Molecular characterization and comparative analysis of two waxy alleles in barley

Two alleles of the barley waxy locus were characterized from non-waxy cultivar Bowman and waxy cultivar CDC Candle, respectively. Their nucleotide and protein sequences were compared with other known waxy genes. The comparison results indicated that there were 100 polymorphic sites, among which 69 were in the non-coding region and 31 were in the coding region. Out of 100 polymorphic sites, 45 were trans-version, 35 were transition and 20 were indels. A 397 bp deletion and a 193 bp insertion in the promoter region and a 15 bp insertion in the coding region were found in CDC Candle, but not in Bowman. A deletion (11 bp) was detected in Bowman, which exhibited no effects on normal waxy expression. In summary, the 397 bp deletion was supposed to account for the reduction of GBSS I, resulting in the low amylose in CDC Candle; whereas other polymorphic sites might be not correlated with amylose synthesis.     

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.     

The molecular basis of mutations at the Waxy locus from Amaranthus caudatus L.: evolution of the waxy phenotype in three species of grain amaranth

Polymorphisms at the Waxy locus of Amaranthus caudatus L. collected from a wide range of regions were used to investigate genetic diversity and mutation sites. A comparison of the Waxy locus revealed a very high level of sequence conservation. This result clearly showed low environmental and evolutionary variability in the Waxy gene. We also performed screening to confirm the mutation sites in the coding sequences of all accessions. The results indicate that one insertion in the coding region of Waxy genes was responsible for the change in perisperm starch leading to the waxy phenotype in all accessions of this species, and thus that a single mutation event altered the regulation of the Waxy gene during the domestication of this crop. In addition, phylogenetic analysis showed that waxy phenotypes within each of three species, A. caudatus, A. cruentus and A. hypochondriacus, originated separately or differentiated from nonwaxy phenotypes of each species through a single mutational event (i.e., a frame shift or base substitution). We also compared obvious structural features of the coding sequence of waxy and nonwaxy phenotypes with those of low-amylose phenotypes in A. caudatus. The Waxy coding sequences of low-amylose phenotypes do not show polymorphisms and are identical with those of waxy phenotypes. This could mean that there is another gene that encodes a key enzyme responsible for amylose synthesis as the elementary quantity in tissues other than perisperm in A. caudatus.     

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.     

Molecular characteristics of two new <Emphasis Type="Italic">waxy</Emphasis> mutations in China waxy maize

The waxy gene mutation causes waxy maize grain to have a sticky quality. China has numerous waxy maize landraces and is thought to be the place of origin of waxy maize. The most abundant waxy maize resources in China are located in the Yunnan province and its surrounding areas. We collected 57 waxy maize landraces from Yunnan province and cloned and sequenced the waxy gene from its fourth to eighth exon. Two new waxy gene mutations, named wx-Cin4 and wx-124, were identified. The wx-Cin4 mutation is a 466-bp retrotransposon inserted into exon six. The wx-124 mutation is a 116-bp miniature inverted-repeat transposable element inserted into exon seven. This is the first time a 124-type mutation has been found in a maize waxy gene. The discovery of the two specific waxy mutations from landraces collected in Yunnan province provides new evidence supporting the hypothesis that China is the origin area for waxy maize.     

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.     

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.     

Identification of novel alleles induced by EMS-mutagenesis in key genes of kernel hardness and starch biosynthesis in wheat by TILLING

To identify novel allelic variations in key genes of wheat quality, the present study used the targeting induced local lesions in genomes platform to detect point mutations in target genes. The wheat variety Longfumai 17 was treated by the mutagen ethyl methanesulfonate to produce a bulk M₂ generation, and the population included 1122 plants. A total length of 3906.80 kb nucleotides was analyzed, and the average mutation density was 1/244.17 kb. The identified mutations included G>A substitutions (43.75%), C>T substitutions (31.25%), A insertions (12.50%), T insertions (6.25%), and deletions (6.25%). These point mutations led to changes in amino acids and thus the encoded protein sequences, ultimately producing 18.75% of missense mutations, 12.50% of frame shift mutations, 6.25% of nonsense mutations, 25.00% of silent mutations and 37.50% of non-coding region mutations. In the kernel hardness gene Pinb and 3 starch synthesis genes waxy, Agp2 and SSIIa-A, we detected 16 different point mutations in 25 mutant lines. The Pinb gene harbored two missense mutations and a nonsense mutation; the C>T missense mutation resulted in a novel allele, this novel allele and the nonsense mutation alerted protein 3D structure; the waxy gene presented missense and frame shift mutations; the Agp2 gene carried a missense mutation; the SSIIa-A incurred a missense mutation and a frame shift mutation that resulted in premature protein termination. All the frame shift mutations, nonsense mutations and the Pinb novel allele resulted in allelic variation of their corresponding genes, which in turn affected their gene functions. The identified mutant lines can be used as intermediate materials in wheat quality improvement schemes.     

Comparison of the primary structure ofwaxy proteins (granule-bound starch synthase) between polyploid wheats and related diploid species

Thewaxy proteins encoded by the genomes A, B, and D in polyploid wheats and related diploid species were isolated by SDS-PAGE. The N-terminal amino acid sequences of mature proteins and V8 protease-induced fragments were determined. A total of five amino acid substitutions was detected in these sequences, which represent about 10% of the whole sequences of thewaxy proteins. A comparison of these sequences in polyploid wheats with those in related diploid species revealed the following: (i)waxy proteins encoded by the A genome of polyploid wheats were identical to that ofTriticum monococcum, (ii) thewaxy protein encoded by the B genome ofT. turgidum was identical to that ofT. searsii, but differed from those ofT. speltoides andT. longissimum by one amino acid substitution, (iii) thewaxy protein encoded by the B genome ofT. aestivum differed from that encoded by the B genome ofT. turgidum by one amino acid substitution, and (iv) thewaxy protein encoded by the D genome ofT. aestivum was identical to that ofT. tauschii.     

Marker-assisted selection for pyramiding the waxy and opaque-16 genes in maize using cross and backcross schemes

The waxy (wx) gene in maize is associated with higher content of amylopectin in the endosperm and better flavor. The opaque-16 (o16) gene is associated with higher lysine content in the endosperm and better nutritional value. To pyramid the wx and o16 genes, cross and backcross populations were constructed using the o16 line QCL3024 and the two waxy lines, QCL5019 and QCL5008, as parents. The linkage marker umc1141 for the o16 gene and the internal marker phi027 for the wx gene were used to select the target genes. Simple sequence repeat markers covering the whole genome were used for background selection in individual progenies of the backcross population. The grain lysine content was determined using the Acid Orange-12 Dye Binding Lysine method. Qualitative and quantitative analyses of the grain content of amylopectin were performed using the I₂-KI procedure and double-wavelength spectrophotometry, respectively. Four lines of the double recessive genotype wxwxo16o16 were obtained from the F4 generation of the cross population and three lines of the same genotype were obtained from the BC2F4 generation of the backcross population. The lysine content of the pyramid lines was 16–27 and 18–28 % higher than the waxy parents QCL5019 and QCL5008, respectively. The pyramid lines had 61–63 % more amylopectin than the high-lysine parent QCL3024. The three pyramid lines from the backcross population had similar genetic background to the waxy parent QCL5008. Our results are of significance for the improvement of maize quality.     

Post-Domestication Selection in the Maize Starch Pathway

Modern crops have usually experienced domestication selection and subsequent genetic improvement (post-domestication selection). Chinese waxy maize, which originated from non-glutinous domesticated maize (Zea mays ssp. mays), provides a unique model for investigating the post-domestication selection of maize. In this study, the genetic diversity of six key genes in the starch pathway was investigated in a glutinous population that included 55 Chinese waxy accessions, and a selective bottleneck that resulted in apparent reductions in diversity in Chinese waxy maize was observed. Significant positive selection in waxy (wx) but not amylose extender1 (ae1) was detected in the glutinous population, in complete contrast to the findings in non-glutinous maize, which indicated a shift in the selection target from ae1 to wx during the improvement of Chinese waxy maize. Our results suggest that an agronomic trait can be quickly improved into a target trait with changes in the selection target among genes in a crop pathway.     

Polymorphism in a histone H1 subtype with a short N-terminal domain in three legume species (Fabaceae, Fabaeae)

A number of alleles of an orthologous gene His6 encoding histone H1 subtype f (H1-6 in pea) accumulated in chromatin of old tissues were sequenced in three legume species: seven alleles in Pisum sativum, four in Vicia unijuga and eight in Lathyrus gmelinii. In the total of 19 alleles sequenced in the three species, 29 non-synonymous substitutions and six indels were found in the coding region; most of amino acid substitutions (26 of 29) and all indels occurred in the C-terminal hydrophilic domain of the encoded protein. All species were polymorphic for some non-synonymous substitutions, V. unijuga was also polymorphic for one and P. sativum for two indels. Three near-isogenic lines of P. sativum bearing different alleles showed differences in many quantitative traits; that in the growth dynamic could be tentatively attributed to the allelic substitution of subtype H1-6. The frequencies of four electromorphs in a sampled locality of V. unijuga were found to be close to those observed 25?years ago, although their rapid change in the past was supposed in the previous study.     

Waxy genes from spelt wheat: new alleles for modern wheat breeding and new phylogenetic inferences about the origin of this species

Background and Aims

Waxy proteins are responsible for amylose synthesis in wheat seeds, being encoded by three waxy genes (Wx-A1, Wx-B1 and Wx-D1) in hexaploid wheat. In addition to their role in starch quality, waxy loci have been used to study the phylogeny of wheat. The origin of European spelt (Triticum aestivum ssp. spelta) is not clear. This study compared waxy gene sequences of a Spanish spelt collection with their homologous genes in emmer (T. turgidum ssp. dicoccum), durum (T. turgidum ssp. durum) and common wheat (T. aestivum ssp. aestivum), together with other Asian and European spelt that could be used to determine the origin of European spelt.

Methods

waxy genes were amplified and sequenced. Geneious Pro software, DNAsp and MEGA5 were used for sequence, nucleotide diversity and phylogenetic analysis, respectively.

Key Results

Three, four and three new alleles were described for the Wx-A1, Wx-B1 and Wx-D1 loci, respectively. Spelt accessions were classified into two groups based on the variation in Wx-B1, which suggests that there were two different origins for the emmer wheat that has been found to be part of the spelt genetic make-up. One of these groups was only detected in Iberian material. No differences were found between the rest of the European spelt and the Asiatic spelt, which suggested that the Iberian material had a different origin from the other spelt sources.

Conclusions

The results suggested that the waxy gene variability present in wheat is undervalued. The evaluation of this variability has permitted the detection of ten new waxy alleles that could affect starch quality and thus could be used in modern wheat breeding. In addition, two different classes of Wx-B1 were detected that could be used for evaluating the phylogenetic relationships and the origins of different types of wheat.     

Molecular characterization of novel haplotypes of eIF4E family in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Two genes coding for eukaryotic translation initiation factors, eIF4E.a and eIF4E.c, were isolated from twelve accessions of Chinese cabbage (Brassica rapa L. ssp. pekinensis). Polymorphism analysis revealed that 94 and 142 polymorphic sites were characterized from allele of BraeIF4E.a and BraeIF4E.c which produced complex haplotype structures. Six novel haplotypes were characterized from the two alleles respectively. Among the six novel haplotypes of BraeIF4E.a, three loss-of-function mutations were identified in which a conserved single nucleotide deletion mutation cause the early termination of BraeIF4E.a coding product; while for six new BraeIF4E.c haplotypes, their coding product show amino acid substitution mutations on non-conservative amino acid residues which might affect TuMV infection in Chinese cabbage.     

Sequence of the 18S-5S ribosomal gene region and the cytochrome oxidase II gene from mtDNA of Zea diploperennis

Summary The coding and flanking sequences of the 18S-5S ribosomal RNA genes and the cytochrome oxidase subunit II gene of Zea diploperennis mitochondrial DNA have been determined and compared to the corresponding sequences of normal maize (Zea mays L.) Both length and substitution mutations are found in the coding region of the 18S rRNA gene, whereas only one substitution mutation is found in the coding region of cytochrome oxidase II. Sequence divergence between maize and Zea diploperennis is about one-tenth of that between wheat and maize. The rate of nucleotide divergence by base substitution is less for plant mitochrondrial genes than for comparable genes in animal mitochondria.     

Partial purification and characterization of granule-bound starch synthases from normal and waxy maize

The granule-bound starch-synthases from normal and waxy maize kernels have been solubilized, partially purified, and characterized. Two broad categories of starch synthases were revealed with representatives in the soluble phase and also on granules from both normal and waxy maize though the activity associated with granules from waxy was small. Data for native molecular weights, kinetic parameters, and immunological relatedness are used to demonstrate that the granule-bound isozymes from normal maize are different from the soluble enzymes. These distinct granule-bound enzymes are missing in waxy maize granules and a further novel form of starch synthase is revealed. These findings are discussed in relation to the type of starch produced in tissue affected by the waxy mutation.     

Complete sequence of the chloroplast genome from pear (Pyrus pyrifolia): genome structure and comparative analysis

The chloroplast genome of Pyrus was found to be 159,922?bp in length which included a pair of inverted repeats (IRs) of 26,392?bp, separated by a small single-copy region of 19,237?bp and a large single-copy region (LSC) of 87,901?bp. A total of 130 predicted genes (113 unique genes and 17 genes, which were duplicated in the IR) including 79 protein-coding genes, four ribosomal RNA genes and 30 tRNA genes were identified based on similarity to homologs from the chloroplast genome of Nicotiana tabacum. Genome organization was very similar to the inferred ancestral angiosperm chloroplast genome. Comparisons between Pyrus, Malus, and Prunus in Rosaceae revealed 220 indels (??10?bp). Excluding ycf1 and ycf2, which contained deletions in the coding region, all of these were detected in the spacer or intron regions. Three insertions and 13 deletions were detected in Pyrus compared to the same loci in Malus and Prunus. After comparing 89 noncoding chloroplast DNA regions in Pyrus and Malus, highly variable regions such as ndhC-trnV and trnR-atpA were identified. In Pyrus and Malus, the IR/LSC borders were 62?bp shorter than those of Prunus. In addition, there were length mutations at the IRa/LSC junction and in trnH. A total of 67 simple sequence repeats (more than 10 repeated motifs) were identified in the Pyrus chloroplast genome. The indels and simple sequence repeats will be useful evolutionary tools at both intra- and interspecific levels. Phylogenetic analysis demonstrated a close relationship between Pyrus and Prunus in the Rosaceae.