomical attributes. Germplasm comprised of fifteen genotypes were genotyped for gluten strength (Glu A1, Glu D1), height reduction (Rht 1), starch quality (Waxy), vernalization (Vrn A1, Vrn B1), photoperiod sensitivity (Ppd 1), Tan spot disease (Tns-1) and leaf rust disease (Lr34). Statistical analysis with endpoint genotyping, graphical display and principal coordinate revealed significant results for KASP assay. Most variable crosses were S3 X I1, S3 X I3, S2 X I1, S2 X I2 and S1 X I1 with maximum segregants. Whereas, variation was observed for Glu-D1, Tsn1, Glu-A1 followed by Lr34 alleles. Therefore, the objective was achieved successfully with the most reliable KASP markers. The diverse attributes dissected, and key hybrids identified in this study would provide guidance for the improvement of the economically important traits and provide understanding of genetic variability in distinct segregating populations. Besides we have identified significantly important partial mutants in the germplasm for Waxy allele. These findings could provide the end user with wheat products with improved quality such as with or without amylose content.

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.

Genotyping-by-sequencing based QTL mapping identified a novel waxy allele contributing to high amylose starch in wheat

Genotyping-by-sequencing based QTL mapping identified a novel waxy allele contributing to high amylose starch in wheat

The origin of Wxla provides new insights into the improvement of grain quality in rice

Appearance and taste are important factors in rice (Oryza sativa) grain quality. Here, we investigated the taste scores and related eating‐quality traits of 533 diverse cultivars to assess the relationships between—and genetic basis of—rice taste and eating‐quality. A genome‐wide association study highlighted the Wx gene as the major factor underlying variation in taste and eating quality. Notably, a novel waxy (Wx) allele, Wx la, which combined two mutations from Wx b and Wx in, exhibited a unique phenotype. Reduced GBSSI activity conferred Wx la rice with both a transparent appearance and good eating quality. Haplotype analysis revealed that Wx la was derived from intragenic recombination. In fact, the recombination rate at the Wx locus was estimated to be 3.34 kb/cM, which was about 75‐fold higher than the genome‐wide mean, indicating that intragenic recombination is a major force driving diversity at the Wx locus. Based on our results, we propose a new network for Wx evolution, noting that new Wx alleles could easily be generated by crossing genotypes with different Wx alleles. This study thus provides insights into the evolution of the Wx locus and facilitates molecular breeding for quality in rice.

Sequence polymorphism of the waxy gene in waxy maize accessions and characterization of a new waxy allele

Waxy maize has many excellent characteristics in terms of its nutritional and economic value. In recent decades, the waxy maize germplasm has increased dramatically as a result of different selection methods. We collected 200 waxy maize inbred accessions from different origins to study their genetic diversity and phylogenetic relationships, and to identify new waxy mutations. A simple sequence repeat (SSR) analysis revealed wide genetic diversity among the 200 waxy maize accessions. The maize accessions were clustered into three groups. We sequenced the waxy gene from the first to the 14th exon. Nucleotide variation analysis of 167 waxy maize and 14 flint maize lines revealed some nucleotide differences in the waxy gene among different waxy maize groups, and much narrower nucleotide diversity in waxy maize than in flint maize. In a phylogenetic analysis, waxy maize carrying the same mutation allele clustered together, and waxy maize carrying different mutation alleles distributed in different groups; waxy maize was intermixed with flint maize in each branch, and wx-D7 waxy maize separated significantly from waxy maize lines carrying wx-D10, wx-124 and wx-hAT mutant alleles. The wx-hAT was a new waxy mutation identified in this study. It consisted of a 2286-bp transposon inserted into the middle of exon three of the waxy gene. A PCR marker specific for the wx-hAT allele was developed. These results will be useful for the utilization and preservation of the waxy maize germplasm, and the PCR marker has potential uses in waxy maize breeding programs.

Ekmeklik Buğdayda Geliştirilen Resiprokal Rekombinant Kendilenmiş Hat Populasyonunda Çavdar Translokasyonu Taşıyan Hatların Yarı Bodurluk, Fotoperiyod, Vernalizasyon ve Waxy Genlerinin Taraması

Bu araştırmanın amacı, çavdar translokasyonu taşıyan rekombinant kendilenmiş hatların yarı bodurluk (Rht), fotoperiyod (Ppd) ve vernalizasyon (Vrn) genleri bakımından genotipik olarak taranmasıdır. Rekombinant kendilenmiş hatlar Tosunbey ile Tahirova-2000 ekmeklik buğday çeşitlerinin melezlenmesi sonucu elde edilmiştir ve bu hatlar içerisinden sadece 1BL.1RS çavdar translokasyonu taşıyan hatlar araştırmada kullanılmıştır. Anaçların ve hatların hepsinin yarı bodurluk alleli olan Rht-B1a ve fotoperiyoda hassasiyet taşımayan allel olan Ppd-D1a alleline sahip olduğu belirlenmiştir. Hatların sahip oldukları vernalizasyon allelleri de (Vrn-A1, Vrn-B1 ve Vrn-D1) belirlenmiştir. Her iki anacın ve hatların vrn-A1 allelini taşıdığı ve Vrn-A1c allelini ise taşımadığı belirlenmiştir. 151 adet hat vrn-B1 allelini ve 154 adet hat Vrn-B1 allelini taşırken, 131 adet hattın vrn-D1 allelini, 174 adet hattın Vrn-D1 allelini taşıdığı saptanmıştır. Çalışmada ayrıca unun kabarması, su tutma kapasitesi ve raf ömrü üzerinde etkili waxy allelleri (Wx-A1, Wx-B1 ve Wx-D1) bakımından da moleküler taramalar yapılmıştır. Taramalar sonucunda 141 adet hattın her üç alleli de taşıdığı ve “normal amilozlu buğday” olduğu, 164 adet hattın ise sadece Wx-A1 ve Wx-D1 allellerine sahip olduğu ve “düşük amilozlu buğday” olduğu belirlenmiştir. Elde edilen sonuçlar, araştırmada kullanılan popülasyonun kuraklık başta olmak üzere bazı araştırmalara konu olabileceğini ve buğday ıslah programları için çavdar translokasyonu taşıyan genetik materyal geliştirmek için kullanılabileceğini göstermektedir.

Identification and validation of mutation points associated with waxy phenotype in cassava

BackgroundThe granule-bound starch synthase I (GBSSI) enzyme is responsible for the synthesis of amylose, and therefore, its absence results in individuals with a waxy starch phenotype in various amylaceous crops. The validation of mutation points previously associated with the waxy starch phenotype in cassava, as well as the identification of alternative mutant alleles in the GBSSI gene, can allow the development of molecular-assisted selection to introgress the waxy starch mutation into cassava breeding populations.ResultsA waxy cassava allele has been identified previously, associated with several SNPs. A particular SNP (intron 11) was used to develop SNAP markers for screening heterozygote types in cassava germplasm. Although the molecular segregation corresponds to the expected segregation at 3:1 ratio (dominant gene for the presence of amylose), the homozygotes containing the SNP associated with the waxy mutation did not show waxy phenotypes. To identify more markers, we sequenced the GBSS gene from 89 genotypes, including some that were segregated from a cross with a line carrying the known waxy allele. As a result, 17 mutations in the GBSSI gene were identified, in which only the deletion in exon 6 (MeWxEx6-del-C) was correlated with the waxy phenotype. The evaluation of mutation points by discriminant analysis of principal component analysis (DAPC) also did not completely discriminate the waxy individuals. Therefore, we developed Kompetitive Allele Specific PCR (KASP) markers that allowed discrimination between WX and wx alleles. The results demonstrated the non-existence of heterozygous individuals of the MeWxEx6-del-C deletion in the analyzed germplasm. Therefore, the deletion MeWxEx6-del-C should not be used for assisted selection in genetic backgrounds different from the original source of waxy starch. Also, the alternative SNPs identified in this study were not associated with the waxy phenotype when compared to a panel of accessions with high genetic diversity.ConclusionAlthough the GBSSI gene can exhibit several mutations in cassava, only the deletion in exon 6 (MeWxEx6-del-C) was correlated with the waxy phenotype in the original AM206–5 source.

Characterization of eating quality and starch properties of two Wx alleles japonica rice cultivars under different nitrogen treatments

To understand the effect of nitrogen (N) fertilizer on rice (Oryza sativa L.) eating and cooking quality (ECQ). Here, we investigated the ECQ attributes, physicochemical foundation of ECQ, and amylopectin fine structure of two Waxy (Wx) alleles japonica rice cultivars Nanjing 9108 (NJ9108) and Huaidao 5 (HD5) under four N rates (0, 150, 300, and 450 kg ha−1). Sensory and pasting properties of the two cultivars varied depending on N rates. Compared with the control (0 kg ha−1), the overall eating quality and sensory value were significantly decreased under the N rates of 300 and 450 kg ha−1. Further, conventional descriptive analysis showed that the stickiness and retrogradation of cooked rice were significantly decreased. These results indicated that application of N fertilizer seems to affect the texture of cooked rice, causing it to be less sticky, lowering its retrogradation, and consequently reducing its palatability. Results from rapid visco analyzer (RVA) revealed that the peak and breakdown viscosities significantly decreased, while the setback viscosity and peak time increased under the N rates of 300 and 450 kg ha−1. However, no significant difference was observed when the N rate was 150 kg ha−1, indicating that less N fertilization can maintain rice ECQ. As the N rate increasing, protein content increased, whereas apparent amylose content, starch content, and gel consistency almost unchanged. Interestingly, compared with the control, under N treatments, the percentage of short amylopectin branches in NJ9108 was decreased, but increased in HD5, as controlled by amylopectin synthesis-related genes. Notably, SSI and BEIIb were down-regulated in NJ9108, whereas BEIIb was up-regulated in HD5. Thus, the palatability of both rice cultivars was significantly decreased under excessive N fertilization as a consequence of reduced stickiness and retrogradation of the cooked rice, which might have resulted from an elevated protein content and altered amylopectin fine structure. In addition, amylopectin synthesis appeared to be affected by N fertilizer and the genotype of the rice cultivar.

Superior field performance of waxy corn engineered using CRISPR-Cas9.

We created waxy corn hybrids by CRISPR-Cas9 editing of a waxy allele in 12 elite inbred maize lines, a process that was more than a year faster than conventional trait introgression using backcrossing and marker-assisted selection. Field trials at 25 locations showed that CRISPR-waxy hybrids were agronomically superior to introgressed hybrids, producing on average 5.5 bushels per acre higher yield.

New Waxy allele wx-Reina found in Chinese waxy maize

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