Identification of SNPs in the<Emphasis Type="Italic"> waxy</Emphasis> gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice

Common non-waxy (Wx) rice cultivars contain two different alleles at the waxy locus, designated Wx ^a and Wx ^b, which encode different levels of granule-bound starch synthases and are hence involved in the control of endosperm amylose content. The Wx ^a allele was predominant in non-waxy indica cultivars, whereas the Wx ^b allele was common to the non-waxy japonica variety. Recently, some of the molecular mechanisms underlying the differentiation of Wx ^a from Wx ^b have been characterized. One structural difference between these two alleles was shown to be due to alternative splicing caused by a single-base substitution (AGGT to AGTT) at a donor site of the first intron within the Wx gene. In the case of waxy (wx) rice, it was not possible to distinguish whether the each wx allele was derived from Wx ^a or Wx ^b alleles by phenotypic analysis. However, we succeeded in developing a derived cleaved amplified polymorphic sequence (dCAPS) marker for the detection of the one-base splicing mutation without the need for sequencing. A mismatch primer was used to generate a restriction site in the Wx ^a allele (AGGT) but not in the Wx ^b allele (AGTT). Three hundred fifty-three waxy rice strains that are widely found in Asia were then employed for analysis using this dCAPS marker. Our findings suggested that waxy rice strains have both Wx ^a- and Wx ^b-derived alleles, but that the Wx ^b-derived allele was predominant, and its distribution was independent of indica-japonica differentiation. The wild relatives of cultivated rice all possessed the AGGT allele. It was concluded that the waxy mutations, and the corresponding rice cultivation, originated from japonica during the evolution and domestication process of rice and was preferentially selected by most Asian peoples.Communicated by J. Heslop-Harrison     

Detection of allelic variation at the Wx locus with single-segment substitution lines in rice (Oryza sativa L.)

Apparent amylose content (AAC) is a key determinant of eating and cooking quality in rice and it is mainly controlled by the Wx gene which encodes a granule-bound starch synthase (GBSS). In this study, sixteen single-segment substitution lines harboring the Wx gene from 16 different donors and their recipient HJX74 were used to detect the naturally occurring allelic variation at the Wx locus. The AAC in the materials varied widely and could be grouped into glutinous, low, intermediate, and two high AAC sub-classes, high I (24.36?C25.20%) and high II (25.81?C26.19%), under different experimental environments, which showed a positive correlation with the enzymatic activity of GBSS. One insertion/deletion (InDel) and three single nucleotide polymorphisms in the Wx gene were detected and their combinations resulted in the variation of five classes of AAC. Based on the results of AAC phenotypes, GBSS activities and cDNA sequences, five Wx alleles, wx, Wx ^t, Wx ^g1, Wx ^g2, and Wx ^g3, were identified, two of which, Wx ^g2 and Wx ^g3, are separated for the first time in this study. Under different cropping seasons, the AAC differed significantly for the Wx ^t and Wx ^g1 alleles, with higher AAC in the fall season than in the spring season, but did not differ significantly for the wx, Wx ^g2, and Wx ^g3 alleles. In conclusion, the present results might contribute to our understanding of the naturally occurring allelic variation at the Wx locus and will facilitate the improvement of rice quality by marker-assisted selection.     

Two distinct <Emphasis Type="Italic">waxy</Emphasis> alleles impact the granule-bound starch synthase in sorghum

The granule-bound starch synthase (GBSS) is the enzyme responsible for amylose synthesis in starch granules. Loss of GBSS activity results in starch granules containing mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles were identified in sorghum (Sorghum bicolor L. Moench) characterized by the absence (waxy ^a ; wx ^a ) or presence (waxy ^b ; wx ^b ) of the GBSS protein in the endosperm. To characterize these alleles, we examined endosperm architecture using scanning electron microscopy (SEM), assayed GBSS enzymatic activities, and identified DNA lesions associated with the mutations in the GBSS (Sb10g002140) gene. wx ^a , the allele present in B Tx630 and R Tx2907, contained a large insertion in the third exon, which was consistent with the absence of the GBSS protein previously observed. wx ^b , the allele present in B 9307 and B TxARG1, contained a missense mutation that resulted in conversion of glutamine 268 to histidine in a conserved domain in starch synthases. In wx ^b , GBSS activity was less than 25% that of the non-waxy line B Wheatland, and GBSS activity was not detected in wx ^a . SEM showed that endosperm architecture was very similar in both wx ^a and wx ^b alleles, but altered in comparison to non-waxy lines R Tx430 and B Wheatland. Both alleles may have a range of potential applications in grain sorghum because of low amylose content in their starch and the presence or absence of the GBSS protein. PCR based markers were developed for both the wx ^a and the wx ^b alleles to aid in molecular breeding of low amylose sorghum.     

Differential regulation of waxy gene expression in rice endosperm

Summary In order to examine the effects of different alleles on the gene expression at the waxy locus, the Wx gene product which controls the synthesis of amylose was isolated from endosperm starch of rice plants and analysed by electrophoretic techniques. The major protein bound to starch granules was absent in most of waxy strains and increased with the number of Wx alleles in triploid endosperms, suggesting that the major protein is the Wx gene product. In addition to wx alleles which result in the absence or drastic reduction of the Wx gene product and amylose, differentiation of Wx alleles seemed to have occurred among nonwaxy rice strains. At least two Wx alleles with different efficiencies in the production of the major protein as well as amylose were detected. These alleles are discussed in relation to regulation of the gene expression.     

Mutation loci and intragenic selection marker of the granule-bound starch synthase gene in waxy maize

Four pairs of specific PCR primers have been designed on the basis of the sequence of the granule-bound starch synthase gene (GBSS; dominant non-waxy gene Wx) and used to amplify its homologous sequence from thirteen waxy and two non-waxy inbred lines. Results from electrophoresis indicated that the recessive waxy gene was wx, derived from the dominant non-waxy gene Wx by mutation at its 3′ end. The sequence of the mutated 3′ end was amplified by the TAIL-PCR technique. Sequence alignment showed that the mutation of the wx gene was caused by transposition of the aldehyde dehydrogenase gene rf2. Two pairs of specific primers were designed on the basis of the sequence difference between the dominant gene Wx and its mutated recessive allele wx and used as intragenic selection markers to identify individual plants of genotypes WxWx, Wxwx, and wxwx by PCR amplification from the segregating population of the F₂ generation crossed between waxy and non-waxy inbred lines. Iodine solution staining and starch component assay showed that all the 35 F₂ plants identified as genotype WxWx produced non-waxy kernels of the F₃ generation and that all 33 F₂ plants identified as genotype wxwx produced waxy kernels of the F₃ generation. This result can be used to improve the selection efficiency of waxy maize breeding and for selection of other single genes and major polygenes.     

Characterization of Starch Granules from Waxy,Nonwaxy, and Hybrid Seeds of Amaranthus hypochondriacus L.

Perisperm starch granules of the dicotyledonous plant Amaranthus hypochondriacus L. were prepared from two homozygous lines (WxWx and wxwx) and their hybrid (Wxwx). The hybrid line was obtained by natural hybridization. By Sephadex G-75 column chromatography of isoamylase-debranched starches, the amylose content of WxWx starch was 16.9%, that of Wxwx was 10.7, and wxwx was zero. SDS-polyacrylamide gel electrophoresis showed that starch granules from two genotypes (WxWx and Wxwx) contained a Wx protein (MW = 68,000) which was supposed to be a starch granule-bound starch synthase and was associated with amylose synthesis, as observed in nonwaxy maize. The intensities of the stained protein bands were apparently correlated with the number of the Wx alleles. The Wx protein was not detected in the wxwx starch. These findings suggest that the Wx allele produces the Wx protein and amylose in the perisperm of A. hypochondriacus, with incomplete dominance over the wx allele. The Wx allele did not affect the fine structure of amylopectin and had little if any effect on susceptibility to glucoamylase and pasting properties of starch granules from these genotypes.     

Large-scale DNA polymorphism study of <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Emphasis Type="Italic">O. rufipogon</Emphasis> reveals the origin and divergence of Asian rice

Polymorphism over ∼26 kb of DNA sequence spanning 22 loci and one region distributed on chromosomes 1, 2, 3 and 4 was studied in 30 accessions of cultivated rice, Oryza sativa, and its wild relatives. Phylogenetic analysis using all the DNA sequences suggested that O. sativa ssp. indica and ssp. japonica were independently domesticated from a wild species O. rufipogon. O. sativa ssp. indica contained substantial genetic diversity (π = 0.0024), whereas ssp. japonica exhibited extremely low nucleotide diversity (π = 0.0001) suggesting the origin of the latter from a small number of founders. O. sativa ssp. japonica contained a larger number of derived and fixed non-synonymous substitutions as compared to ssp. indica. Nucleotide diversity and genealogical history substantially varied across the 22 loci. A locus, RLD15 on chromosome 2, showed a distinct genealogy with ssp. japonica sequences distantly separated from those of O. rufipogon and O. sativa ssp. indica. Linkage disequilibrium (LD) was analyzed in two different regions. LD in O. rufipogon decays within 5 kb, whereas it extends to ∼50 kb in O. sativa ssp. indica. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Modulation of amylose content by structure‐based modification of OsGBSS1 activity in rice (Oryza sativa L.)

The rice Waxy (Wx) gene encodes granule‐bound starch synthase 1 (EC 2.4.1.242), OsGBSS1, which is responsible for amylose synthesis in rice seed endosperm. In this study, we determined the functional contribution of eight amino acids on the activity of OsGBSS1 by introducing site‐directed mutated Wx gene constructs into the wx mutant glutinous rice. The eight amino acid residues are suspected to play roles in OsGBSS1 structure maintenance or function based on homologous enzyme sequence alignment and homology modelling. Both OsGBSS1 activity and amylose content were analysed in homozygous transgenic lines carrying the mutated OsGBSS1 (Wx) genes. Our results indicate that mutations at diverse sites in OsGBSS1 reduces its activity by affecting its starch‐binding capacity, its ADP‐glucose‐binding capability or its protein stability. Our results shed new light on the structural basis of OsGBSS1 activity and the mechanisms of OsGBSS1 activity on amylose synthesis in vivo. This study also demonstrates that it is feasible to finely modulate amylose content in rice grains by modifying the OsGBSS1 activity.     

Identification of neutral alleles at pollen sterility gene loci of cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) from wild rice (<Emphasis Type="Italic">O. rufipogon</Emphasis> Griff.)

Oryza rufipogon Griff. is the ancestor of Asian cultivated rice (O. sativa L.) and possesses valuable genes for rice breeding. Pollen abortion is one of the major causes of indica–japonica hybrid sterility in rice and it happens due to allelic interaction at the pollen sterility gene loci. A total of six loci (Sa, Sb, Sc, Sd, Se, and Sf) have been found to be associated with F₁ pollen sterility between indica and japonica rice, and five of them (all except Sf) have been mapped. Neutral alleles (S ⁿ ) at each locus have the potential to overcome the pollen sterility associated with the respective locus. Therefore, exploitation and utilization of neutral alleles have significant importance in overcoming indica–japonica hybrid sterility. In this study, an accession (IRW28) of O. rufipogon, native to China, was selected as paternal to cross with typical japonica (Taichung 65) and indica (Guanglu’ai 4) tester lines, and two F₂ populations were developed. The simple sequence repeat markers tightly linked to five pollen sterility loci were applied for genotyping the F₂ populations. Chi-squared tests were applied to examine the normal segregation/distortion at each locus. The expected and observed pollen sterility for each locus were estimated. As a result, the genotypes at five pollen sterility gene loci for IRW28 were identified as: Sa ⁱ⁻¹/Sa ⁱ⁻¹, Sb ⁿ /Sb ⁿ , Sc ⁱ⁻²/Sc ⁱ⁻², Sd ⁿ /Sd ⁿ and Se ⁿ /Se ⁿ . Our results suggest that IRW28 (O. rufipogon) has the neutral alleles for pollen fertility at the Sb, Sd and Se loci, and these alleles have a good affinity with indica and japonica rice. These neutral alleles provide valuable gene resources to overcome the inter-subspecific hybrid pollen sterility in rice.     

Wx gene in diploid wheat: molecular characterization of five novel alleles from einkorn (Triticum monococcum L. ssp. monococcum) and T. urartu

The Wx gene encodes the granule-bound starch synthase I or waxy protein, which is the sole enzyme responsible for amylose synthesis in wheat seeds. Triticum urartu and einkorn (T. monococcum L. ssp. monococcum), which are related to the A genome of bread wheat, could be important sources of variation for this gene. This study evaluated the Wx gene variability in 52 accessions of these species and compared their nucleotide sequences with the Wx-A1a allele of bread wheat. The level of polymorphism found was high, although not distributed equally between the two species. Five different alleles were found in T. urartu, of which four were novel (Wx-A ^u 1b, -A ^u 1c, -A ^u 1d and -A ^u 1e). All einkorn accessions had the same allele, which was also novel and was named Wx-A ^m 1a. A comparison between the proteins deduced from the novel alleles and the Wx-A1a protein showed that there were up to 33 amino acid changes in both the transit peptide and the mature protein. These results showed that these species, especially T. urartu, are a potential source of novel waxy variants.     

Geographic distribution and multilocus organization of isozyme variation of rice (Oryza sativa L.)

Genetic organization of isozyme variation in rice (Oryza sativa L.) was investigated based on 17 polymorphic isozyme loci using a sample of 511 accessions of worldwide origin. The genetic diversity within the species was very high (H=0.36 with 4.82 alleles per locus), as compared with most selfing plant species. Three diversity centers were detected for isozyme variation including South Asia, China and Southeast Asia. The accessions were classified into three well-differentiated cultivar groups corresponding to the indica and japonica subspecies, and a new unnamed group. Variation within the cultivar groups accounted for 80% of the total isozyme variation. Within-country variation accounted for 58% of the total variation while among-region and among-country variation within the cultivar groups accounted for only 14% and 8% of the total variation. Analyses using log-linear models revealed that pronounced non-random associations between and among alleles at many unlinked isozyme loci were organized in a non-hierarchical pattern, and subspecific and macro-geographic differentiation was much more pronounced in multilocus phenotype frequencies than in allelic frequencies at individual loci. These results suggest that selection on multilocus gene complexes was largely responsible for the maintenance of the extensive isozyme variation within the species and the indica-japonica differentiation. Our results further suggest the independent domestication of indica and japonica, the dual origins of the indica rice from China and South Asia (India), and the differentiation of the ecotypes ’javanica’ and the ’temperate japonica’ within the japonica subspecies. Received: 5 August 1999 / Accepted: 13 December 1999     

Comparative analysis of microsatellite DNA polymorphism in landraces and cultivars of rice

Genetic polymorphisms of ten microsatellite DNA loci were examined among 238 accessions of landraces and cultivars that represent a significant portion of the distribution range for both indica and japonica groups of cultivated rice. In all, 93 alleles were identified with these ten markers. The number of alleles varied from a low of 3 or 4 at each of four loci, to an intermediate value of 9–14 at five loci, and to an extra-ordinarily high 25 at one locus. The numbers of alleles per locus are much larger than those detected using other types of markers. The number of alleles detected at a locus is significantly correlated with the number of simple sequence repeats in the targeted microsatellite DNA. Indica rice has about 14% more alleles than japonica rice, and such allele number differences are more pronounced in landraces than in cultivars. The indica-japonica differentiation component accounted for about 10% of the diversity in the total sample, and twice as much differentiation was detected in cultivars as in landraces. About two-thirds as many alleles were observed in cultivars as in landraces; another two-thirds of the alleles in the cultivar group were found in modern elite cultivars or parents of hybrid rice. The majority of the simple sequence repeat (SSR) alleles that were present in high or intermediate frequencies in landraces ultimately survived into modern elite cultivars and hybrids. The greater resolving power and the efficient production of massive amounts of SSR data may be particularly useful for germplasm assessment and evolutionary studies of crop plants.     

Cloning and Characterization of the Wx Gene Encoding a Granule-Bound Starch Synthase in Lotus (Nelumbo nucifera Gaertn)

The granule-bound starch synthase (GBSS) proteins were widely considered as one of the most important enzymes in plant amylose synthesis. However, understanding of the molecular basis of the GBSS protein in lotus remains fragmented. In this work, a lotus Wx gene, encoding a GBSS (GenBank accession no. EU938541), was isolated and characterized. This gene comprises 13 exons and 12 introns and covers 4152?bp (GenBank accession no. FJ602702). The exons of Wx gene have similar lengths, while the introns vary greatly. Phylogenetic tree indicated that the lotus GBSS protein belongs to a GBSS I subgroup. The expression of the Wx gene varies in different organs of the lotus during its development process and is also expressed differently in different cultivars. The Wx gene is expressed at a higher level in the rhizomes of cultivar Meirenhong than in those of cultivar Elian 4. This study elucidates more molecular information about the Wx gene in lotus and provides a theoretical foundation for the genes regulation and the modification of starch quality.     

Multiple introgression events surrounding the Hd1 flowering-time gene in cultivated rice, Oryza sativa L.

Flowering time is a major determinant for the local adaptation of crops. Hd1 is a key flowering-time gene in rice and is orthologous to the Arabidopsis CONSTANS gene. To elucidate the role of Hd1 in selection, we examined the Hd1 alleles of 60 landraces of Asian cultivated rice (Oryza sativa L.) originating from all regions of Asia, which comprised three cultivar groups, indica, japonica, and aus. The identified alleles were classified into four allele groups. The functional Hd1 alleles in allele groups I and II corresponded to indica and japonica, respectively. Non-functional alleles in these groups were not clearly associated with cultivar groups or locations. Allele groups III and IV corresponded to the aus cultivar group. The ancestry of each cultivar group was identified by the coalescent approach for Hd1 molecular evolution using the haplotype patterns of 14 regions over the 1.1 Mb chromosomal region surrounding Hd1 and the pSINE patterns of two loci, 1.4 and 4.4 Mb apart from Hd1. The haplotype patterns clearly revealed that Hd1 allele migration was caused by multiple and complex introgression events between cultivar groups. The Hd1 haplotypes among dozens of accessions of the wild species O. rufipogon were strongly divergent and only two of the haplotype clusters in O. rufipogon were closely related to those in cultivated rice. This strongly suggested that multiple introgression events have played an important role in the shaping and diversification of adaptation in addition to primary selection steps at the beginning of domestication.     

Molecular characterisation of the Wx-B1 allelic variants identified in cultivated emmer wheat and comparison with those of durum wheat

Emmer wheat is a neglected crop that could be used in the breeding of modern durum wheat for quality, one important aspect of which is the starch composition that is related to the waxy proteins. A collection of 87 accessions of Spanish emmer wheat was analysed for waxy protein composition by SDS?CPAGE. No polymorphism was found for the Wx-A1 gene. However, for the Wx-B1 gene, three alleles were detected, two of them new. The whole gene sequence of these alleles was amplified by PCR in three fragments, which were digested with several endonucleases to determine internal differences in the sequence. These variants were also compared with the Wx alleles present in durum wheat. Differences in size and restriction sites were detected. DNA sequence analysis confirmed that the alleles found in emmer wheat are different from those in durum wheat. The first data suggested that these alleles showed a different influence on the amylose content of these lines. The variation found could be used to enlarge the gene pool of durum and emmer wheat, and design new materials with different amylose content.     

Major QTLs reduce the deleterious effects of high temperature on rice amylose content by increasing splicing efficiency of Wx pre-mRNA

Key message

We discovered four QTLs that maintain proper rice amylose content at high temperature by increasing the splicing efficiency of Wx gene.

Abstract

Amylose content mainly controlled by Wx gene is a key physicochemical property for eating and cooking quality in rice. During the grain filling stage, high temperature can harm rice grain quality by significantly reducing the amylose content in many rice varieties. Here, we provide genetic evidences between Wx gene expression and rice amylose content at high temperature, and identified several quantitative trait loci (QTLs) in this pathway. We performed a genome-wide survey on a set of chromosome segment substitution lines (CSSLs) which carried chromosomal segments from the heat resistant indica 9311 in the heat-sensitive japonica Nipponbare background. Four QTLs, qHAC4, qHAC8a, qHAC8b and qHAC10, which can reduce the deleterious effects of amylose content at high temperature, were identified and mapped to chromosome 4, 8, 8 and 10, respectively. The major QTL qHAC8a, with the highest LOD score of 6.196, was physically mapped to a small chromosome segment (~300 kb). The CSSLs carrying the qHAC8a, qHAC8b and/or qHAC4 from 9311 have the high pre-mRNA splicing efficiency of Wx gene and likely lead to stable amylose content at high temperature. Thus, increasing pre-mRNA processing efficiency of Wx gene could be an important regulation mechanism for maintaining stable amylose content in rice seeds at high temperature. In addition, our results provide a theoretical basis for breeding heat-stable grain in rice.