Origin and evolution of the waxy phenotype in Amaranthus hypochondriacus: evidence from the genetic diversity in the Waxy locus

The existence of polymorphism in the Waxy locus in a large gene pool of 53 strains with various waxy phenotypes from samples of Amaranthus hypochondriacus collected from different regions was investigated in an origin-and-evolution study. First, we screened all strains for a mutation point (G–A polymorphism in exon 6) by using PCR–RFLP and/or direct sequence analysis. The results showed that the nonsense mutation in the coding region (exon 6) of the Waxy gene was responsible for the change in perisperm starch, leading to a waxy phenotype in all strains. Second, phylogenetic analysis, which was based on the Waxy variation, indicated diverse waxy types occurring separately and independently in certain domesticated regions in Mexico. Finally, we designated nine molecular types by comparing obvious structural variations in the coding region of the Waxy gene. Among the molecular types, A. hypochondriacus contained Type III in three subtypes with the waxy phenotype, with evolutionary routes that could originate from Type II in accordance with G–A polymorphism. In addition, these types had the same mutation points by which the Waxy gene was converted into the waxy phenotype. Therefore, the present results showed that the nonsense mutation is a unique event in the evolution of waxy phenotypes in this crop. This study will provide useful information for understanding the evolutionary process of the waxy phenotype.     

Deletion commonly found in Waxy gene of Japanese and Korean cultivars of Job’s tears (Coix lacryma-jobi L.)

We isolated the entire sequence of the coding region of Waxy gene of a non-waxy accession of Job??s tears (Coix lacryma-jobi) by PCR-based methods. We also compared the entire sequences of the gene between two non-waxy accessions and three waxy cultivars and found a 275-bp deletion in the coding region (exons 10?C11) of this gene specific to waxy cultivars. We showed by PCR genotyping that this deletion is commonly found in Japanese and Korean cultivars and confirmed that this deletion resulted in lack of Wx protein. We also confirmed that this polymorphism of the gene co-segregates with phenotypes in endosperm and pollen. These results suggest that this PCR-based marker will be useful in breeding of Job??s tears and that genetic information obtained in other grass species will be also useful in genetics and breeding of Job??s tears.     

Waxy strains of three amaranth grains raised by different mutations in the coding region

Waxy strains raised by waxy mutation have been found for three amaranth grains. Three genes encoding waxy protein were isolated from Amaranthus caudatus (Wx-ca), A. cruentus (Wx-cr), and A. hypochondriacus (Wx-hy). Sequence analysis indicated that the Wx-ca, Wx-cr, and Wx-hy genes contained the same exon (13 exons) and intron (12 introns) structure. The lengths of the Wx-ca, Wx-cr, and Wx-hy genes were 3,236, 3,237, and 3,225 bp, respectively. The alignment of the coding sequence of the three Waxy genes showed 12 polymorphic sites, including 11 single nucleotide polymorphisms (SNPs) (in exons 10 and 12 and introns 1, 3, 4, 9, and 11) and 5 deletions or insertions (indels) (in introns 4, 9, and 11). In particular, major polymorphism was detected in 8- and 3-bp indels in intron 4. Moreover, the mutation in the waxy alleles (wx-ca, wx-cr, and wx-hy) of all three species was also isolated and characterized. Comparison of coding sequences of the three Waxy genes and their waxy alleles indicated one base insertion (wx-ca: insert of T base in exon 8) and a base substitution (wx-cr: a G-to-T base substitution in exon 10; wx-hy: a G-to-A base substitution in exon 6), which occurred as internal termination codon in the three Waxy genes, suggesting the involvement of a nonsense or frameshift mutation. Therefore, these different mutations in coding regions were considered to be the cause of the waxy (amylose-free) phenotype.     

Molecular evidence for post-domestication selection in the <Emphasis Type="Italic">Waxy</Emphasis> gene of Chinese waxy maize

Waxy maize was first reported in China in 1909 and is mainly used in food production in Asia. The evidence for strong domestication selection in the Waxy locus of rice and a selective sweep around its genomic region make us to wonder whether there has been similar selection in Waxy in glutinous maize. To address this issue, DNA sequences of Waxy, three flanking genes and an unlinked gene (Adh1) of 30 accessions sampled from Chinese waxy maize accessions, including representative landraces and inbred lines, were determined in this study. Sharp reduction of nucleotide diversity and significant neutrality tests (Tajima’s D and Fu and Li’s F*) were observed in the Waxy locus in Chinese waxy maize but not in nonglutinous maize; comparison with the unlinked gene confirmed that this pattern was different to Waxy. Sequence analysis across a 143 kb genomic segment centered on the Waxy locus revealed patterns consistent with a selective sweep in the upstream region of Waxy. The selective sweep detected based on current limited genomic sequences exceeded over 50 kb, indicating strong selection in this or a bigger region. However, No sweep effect was detected in the repetitive downstream region of Waxy. Phylogenetic analysis indicated that Chinese waxy maize was domesticated from the cultivated flint maize (Zea mays ssp. mays) that was introduced from the new world. At least two independent deletions in exon 7 (30 bp) and 10 (15 bp) were identified in the Chinese accessions respectively. These findings demonstrate a similar pattern of domestication selection in the Waxy genomic region in both glutinous maize and rice, suggesting that this pattern in the rise of glutinous phenotype is likely in other cereal crops.     

Molecular characterization of spontaneous and induced mutations in the three homoeologous waxy genes of Japanese barnyard millet [Echinochloa esculenta (A. Braun) H. Scholz]

Japanese barnyard millet is an important food source in East Asian countries. However its crumbly texture limits desirability and consumption. Controlling amylose level in the endosperm is important to improve the eating quality of the millet. Because it is well known that the waxy gene determines the amylose level in the endosperm, we conducted a molecular analysis of the gene. Segregation analysis revealed that wild-type cultivars had three functional genes while low-amylose cultivars had one. We determined complete sequences of the three homoeologous waxy structural genes, EeWx1, EeWx2 and EeWx3, in a wild-type cultivar. These sequences showed high homology in the exon regions (97 %), and lower homology in the introns (82 %). Two spontaneous mutations were characterized in the low-amylose cultivars. In addition, one induced mutation was found in the fully waxy cultivar, Chojuromochi. Spontaneous mutations are deletions of whole and terminal regions in the EeWx2 and EeWx3 alleles, respectively. The induced mutation is a single-base deletion that led to a premature termination codon in EeWx1. These findings led us to develop useful markers for selecting low-amylose and waxy lines in millet.     

Genetic analysis of waxy locus in rice (Oryza sativa L.)

Summary Inheritance of waxy locus was studied in crosses of a waxy variety with four non-waxy parents having high-, intermediate-, low- or very low-amylose content. The analysis for amylose content was done on a single grain basis in parents, F₁, F₂, B₁F₁, and B₂F₁ seeds. The waxy parent lacking synthesis of amylose content was found to differ from the ones having high-, intermediate-, low- or very low-amylose content by one gene with major effect. Dosage effects for amylose content were observed to have great influence on segregation pattern and efficiency of selection. Selection efficiency for amylose content can be enhanced by selecting for endosperm appearance in early segregating generations.     

Evidence for the Inclusion of Controlling Elements within the Structural Gene at the Waxy Locus in Maize

Minimal limits for the structural gene at the waxy locus have been set by investigations of the protein product of the gene. An altered protein is produced by four of the waxy mutants including B3, a controlling-element mutation. All are similar to wild type in molecular weight as determined by electrophoresis in SDS acrylamide gels. At least three of the five wx controlling-element mutations studied have been shown to lie within the limits of the structural gene.     

Structural variation in the Waxy gene and differentiation in foxtail millet [Setaria italica (L.) P. Beauv.]: implications for multiple origins of the waxy phenotype

The origin and evolution of the waxy type of foxtail millet [Setaria italica (L.) P. Beauv] were studied by analyzing structural variation in the Waxy gene. Initially, the Waxy gene was amplified by RT-PCR, RACE and genomic PCR from a non-waxy strain to determine the structure of the wild-type gene. Secondly, we screened by PCR for polymorphisms at the Waxy locus in 79 strains with various waxy phenotypes. We then carried out genomic Southern analysis on 67 strains and identified seven RFLP classes which were designated as types I-VII. RFLP type was correlated with phenotype, such that types I and II corresponded to non-waxy, types III and VI to low-amylose, and types IV, V and VII to waxy phenotypes. The differences between RFLP types could be attributed to insertions in the Waxy gene. Types II and VI were caused by the insertion of a Tourist element into intron 1 and a SINE-like sequence into intron 12, respectively. Types III, IV, V and VII were characterized by the insertion of large sequences into the Waxy gene that may alter the expression of the gene. Thus, multiple, independent insertions in the Waxy gene appear to have caused the loss-of-function waxy phenotypes. Furthermore, the geographical distributions of the three RFLP types associated with the waxy phenotype (types IV, V and VII) were distinct, with type IV being found mainly in Taiwan and Japan, type V in Korea, and type VII in Myanmar. These results indicate a polyphyletic origin for the waxy phenotype in landraces of foxtail millet.     

Diverse origins of waxy foxtail millet crops in East and Southeast Asia mediated by multiple transposable element insertions

The naturally occurring waxy and low-amylose variants of foxtail millet and other cereals, like rice and barley, originated in East and Southeast Asia under human selection for sticky foods. Mutations in the GBSS1 gene for granule-bound starch synthase 1 are known to be associated with these traits. We have analyzed the gene in foxtail millet, and found that, in this species, these traits were originated by multiple independent insertions of transposable elements and by subsequent secondary insertions into these elements or deletion of parts of the elements. The structural analysis of transposable elements inserted in the GBSS1 gene revealed that the non-waxy was converted to the low-amylose phenotype once, while shifts from non-waxy to waxy occurred three times, from low amylose to waxy once and from waxy to low amylose once. The present results, and the geographical distribution of different waxy molecular types, strongly suggest that these types originated independently and were dispersed into their current distribution areas. The patterns of GBSS1 variation revealed here suggest that foxtail millet may serve as a key to solving the mystery of the origin of waxy-type cereals in Asia. The GBSS1 gene in foxtail millet provides a new example of the evolution of a gene involved in the processes of domestication and its post-domestication fate under the influence of human selection. Electronic Supplementary Material Supplementary material is available for this article at     

Genomic and phenotypic evidence for an incomplete domestication of South American grain amaranth (Amaranthus caudatus)

The domestication syndrome comprises phenotypic changes that differentiate crops from their wild ancestors. We compared the genomic variation and phenotypic differentiation of the two putative domestication traits seed size and seed colour of the grain amaranth Amaranthus caudatus, which is an ancient crop of South America, and its two close wild relatives and putative ancestors A. hybridus and A. quitensis. Genotyping 119 accessions of the three species from the Andean region using genotyping by sequencing (GBS) resulted in 9485 SNPs that revealed a strong genetic differentiation of cultivated A. caudatus from its two relatives. A. quitensis and A. hybridus accessions did not cluster by their species assignment but formed mixed groups according to their geographic origin in Ecuador and Peru, respectively. A. caudatus had a higher genetic diversity than its close relatives and shared a high proportion of polymorphisms with their wild relatives consistent with the absence of a strong bottleneck or a high level of recent gene flow. Genome sizes and seed sizes were not significantly different between A. caudatus and its relatives, although a genetically distinct group of A. caudatus from Bolivia had significantly larger seeds. We conclude that despite a long history of human cultivation and selection for white grain colour, A. caudatus shows a weak genomic and phenotypic domestication syndrome and proposes that it is an incompletely domesticated crop species either because of weak selection or high levels of gene flow from its sympatric close undomesticated relatives that counteracted the fixation of key domestication traits.     

A Novel Role for Mc1r in the Parallel Evolution of Depigmentation in Independent Populations of the Cavefish Astyanax mexicanus

The evolution of degenerate characteristics remains a poorly understood phenomenon. Only recently has the identification of mutations underlying regressive phenotypes become accessible through the use of genetic analyses. Focusing on the Mexican cave tetra Astyanax mexicanus, we describe, here, an analysis of the brown mutation, which was first described in the literature nearly 40 years ago. This phenotype causes reduced melanin content, decreased melanophore number, and brownish eyes in convergent cave forms of A. mexicanus. Crosses demonstrate non-complementation of the brown phenotype in F₂ individuals derived from two independent cave populations: Pachón and the linked Yerbaniz and Japonés caves, indicating the same locus is responsible for reduced pigmentation in these fish. While the brown mutant phenotype arose prior to the fixation of albinism in Pachón cave individuals, it is unclear whether the brown mutation arose before or after the fixation of albinism in the linked Yerbaniz/Japonés caves. Using a QTL approach combined with sequence and functional analyses, we have discovered that two distinct genetic alterations in the coding sequence of the gene Mc1r cause reduced pigmentation associated with the brown mutant phenotype in these caves. Our analysis identifies a novel role for Mc1r in the evolution of degenerative phenotypes in blind Mexican cavefish. Further, the brown phenotype has arisen independently in geographically separate caves, mediated through different mutations of the same gene. This example of parallelism indicates that certain genes are frequent targets of mutation in the repeated evolution of regressive phenotypes in cave-adapted species.     

Evolutionary dynamics of Waxy and the origin of hexaploid Spartina species (Poaceae)

We investigated the evolutionary dynamics of duplicated copies of the granule-bound starch synthase I gene (GBSSI or Waxy) within polyploid Spartina species. Molecular cloning, sequencing, and phylogenetic analyses revealed incongruences between the expected species phylogeny and the inferred gene trees. Some genes within species were more divergent than expected from ploidy level alone, suggesting the existence of paralogous sets of Waxy loci in Spartina. Phylogenetic analyses indicate that this paralogy originated from a duplication that occurred prior to the divergence of Spartina from other Chloridoideae. Gene tree topologies revealed three divergent homoeologous sequences in the hexaploid S. alterniflora that are consistent with the proposal of an allopolyploid origin of the hexaploid clade. Waxy sequences differ in insertion–deletion events in introns, which may be used to diagnose gene copies. Both paralogous and homoeologous coding regions appear to evolving under selective constraints.     

Genetic Diversity and Molecular Evolution of Chinese Waxy Maize Germplasm

Waxy maize (Zea mays L. var. certaina Kulesh), with many excellent characters in terms of starch composition and economic value, has grown in China for a long history and its production has increased dramatically in recent decades. However, the evolution and origin of waxy maize still remains unclear. We studied the genetic diversity of Chinese waxy maize including typical landraces and inbred lines by SSR analysis and the results showed a wide genetic diversity in the Chinese waxy maize germplasm. We analyzed the origin and evolution of waxy maize by sequencing 108 samples, and downloading 52 sequences from GenBank for the waxy locus in a number of accessions from genus Zea. A sharp reduction of nucleotide diversity and significant neutrality tests (Tajima’s D and Fu and Li’s F*) were observed at the waxy locus in Chinese waxy maize but not in nonglutinous maize. Phylogenetic analysis indicated that Chinese waxy maize originated from the cultivated flint maize and most of the modern waxy maize inbred lines showed a distinct independent origin and evolution process compared with the germplasm from Southwest China. The results indicated that an agronomic trait can be quickly improved to meet production demand by selection.     

Molecular Evolution of the TAC1 Gene from Rice （Oryza sativa L.）

Tiller angle is a key feature of the architecture of cultivated rice(Oryza sativa),since it determines planting density and influences rice yield.Our previous work identified Tiller Angle Control 1(TACl) as a major quantitative trait locus that controls rice tiller angle.To further clarify the evolutionary characterization of the TACl gene,we compared a TACl-containing 3164-bp genomic region among 113 cultivated varieties and 48 accessions of wild rice,including 43 accessions of O.rufipogon and five accessions of O.nivara.Only one single nucleotide polymorphism(SNP),a synonymous substitution,was detected in TACl coding regions of the cultivated rice varieties, whereas one synonymous and one nonsynonymous SNP were detected among the TACl coding regions of wild rice accessions.These data indicate that little natural mutation and modification in the TACl coding region occurred within the cultivated rice and its progenitor during evolution.Nucleotide diversities in the TACl gene regions of O.sativa and O.rufipogon of 0.00116 and 0.00112,respectively, further indicate that TACl has been highly conserved during the course of rice domestication.A functional nucleotide polymorphism (FNP) of TACl was only found in the japonica rice group.A neutrality test revealed strong selection,especially in the 3’-flanking region of the TACl coding region containing the FNP in the japonica rice group.However,no selection occurred in the indica and wild-rice groups.A phylogenetic tree derived from TACl sequence analysis suggests that the indica and japonica subspecies arose independently during the domestication of wild rice.     

Seed coat variation in Glycine Willd. subgenus Glycine (Leguminosae) by SEM

Seed of the genusGlycine Willd. typically exhibits a muriculate appearance resulting from adherence to the true seed coat of the perisperm or inner pod wall layer. Thickened cell walls of the perisperm superimpose a reticulate network on the seed coat, the type of network ranging from alveolate to stellate depending on the shape of the perisperm cells. Tubercles distributed at intervals give the seed its roughened appearance. Seed lacking an attached perisperm appears smooth and shiny. Seed morphology of 62 collections representing the six species of the subgenusGlycine is examined in detail to elucidate inter-and intraspecific variability. Seed perisperm pattern appears to be characteristic for each species, but there are exceptions.Glycine canescens F. J. Herrn. andG. clandestina Willd. seeds possess a reticulate network and tubercles of irregular shape, the perisperm appearing granular inG. clandestina. Seeds ofG. latrobeana (Meissn.) Benth. andG. tabacina (Labill.) Benth. lack a distinct network and have stellate tubercles; the perisperm is granular inG. latrobeana and some plants ofG. tabacina. A few collections ofG. clandestina approachG. tabacina in seed appearance.Glycine tomentella Hayata seeds exhibit a regularly alveolate arrangement, while those ofG. falcata Benth. lack a perisperm layer altogether. Variation in seed coat within a species can usually be linked to differences in chromosome number or some aspect of gross morphology. Diploid collections ofG. tomentella (2n = 40) exhibit recognizable differences in seed morphology compared with tetraploids (2n = 80), coincident with other striking dissimilarities in gross morphology. An incompletely attached perisperm is accompanied by aneuploidy in severalG. tomentella accessions, while other 78 and 38 chromosome aneuploids produce normal seeds.     

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.     

Natural Variation in the Flag Leaf Morphology of Rice Due to a Mutation of the NARROW LEAF 1 Gene in Oryza sativa L.

We investigated the natural variations in the flag leaf morphology of rice. We conducted a principal component analysis based on nine flag leaf morphology traits using 103 accessions from the National Institute of Agrobiological Sciences Core Collection. The first component explained 39% of total variance, and the variable with highest loading was the width of the flag leaf (WFL). A genome-wide association analysis of 102 diverse Japanese accessions revealed that marker RM6992 on chromosome 4 was highly associated with WFL. In analyses of progenies derived from a cross between Takanari and Akenohoshi, the most significant quantitative trait locus (QTL) for WFL was in a 10.3-kb region containing the NARROW LEAF 1 (NAL1) gene, located 0.4 Mb downstream of RM6992. Analyses of chromosomal segment substitution lines indicated that a mutation (G1509A single-nucleotide mutation, causing an R233H amino acid substitution in NAL1) was present at the QTL. This explained 13 and 20% of total variability in WFL and the distance between small vascular bundles, respectively. The mutation apparently occurred during rice domestication and spread into japonica, tropical japonica, and indica subgroups. Notably, one accession, Phulba, had a NAL1 allele encoding only the N-terminal, or one-fourth, of the wild-type peptide. Given that the Phulba allele and the histidine-type allele showed essentially the same phenotype, the histidine-type allele was regarded as malfunctional. The phenotypes of transgenic plants varied depending on the ratio of histidine-type alleles to arginine-type alleles, raising the possibility that H²³³-type products function differently from and compete with R²³³-type products.     

Molecular characterization and expression analysis of a gene encoding an isoamylase-type starch debranching enzyme 3 (ISA3) in grain amaranths

A cDNA clone from amaranth perisperm that encodes an isoamylase (ISA)-type starch debranching enzyme 3 was isolated and analyzed for the first time. The cDNA consisted of 2,715 bp with a single open reading frame of 2,346 bp, encoding a protein of 781 amino acid residues. The deduced amino acid sequence of CrISA3 shared 63–71 % identity with those of other plant ISA3s. We also investigated the genetic diversity of ISA3 in three species of grain amaranth. A comparison of their ISA3 coding sequences revealed an extremely high level of conservation and only 11 single nucleotide polymorphisms were detected. The expression of the CrISA3 gene in amaranth developmental seeds and several tissues was investigated by qRT-PCR analysis. The results showed that CrISA3 was rapidly expressed at the early stage during seed maturation. It was also expressed in non-storage tissues (leaf, petiole, stem, and root) as well as in storage tissue. This observation demonstrates that CrISA3 may play an important role in perisperm starch accumulation at the early developmental stages. In addition, our results indicate that CrISA3 plays important roles in the synthesis of storage and transitory starches. The characterization of the CrISA3 gene will contribute to further studies on starch biosynthesis in Amaranthus.