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 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.     

High-resolution cytological mapping of the long arm of chromosome 5A in common wheat using a series of deletion lines induced by gametocidal (Gc) genes of Aegilops speltoides

Gametocidal (Gc) genes of Aegilops in the background of the wheat genome lead to breakage of wheat chromosomes. The Q gene of wheat was used as a marker to select 19 deletion lines for the long arm of chromosome 5A of common wheat, Triticum aestivum cv. Chinese Spring (CS). The extents of deleted segments were cytologically estimated by the C-banding technique. The DNAs of deletion lines were hybridized with 22 DNA probes recognizing sites on the long arm of the chromosome (5AL) to determine their physical order. Based on the breeding behavior of the deletion lines, the location of a novel gene (Pv, pollen viability) affecting the viability of the male gamete was deduced. The segment translocated from 4AL to 5AL in CS was cytologically estimated to represent 13% of the total length of 5AL. Although DNA markers were almost randomly distributed along the chromosome arm, DNA markers located around the centromere and C-banded regions were obtained only rarely. Some deletion lines were highly rearranged in chromosome structure due to the effect(s) of the Gc gene. Applications of Gc genes for manipulating wheat chromosomes are discussed.     

Overexpression of Puroindoline a gene in transgenic durum wheat (Triticum turgidum ssp. durum) leads to a medium–hard kernel texture

Durum wheat is the second-most widely grown wheat species, and is primarily used in the production of pasta and couscous. The grain utilization of durum wheat is partly related to its very hard kernel texture because of the lack of the D genome and consequentially the Puroindoline genes. Our previous study reported the transformation of durum wheat with the Puroindoline a (Pina) gene. Here, we characterized the transgenic durum wheat lines expressing the Pina gene, and studied the effects of PINA on grain texture and other kernel characteristics. SDS-PAGE and Western blotting results demonstrated that starch-bound PINA levels of Pina-overexpressing lines were lower than that of Pina-positive control, common wheat cv. Chinese Spring, suggesting a weak association of PINA protein with starch granules in the absence of Pinb. Grain hardness analysis and flour milling tests indicated that the overexpression of PINA resulted in decreased grain hardness and increased flour yield in transgenic durum wheat lines. The agronomic performance of the transgenic and control lines was also examined and it was found that no significant differences in measured traits were observed between Pina-overexpressing and control lines in the 2-year field trials. Since grain hardness strongly affects milling and end-use qualities, the development of medium–hard-textured durum wheat lines is not only of significance for our knowledge of grain hardness and Puroindolines, but also has practical implications for plant breeders and food technologists for the expansion of utilization of durum wheat.     

Physical localization of a novel blue-grained gene derived from Thinopyrum bessarabicum

Blue wheat grain contains different groups of pigments that can be used for making specialty foods or as food colorants. Thinopyrum bessarabicum, a wild relative of wheat, carries a blue-grained gene on chromosome 4J. In this study, we analyzed the mitotic chromosomes of 159 F7 lines derived from the cross between Triticum aestivum cv. Chinese Spring (CS) and a CS–Th. bessarabicum amphiploid by using multi-color fluorescence in situ hybridization, genomic in situ hybridization, and newly developed chromosome 4J-specific DNA markers. Intact chromosome 4J and various 4J chromosomal segments were identified in the 159 lines. The blue-grained gene of Th. bessarabicum was physically localized to the region between the centromere and FL0.52 on chromosome arm 4JL. The chromosomal location of this gene differed from the location of previously reported blue-grained genes. In addition, a strong dosage effect was observed with this gene. These results suggest that the blue-grained gene in Th. bessarabicum represents a novel gene locus for blue aleurone, designated BaThb. The wheat lines and 4J chromosome-specific molecular markers developed in this study will facilitate the introgression and utilization of BaThb for wheat nutritional quality improvement.     

Mapping of the loose smut resistance gene Ut6 in wheat (Triticum aestivum L.)

Loose smut of wheat (Triticum aestivum L.) caused by Ustilago tritici (Pers.) Rostr. can cause considerable yield losses in the absence of appropriate management practices. The use of wheat varieties with loose smut resistance is an efficient and effective control technique. However, the development of commercial wheat lines with resistance to loose smut is time- and labour-consuming. DNA markers linked to loose smut resistance gene(s) would assist the development of loose smut resistant genotypes. The genetics of loose smut resistance was studied in an F5‐derived recombinant inbred line (RIL) population of 94 lines from the cross BW278/AC Foremost. The line AC Foremost is resistant and line BW278 is susceptible to U. tritici race T10. Phenotypic assessment revealed that a single gene, designated Ut6, segregated for resistance to race T10 in the RIL population. A modified bulked segregant analysis identified a microsatellite marker linked to Ut6. A linkage map was developed consisting of linked microsatellite loci and the resistance gene. The loose smut resistance gene Ut6 mapped to the long arm of chromosome 5B. Five microsatellite markers mapped within 6.7 cM of Ut6. The microsatellite markers gpw5029 and barc232 flanked Ut6 at distances of 1.3 and 2.8 cM on the distal and proximal sides, respectively. A diverse set of wheat lines was haplotyped for Ut6 using the linked microsatellite markers gpw5029 and barc232. The haplotype analysis suggested that the microsatellite markers associated with Ut6 will be useful for marker-assisted selection of loose smut resistant wheat lines.     

Genetic control of amylose content in wheat endosperm starch and differential effects of three Wx genes

The endosperm starch of the wheat grain is composed of amylose and amylopectin. Genetic manipulation of the ratio of amylose to amylopectin or the amylose content could bring about improved texture and quality of wheat flour. The chromosomal locations of genes affecting amylose content were investigated using a monosomic series of Chinese Spring (CS) and a set of Cheyenne (CNN) chromosome substitution lines in the CS genetic background. Trials over three seasons revealed that a decrease in amylose content occurred in monosomic 4A and an increase in monosomic 7B. Allelic variation between CS and CNN was suggested for the genes on chromosomes 4A and 7B. To examine the effects of three Waxy (Wx) genes which encode a granule-bound starch synthase (Wx protein), the Wx proteins from CS monosomics of interest were analyzed using SDS-PAGE. The amount of the Wx protein coded by the Wx-B1 gene on chromosome arm 4AL was reduced in monosomic 4A, and thus accounted for its decreased amylose content. The amounts of two other Wx proteins coded by the Wx-A1 and Wx-D1 genes on chromosome arms 7AS and 7DS, respectively, showed low levels of protein in the monosomics but no effect on amylose content. The effect of chromosome 7B on the level of amylose suggested the presence of a regulator gene which suppresses the activities of the Wx genes.     

Thinopyrum 7Ai-1-derived small chromatin with Barley Yellow Dwarf Virus (BYDV) resistance gene integrated into the wheat genome with retrotransposon

Thinopyrum intermedium is a useful source of resistance genes for Barley Yellow Dwarf Virus (BYDV), one of the most damaging wheat diseases. In this study, wheat/Th. intermedium translocation lines with a BYDV resistance gene were developed using the Th. intermedium 7Ai-1 chromosome. Genomic in situ hybridization (GISH), using a Th. intermedium total genomic DNA probe, enabled detection of 7Ai-1-derived small chro-matins containing a BYDV resistance gene, which were translocated onto the end of wheat chromosomes in the lines Y95011 and Y960843. Random amplified polymorphic DNA (RAPD) analyses using 120 random 10-mer primers were conducted to compare the BYDV-resistant translocation lines with susceptible lines. Two primers amplified the DNA fragments specific to the resistant line that would be useful as molecular markers to identify 7Ai-1-derived BYDV resistance chromatin in the wheat genome. Additionally, the isolated Th. intermedium-specific retrotransposon-like sequence pTi28 can be used to identify Th. intermedium chromatin transferred to the wheat genome.     

The Bxb1 Recombinase Mediates Site-Specific Deletion in Transgenic Wheat

The utility and commercial potential of genetically engineered (GE) plants would benefit from the use of site-specific recombination systems that enable efficient excision of the marker genes used to identify transformants. Although wheat is one of the most important food crops in the world, GE varieties have yet to be put into commercial production. To develop the Bxb1 recombination system (derived from the Mycobacterium smegmati bacteriophage Bxb1) for site-specific marker gene removal in transgenic wheat, we used biolistics to introduce into the wheat genome a codon optimized Bxb1 recombinase gene (BxbNom) under the control of the maize ubiquitin-1 promoter (Ubi1). Recombinase activity was monitored using a GUSPlus reporter gene activation assay. BxbNom recombinase-mediated excision of an att site-flanked stuffer DNA fragment activated ??-glucuronidase reporter activity in callus, endosperm, and leaves in transient assays. The system also detected activity in leaves and endosperm of progeny of multiple independent transgenic wheat lines stably expressing BxbNom. Our results demonstrate that the Bxb1 recombinase is heritable in transgenic wheat plants and performs site-specific excision, providing a useful tool for generating marker-free GE plants. Establishment of wheat lines capable of efficiently excising unneeded marker genes removes one potential barrier to commercial deployment of GE wheat.     

Cytoplasmic effects on DNA methylation between male sterile lines and the maintainer in wheat (Triticum aestivum L.)

Male sterile cytoplasm plays an important role in hybrid wheat, and three-line system including male sterile (A line), its maintainer (B line) and restoring (R line) has played a major role in wheat hybrid production. It is well known that DNA methylation plays an important role in gene expression regulation during biological development in wheat. However, no reports are available on DNA methylation affected by different male sterile cytoplasms in hybrid wheat. We employed a methylation-sensitive amplified polymorphism technique to characterize nuclear DNA methylation in three male sterile cytoplasms. A and B lines share the same nucleus, but have different cytoplasms which is male sterile for the A and fertile for the B. The results revealed a relationship of DNA methylation at these sites specifically with male sterile cytoplasms, as well as male sterility, since the only difference between the A lines and B line was the cytoplasm. The DNA methylation was markedly affected by male sterile cytoplasms. K-type cytoplasm affected the methylation to a much greater degree than T-type and S-type cytoplasms, as indicated by the ratio of methylated sites, ratio of fully methylated sites, and polymorphism between A lines and B line for these cytoplasms. The genetic distance between the cytoplasm and nucleus for the K-type is much greater than for the T- and S-types because the former is between Aegilops genus and Triticum genus and the latter is within Triticum genus between Triticum spelta and Triticum timopheevii species. Thus, this difference in genetic distance may be responsible for the variation in methylation that we observed.     

Molecular mapping of a gene for stripe rust resistance in spring wheat cultivar IDO377s

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases of wheat worldwide. The best strategy to control stripe rust is to grow resistant cultivars. One such cultivar resistant to most races in North America is ‘IDO377s’. To study the genetics of its resistance this spring wheat cultivar was crossed with ‘Avocet Susceptible’ (AvS). Seedlings of the parents, F₂ plants, and F₃ lines were tested under controlled greenhouse conditions with races PST-43 and PST-45 of P. striiformis f. sp. tritici. IDO377s carries a single dominant gene for resistance. Resistance gene analog polymorphism (RGAP) and simple sequence repeat (SSR) techniques were used to identify molecular markers linked to the resistance gene. A total of ten markers were identified, two of which flanked the locus at 4.4 and 5.5 cM. These flanking RGAP markers were located on chromosome 2B with nulli-tetrasomic lines of ‘Chinese Spring’. Their presence in the ditelosomic 2BL line localized them to the long arm. The chromosomal location of the resistance gene was further confirmed with two 2BL-specific SSR markers and a sequence tagged site (STS) marker previously mapped to 2BL. Based on the chromosomal location, reactions to various races of the pathogen and tests of allelism, the IDO377s gene is different from all previously designated genes for stripe rust resistance, and is therefore designated Yr43. A total of 108 wheat breeding lines and cultivars with IDO377s or related cultivars in their parentage were assayed to assess the status of the closest flanking markers and to select lines carrying Yr43. The results showed that the flanking markers were reliable for assisting selection of breeding lines carrying the resistance gene. A linked stripe rust resistance gene, previously identified as YrZak, in cultivar Zak was designated Yr44.     

Hybrid dwarfness in crosses between wheat (Triticum aestivum L.) and rye (Secale cereale L.): a new look at an old phenomenon

The existence of hybrid dwarfs from intraspecific crosses in wheat (Triticum aestivum) was described 100 years ago, and the genetics underlying hybrid dwarfness are well understood. In this study, we report a dwarf phenotype in interspecific hybrids between wheat and rye (Secale cereale). We identified two rye lines that produce hybrid dwarfs with wheat and have none of the hitherto known hybrid dwarfing genes. Genetic analyses revealed that both rye lines carry a single allelic gene responsible for the dwarf phenotype. This gene was designated Hdw‐R1 (Hybrid dwarf‐R1). Application of gibberellic acid (GA₃) to both intraspecific (wheat–wheat) and interspecific (wheat–rye) hybrids showed that hybrid dwarfness cannot be overcome by treatment with this phytohormone. Histological analysis of shoot apices showed that wheat–rye hybrids with the dwarf phenotype at 21 and 45 days after germination failed to develop further. Shoot apices of dwarf plants did not elongate, did not form new primordia and had a dome‐shaped appearance in the seed. The possible relationship between hybrid dwarfness and the genes responsible for the transition from vegetative to generative growth stage is discussed.     

Map-based analysis of the tenacious glume gene Tg-B1 of wild emmer and its role in wheat domestication

The domestication of wheat was instrumental in spawning the civilization of humankind, and it occurred through genetic mutations that gave rise to types with non-fragile rachises, soft glumes, and free-threshing seed. Wild emmer (Triticum turgidum ssp. dicoccoides), the tetraploid AB-genome progenitor of domesticated wheat has genes that confer tenacious glumes (Tg) that underwent genetic mutations to give rise to free-threshing wheat. Here, we evaluated disomic substitution lines involving chromosomes 2A and 2B of wild emmer accessions substituted for homologous chromosomes in tetraploid and hexaploid backgrounds. The results suggested that both chromosomes 2A and 2B of wild emmer possess genes that inhibit threshability. A population of recombinant inbred lines derived from the tetraploid durum wheat variety Langdon crossed with a Langdon — T. turgidum ssp. dicoccoides accession PI 481521 chromosome 2B disomic substitution line was used to develop a genetic linkage map of 2B, evaluate the genetics of threshability, and map the gene derived from PI 481521 that inhibited threshability. A 2BS linkage map comprised of 58 markers was developed, and markers delineated the gene to a 2.3 cM interval. Comparative analysis with maps containing the tenacious glume gene Tg-D1 on chromosome arm 2DS from Aegilops tauschii, the D genome progenitor of hexaploid wheat, revealed that the gene inhibiting threshability in wild emmer was homoeologous to Tg-D1 and therefore designated Tg-B1. Comparative analysis with rice and Brachypodium distachyon indicated a high level of divergence and poorly conserved colinearity, particularly near the Tg-B1 locus. These results provide a foundation for further studies involving Tg-B1, which, together with Tg-D1, had profound influences on wheat domestication.     

Selection and identification of a spontaneous alien chromosome translocation in wheat

A wheat (Triticum aestivum L. emend Thell) disomic addition line (2n = 6x = 44), SH1–152–2, with a pair of Elytrigia pontica (Podp.) Holub 2n = 10x = 70 [syn. Agropyron elongatum (Host) P.B.] chromosomes controlling blue aleurone color was crossed with a short-statured spring wheat `Sonora 64' (T. aestivum). Isoline pairs of blue-disomic addition lines and nonblue euploid lines were produced by selecting plants segregating for blue aleurone for 12 generations. Nineteen of 20 blue aleurone lines were 2n = 44 addition lines, and one had 2n = 42 chromosomes. Several lines of evidence showed that this line had a spontaneous translocation in which the β arm of wheat chromosome 4A was replaced by an Elytrigia chromosome arm carrying the blue aleurone gene. The Elytrigia chromosome in SH1–152–2 appeared to be homologous with E. pontica chromosome 4el₁, which also carries the blue aleurone gene. It was concluded that the spontaneous translocation originated from simultaneous misdivision of univalents and subsequent reunion at the centromere of chromosome arm 4Aα with the Elytrigia chromosome arm.