A New Strategy for Specific Qualities of Waxy Rice Breeding by Analyzing Physicochemical Properties Between Five Waxy Mutants and Corresponding Their Wild Types

Waxy rice is an essential mutant type of rice, which quality is controlled by amylopectin fine structure and apparent amylose content (AAC). The influence of amylopectin structure and AAC on quality and the waxy rice can be obtained by editing the Waxy (Wx) gene have been elucidated. However, the quality of waxy rice cannot be predicted before breeding, especially how to determine the quality of waxy (wx) mutants by wild types (WT) quality remains unclear. Herein, the quality of waxy rice has been successfully predicted through analyzing the association in physicochemical properties before and after Wx gene knockout. We demonstrated that the higher amylose WT would obtain higher amylose wx mutants, and wx mutants were endowed gelatinization temperature, amylopectin chain ratio and agronomic traits similar to WT. These data indicate that the quality of wild varieties played a decisive role in waxy rice breeding. Overall, we provide a new strategy for the specific quality breeding of waxy rice, which can get waxy rice of prescribed quality and contribute to expanding the particular type of waxy rice germplasm resources.

Generation of a New Glutinous Photothermosensitive Genic-Male-Sterile (PTGMS) Line by CRISPR/Cas9-Directed Mutagenesis of Wx in Rice (Oryza sativa L.)

The Photothermosensitive Genic-Male-Sterile (PTGMS) line, Y58S, an indica rice variety, combines high-quality and high-light-efficiency use, disease and stress resistance, and excellent plant type and mating force. Y58S is widely used to assemble two-line hybrid rice varieties, especially super hybrids. The Wx gene is the main effector gene for controlling amylose synthesis, which determines the amylose content (AC) of rice grains. By editing this gene, a glutinous line with a low AC can be obtained. In this study, the CRISPR/Cas9 system was used to mediate the editing of the Wx gene, which caused ultra-low AC mutations that produced a PTGMS glutinous rice strain with excellent waxiness. The results showed that 18 positively transformed plants were obtained from the T0 generation, with a mutation rate of 64.29%, of which six were homozygous mutant plants, indicating that the gene-editing target had a higher targeting efficiency and a higher homozygosity mutation rate. Compared to the wild type, the AC of the mutants was significantly lower. Through molecular marker detection and screening of T1 and T2 generations, five homozygous T-DNA-free mutant strains were identified that were consistent with Y58S in fertility and other agronomic traits except for AC. Among these, the AC of the W-1-B-5 homozygous mutant, the glutinous PTGMS line wx-Y58S, was as low as 0.6%. Our research revealed that the Wx gene of excellent PTGMS rice can be edited to generate a new waxy PTGMS line using the CRISPR/Cas9 system. This study provided a simple and effective strategy for breeding high-yield, high-quality, and glutinous two-line hybrid rice, and provided excellent sterile lines for their large-scale application. Once put into use, waxy hybrid rice will greatly improve the yield of glutinous rice and increase social benefits.

Waxy Gene-Orthologs in Wheat × Thinopyrum Amphidiploids

Starch, as the main component of grain in cereals, serves as the major source of calories in staple food and as a raw material for industry. As the technological and digestive properties of starch depend on its content, the management of its components, amylose and amylopectin, is of great importance. The starch properties of wheat grain can be attuned using allelic variations of genes, including granule-bound starch synthase I (GBSS I), or Wx. The tertiary gene pool, including wheatgrass (Thinopyrum) species, provides a wide spectrum of genes-orthologs that can be used to increase the allelic diversity of wheat genes by wide hybridization. Octaploid partial wheat–wheatgrass hybrids (amphidiploids, WWGHs) combine the complete genome of bread wheat (BBAADD), and a mixed genome from the chromosomes of intermediate wheatgrass (Thinopyrum intermedium, genomic composition JrJrJvsJvsStSt) and tall wheatgrass (Th. ponticum, JJJJJJJsJsJsJs). Thus, WWGHs may carry Wx genes not only of wheat (Wx-B1, Wx-A1 and Wx-D1) but also of wheatgrass origin. We aimed to assess the level of amylose in starch and investigate the polymorphism of Wx genes in 12 accessions of WWGHs. Additionally, we characterized orthologous Wx genes in the genomes of wild wheat-related species involved in the development of the studied WWGHs, Th. intermedium and Th. ponticum, as well as in the putative donors of their subgenomes, bessarabian wheatgrass (Th. bessarabicum, JbJb) and bluebunch wheatgrass (Pseudoroegneria stipifolia, St1St1St2St2). Although no significant differences in amylose content were found between different WWGH accessions, SDS-PAGE demonstrated that at least two WWGHs have an additional band. We sequenced the Wx gene-orthologs in Th. bessarabicum, P. stipifolia, Th. intermedium and Th. ponticum, and developed a WXTH marker that can discriminate the Thinopyrum Wx gene in the wheat background, and localized it to the 7E chromosome in Th. elongatum. Using the WXTH marker we revealed the allelic polymorphism of the Thinopyrum Wx gene in the studied WWGHs. The applicability of Thinopyrum Wx genes in wheat breeding and their effect on starch quality are discussed.

Wx gene polymorphism in winter triticale collection samples

The purpose of the study was to identify the collection of winter triticale in the allelic state of the waxi-genes and to identify sources with the presence of waxi-alleles for these genes. The surveys were conducted over 2017–2019 at the NSc Institute of Agriculture. The subject of the research are 43 collection samples of winter triticale, 29 of which are numbers of own breeding, 14 – breeding varieties of the National Institute of Agriculture of NAAS (9) and scientifi c institutions of Poland (1) and the Russian Federation (4). For control, we used soft winter waxy-wheat Sofi yka and wheat with wild of starch Oksana. Field, laboratory (infrared spectrometry, light microscopy, polymerase chain reaction (PCR)) methods, weights and mathematical and statistical methods of research were used to evaluate the collection material. According to the results of molecular genetic analysis of the Wx gene polymorphism in the winter triticale collection samples, it was found that all the tested samples had wild type alleles according to the Wx-B1 gene and were characterized by the absence of the Wx-D1 gene. The Wx-A1 gene revealed samples with both wild-type alleles and presence in the genome of the wax-allele. 8 collections with Wx-A1 gene alleles were selected: selection numbers 141, 153, 201, 223, 229 and varieties Lubomir, Petrol and Poliskii 7. The selected samples varied signifi cantly in terms of such characteristics as grain productivity, weight of 1000 grains, starch content. The tendency to decrease the size of the granules and increase the evenness of the granulometric structure of the starch in the samples with the presence of the wax-allele of the Wx-A1 gene was established. Wx-A1 gene allele samples are valuable starting material for the creation of new winter triticale varieties with increased amylopectin starch suitable for bioethanol processing. Key words: winter triticale, bioethanol, starch, polymerase chain reaction, amylopectin, amylose, allelic state of wax genes, waxi-allele, wild type.

Characterization of the Common Japonica-Originated Genomic Regions in the High-Yielding Varieties Developed from Inter-Subspecific Crosses in Temperate Rice (Oryza sativa L.)

The inter-subspecific crossing between indica and japonica subspecies in rice have been utilized to improve the yield potential of temperate rice. In this study, a comparative study of the genomic regions in the eight high-yielding varieties (HYVs) was conducted with those of the four non-HYVs. The Next-Generation Sequencing (NGS) mapping on the Nipponbare reference genome identified a total of 14 common genomic regions of japonica-originated alleles. Interestingly, the HYVs shared japonica-originated genomic regions on nine chromosomes, although they were developed through different breeding programs. A panel of 94 varieties was classified into four varietal groups with 38 single nucleotide polymorphism (SNP) markers from 38 genes residing in the japonica-originated genomic regions and 16 additional trait-specific SNPs. As expected, the japonica-originated genomic regions were only present in the japonica (JAP) and HYV groups, except for Chr4-1 and Chr4-2. The Wx gene, located within Chr6-1, was present in the HYV and JAP variety groups, while the yield-related genes were conserved as indica alleles in HYVs. The japonica-originated genomic regions and alleles shared by HYVs can be employed in molecular breeding programs to further develop the HYVs in temperate rice.

Characterization of the Common japonica-Originated Genomic Regions in the High Yielding Varieties Developed from Inter-Subspecific Crosses in Rice (Oryza sativa L.)

The inter-subspecific crossing between indica and japonica subspecies in rice have been utilized to improve yield potential in temperate rice. In this study, a comparative study of the genomic regions in the eight high yielding varieties (HYVs) was conducted with those of the four non-HYV varieties. NGS mapping on the Nipponbare reference genome identified a total of 14 common genomic regions of japonica-originated alleles. Interestingly, the HYVs shared the japonica-originated genomic regions on the nine chromosomes, although they were developed from different breeding programs. A panel of 94 varieties was classified into four varietal groups with the 39 SNP markers from 39 genes residing the japonica-originated genomic regions and 16 additional trait-specific SNPs. As expected, the japonica originated genomic regions were present only in JAP and HYV groups with exceptions for Chr4-1 and Chr4-2. The Wx gene located within Chr6-1 was present in HYV and JAP variety groups, while the yield-related genes were conserved as indica alleles in HYVs. The japonica-originated genomic regions and alleles shared by HYVs can be employed in molecular breeding programs for further development of HYVs in rice.

Wxlv, the Ancestral Allele of Rice Waxy Gene

In rice endosperms, the Waxy (Wx) gene is important for amylose synthesis, and various Wx alleles control the amylose content and affect the taste of cooked rice. Herein, we report the cloning of the ancestral allele Wxlv of the Wx locus, which affects the mouthfeel of rice grains by modulating the size of amylose molecules. Using evolutionary analysis, we demonstrated that Wxlv originated directly from wild rice, and the three major Wx alleles in cultivated rice (Wxb, Wxa, and Wxin) differentiated after the substitution of one base pair at the functional sites. These data indicate that the Wxlv allele played an important role in artificial selection and domestication. The findings also shed light on the evolution of various Wx alleles, which have greatly contributed to improving the eating and cooking quality of rice.

Wx Gene in Hordeum chilense: Chromosomal Location and Characterisation of the Allelic Variation in the Two Main Ecotypes of the Species

Starch, as the main grain component, has great importance in wheat quality, with the ratio between the two formed polymers, amylose and amylopectin, determining the starch properties. Granule-bound starch synthase I (GBSSI), or waxy protein, encoded by the Wx gene is the sole enzyme responsible for amylose synthesis. The current study evaluated the variability in Wx genes in two representative lines of Hordeum chilense Roem. et Schult., a wild barley species that was used in the development of tritordeum (×Tritordeum Ascherson et Graebner). Two novel alleles, Wx-Hch1a and Wx-Hch1b, were detected in this material. Molecular characterizations of these alleles revealed that the gene is more similar to the Wx gene of barley than that of wheat, which was confirmed by phylogenetic studies. However, the enzymatic function should be similar in all species, and, consequently, the variation present in H. chilense could be utilized in wheat breeding by using tritordeum as a bridge species.