Production of novel allelic variation for genes involved in starch biosynthesis through mutagenesis

Given the important role that starch plays in food and non-food uses of many crops, particularly wheat, efforts are being made to manipulate its composition through modification of the amylose/amylopectin ratio. Approaches used to achieve this goal include the manipulation of the genes involved in the starch biosynthetic pathway using natural or induced mutations and transgenic methods. The use of mutagenesis to produce novel allelic variation represents a powerful tool to increase genetic diversity and this approach seems particularly appropriate for starch synthase genes for which limited variation exists. In this work, an EMS-mutagenised population of bread wheat cv. Cadenza has been screened by combining SDS–PAGE analysis of granule bound starch proteins with a TILLING (Targeting Induced Local Lesions IN Genomes) approach at the gene level. In particular we have focused on two groups of synthase genes, those encoding the starch synthase II (Sgp-1) and those corresponding to the waxy proteins (Wx). SDS–PAGE analysis of granule bound proteins allowed the identification of single null genotypes associated with each of the three homoeologous loci. Molecular characterization of induced mutants has been performed using genome specific primer pairs for Sgp-1 and Wx genes. Additional novel allelic variation has also been detected at the different Sgp-1 homoeoloci by using a reverse genetic approach (TILLING). In particular single nucleotide substitutions, introducing a premature stop codon and creating amino acid substitutions, have been identified.     

A high-throughput method for the detection of homoeologous gene deletions in hexaploid wheat

Background  

Mutational inactivation of plant genes is an essential tool in gene function studies. Plants with inactivated or deleted genes may also be exploited for crop improvement if such mutations/deletions produce a desirable agronomical and/or quality phenotype. However, the use of mutational gene inactivation/deletion has been impeded in polyploid plant species by genetic redundancy, as polyploids contain multiple copies of the same genes (homoeologous genes) encoded by each of the ancestral genomes. Similar to many other crop plants, bread wheat (Triticum aestivum L.) is polyploid; specifically allohexaploid possessing three progenitor genomes designated as 'A', 'B', and 'D'. Recently modified TILLING protocols have been developed specifically for mutation detection in wheat. Whilst extremely powerful in detecting single nucleotide changes and small deletions, these methods are not suitable for detecting whole gene deletions. Therefore, high-throughput methods for screening of candidate homoeologous gene deletions are needed for application to wheat populations generated by the use of certain mutagenic agents (e.g. heavy ion irradiation) that frequently generate whole-gene deletions.     

Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae

Key message

A cytogenetic map of wheat was constructed using FISH with cDNA probes. FISH markers detected homoeology and chromosomal rearrangements of wild relatives, an important source of genes for wheat improvement.

Abstract

To transfer agronomically important genes from wild relatives to bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) by induced homoeologous recombination, it is important to know the chromosomal relationships of the species involved. Fluorescence in situ hybridization (FISH) can be used to study chromosome structure. The genomes of allohexaploid bread wheat and other species from the Triticeae tribe are colinear to some extent, i.e., composed of homoeoloci at similar positions along the chromosomes, and with genic regions being highly conserved. To develop cytogenetic markers specific for genic regions of wheat homoeologs, we selected more than 60 full-length wheat cDNAs using BLAST against mapped expressed sequence tags and used them as FISH probes. Most probes produced signals on all three homoeologous chromosomes at the expected positions. We developed a wheat physical map with several cDNA markers located on each of the 14 homoeologous chromosome arms. The FISH markers confirmed chromosome rearrangements within wheat genomes and were successfully used to study chromosome structure and homoeology in wild Triticeae species. FISH analysis detected 1U-6U chromosome translocation in the genome of Aegilops umbellulata, showed colinearity between chromosome A of Ae. caudata and group-1 wheat chromosomes, and between chromosome arm 7S#3L of Thinopyrum intermedium and the long arm of the group-7 wheat chromosomes.     

Development of V chromosome alterations and physical mapping of molecular markers specific to <Emphasis Type="Italic">Dasypyrum villosum</Emphasis>

Wheat-Dasypyrum villosum translocations were induced in the progeny of the amphiploid Triticum durum-D. villosum (AABBVV) by pollen irradiation. The rearranged V genome chromosomes were characterized by genomic/fluorescence in situ hybridization (GISH/FISH) and molecular markers. Twenty wheat-D. villosum translocation chromosomes were selected, including four centric, seven large segments, and nine small segments in a Chinese Spring (CS) background. The four centric translocations were subsequently identified by GISH/FISH and by molecular markers specific to chromosome arms of the Triticeae linkage groups. They were T5DL.4VL, T4BL.7VS, and T4BS.7VL as well as the compensating translocation T7AL.7VS. Using a combination of previously developed V chromosome alterations, 52 translocations or deletions that divided V chromosomes into 42 bins were employed for deletion mapping of molecular markers specific to D. villosum in a wheat background. Ninety-five expressed sequence tag (EST)-sequence-tagged site (STS) and seven SSR markers that were previously reported, as well as 72 STS markers screened in the present study, were physically allocated into 37 of 42 chromosome bins of D. villosum. Multiple loci of EST-STS markers were also mapped using CS nullisomic tetrasomic (NT) and ditelosomic (DT) genetic stocks. Most EST-STS homoeoloci were located on homoeologous chromosomes, suggesting a high degree of homology between the genomes of D. villosum and wheat. Four 4VL-specific markers detected homoeoloci on group 7 chromosomes of wheat, indicating that chromosome 4V of D. villosum shows some affinity to both wheat homoeologous groups 4 and 7. This is the first physical map of D. villosum, which will provide insight into the V genome for molecular breeding.     

Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii

Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4. The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas, land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis.     

Mapping of esterase loci in <Emphasis Type="Italic">Aegilops uniaristata</Emphasis> and homoeologous group 3 chromosomes of wheat

This study was planned to identify the chromosomal location of esterase loci in wheat (Triticum aestivum), in comparison to Aegilops uniaristata, using wheat Ae. uniaristata disomic addition and translocation lines. Two loci (Est-N1 and Est-N8) were identified on 3N chromosome of Ae. uniaristata and their probable homoeoloci were, for the first time, mapped close to three RFLP probes (Xpsr56, Xpsr394, and Xpsr1196) on homoeologous group 3 wheat chromosomes.     

Association of allelic variation in two NPR1-like genes with Fusarium head blight resistance in wheat

Fusarium head blight (FHB) is a destructive disease of wheat and barley. In wheat it is mainly caused by the fungal pathogens Fusarium graminearum and Fusarium culmorum. We report the identification and evaluation of candidate genes for quantitative FHB resistance. These genes showed altered expression levels in the moderately resistant winter wheat genotypes Capo and SVP72017 after inoculation with F. graminearum. Amongst others, a NPR1-like gene was identified. Sequence analysis of this gene fragment revealed a high level of variation between the parents of a doubled haploid population. Single nucleotide polymorphism and polymerase chain reaction markers were developed and two homoeologous genes were mapped on the long arms of chromosomes 2A and 2D, respectively. Markers for both genes had significant effects on FHB resistance in a diverse collection of 178 European winter wheat cultivars evaluated in multi-environmental field trials after spray inoculation with F. culmorum. These results revealed that allelic variation in two homoeologous NPR1-like genes is associated with FHB resistance in European winter wheat. Markers for these genes might therefore be used for marker-assisted breeding programs.     

CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat

Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops.High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat.The emerging effective genome editing tool,CRISPR/Cas9 system,makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles.To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat,we compared four different RNA polymerase(Pol) Ⅲ promoters(TaU3 p,TaU6 p,OsU3 p,and OsU6 p) and three types of sgRNA scaffold in the protoplast system.We show that the TaU3 promoter-driven optimized sgRNA scaffold was most effective.The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles,whose orthologs,OsNP1 in rice and ZmIPE1 in maize,encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility.Triple homozygous mutations in TaNP1 genes result in complete male sterility.We further demonstrated that anyone wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility.Taken together,this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.     

Microsatellite-based deletion bin system for the establishment of genetic-physical map relationships in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Because of polyploidy and large genome size, deletion stocks of bread wheat are an ideal material for physically allocating ESTs and genes to small chromosomal regions for targeted mapping. To enhance the utility of deletion stocks for chromosome bin mapping, we characterized a set of 84 deletion lines covering the 21 chromosomes of wheat using 725 microsatellites. We localized these microsatellite loci to 94 breakpoints in a homozygous state (88 distal deletions, 6 interstitial), and 5 in a heterozygous state representing 159 deletion bins. Chromosomes from homoeologous groups 2 and 5 were the best covered (126 and 125 microsatellites, respectively) while the coverage for group 4 was lower (80 microsatellites). We assigned at least one microsatellite in up to 92% of the bins (mean 4.97 SSR/bin). Only a few discrepancies concerning marker order were observed. The cytogenetic maps revealed small genetic distances over large physical regions around the centromeres and large genetic to physical map ratios close to the telomeres. As SSRs are the markers of choice for many genetic and breeding studies, the mapped microsatellite loci will be useful not only for deletion stock verifications but also for allocating associated QTLs to deletion bins where numerous ESTs that could be potential candidate genes are currently assigned.     

Structural and functional organization of the '1S0.8 gene-rich region' in the Triticeae

Wheat genes are present in physically small, gene-rich regions, interspersed by gene-poor blocks of retrotransposon-like repetitive sequences. One of the largest gene-rich regions is present around fraction length (FL) 0.8 of the short arm of wheat homoeologous group 1 chromosomes and is called `1S0.8 region'. The objective of this study was to reveal the structural and functional organization of the `1S0.8 region' in various Triticeae and other Poaceae species. Consensus genetic linkage maps of the `1S0.8 region' were constructed for wheat, barley, and rye by combining mapping information from 16, 11, and 12 genetic linkage maps, respectively. The consensus genetic linkage maps were compared with each other and with a consensus physical map of wheat homoeologous group 1. Comparative analyses localized 75 agronomically important genes to the `1S0.8 region'. This high-resolution comparison revealed exceptions to the rule of conserved gene synteny, established using low-resolution marker comparisons. Small rearrangements such as duplications, deletions, and inversions were observed among species. Proportion of chromosomal recombination occurring in the `1S0.8 region' was very similar among species. Within the gene-rich region, the extent of recombination was highly variable but the pattern was similar among species. Relative recombination among markers was similar except for a few loci where drastic differences were observed among species. Chromosomal rearrangements did not always change the extent of recombination for the region. Differences in gene order and relative recombination were the least between wheat and barley, and were the highest between wheat and oat.     

Efficient isolation of ion beam-induced mutants for homoeologous loci in common wheat and comparison of the contributions of Glu-1 loci to gluten functionality

Key message

Ion beam mutations can be efficiently isolated and deployed for functional comparison of homoeologous loci in polyploid plants, and Glu - 1 loci differ substantially in their contribution to wheat gluten functionality.

Abstract

To efficiently conduct genetic analysis, it is beneficial to have multiple types of mutants for the genes under investigation. Here, we demonstrate that ion beam-induced deletion mutants can be efficiently isolated for comparing the function of homoeologous loci of common wheat (Triticum aestivum). Through fragment analysis of PCR products from M₂ plants, ion beam mutants lacking homoeologous Glu-A1, Glu-B1 or Glu-D1 loci, which encode high molecular weight glutenin subunits (HMW-GSs) and affect gluten functionality and end-use quality of common wheat, could be isolated simultaneously. Three deletion lines missing Glu-A1, Glu-B1 or Glu-D1 were developed from the original mutants, with the Glu-1 genomic regions deleted in these lines estimated using newly developed DNA markers. Apart from lacking the target HMW-GSs, the three lines all showed decreased accumulation of low molecular weight glutenin subunits (LMW-GSs) and increased amounts of gliadins. Based on the test data of five gluten and glutenin macropolymer (GMP) parameters obtained with grain samples harvested from two environments, we conclude that the genetic effects of Glu-1 loci on gluten functionality can be ranked as Glu-D1 > Glu-B1 > Glu-A1. Furthermore, it is suggested that Glu-1 loci contribute to gluten functionality both directly (by promoting the formation of GMP) and indirectly (through keeping the balance among HMW-GSs, LMW-GSs and gliadins). Finally, the efficient isolation of ion beam mutations for functional comparison of homoeologous loci in polyploid plants and the usefulness of Glu-1 deletion lines for further studying the contribution of Glu-1 loci to gluten functionality are discussed.     

Defining the Sequence Elements and Candidate Genes for the Coloboma Mutation

The chicken coloboma mutation exhibits features similar to human congenital developmental malformations such as ocular coloboma, cleft-palate, dwarfism, and polydactyly. The coloboma-associated region and encoded genes were investigated using advanced genomic, genetic, and gene expression technologies. Initially, the mutation was linked to a 990 kb region encoding 11 genes; the application of the genetic and genomic tools led to a reduction of the linked region to 176 kb and the elimination of 7 genes. Furthermore, bioinformatics analyses of capture array-next generation sequence data identified genetic elements including SNPs, insertions, deletions, gaps, chromosomal rearrangements, and miRNA binding sites within the introgressed causative region relative to the reference genome sequence. Coloboma-specific variants within exons, UTRs, and splice sites were studied for their contribution to the mutant phenotype. Our compiled results suggest three genes for future studies. The three candidate genes, SLC30A5 (a zinc transporter), CENPH (a centromere protein), and CDK7 (a cyclin-dependent kinase), are differentially expressed (compared to normal embryos) at stages and in tissues affected by the coloboma mutation. Of these genes, two (SLC30A5 and CENPH) are considered high-priority candidate based upon studies in other vertebrate model systems.     

Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing

Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.     

The Rad50 genes of diploid and polyploid wheat species. Analysis of homologue and homoeologue expression and interactions with Mre11

The MRN complex plays a central role in the DNA repair pathways of eukaryotic cells and takes part in many other processes, including cell cycle checkpoint signalling, meiosis, DNA replication and telomere maintenance. This complex is formed by the interaction of the products of the Mre11, Rad50 and Nbs1 genes. This paper reports the molecular characterization, expression and interactions of the Rad50 gene in several wheat species with different levels of ploidy. The homoeologous Rad50 wheat genes were found to show a high level of conservation. Most of the RAD50 domains and motifs previously described in other species were also present in wheat RAD50; these proteins are therefore likely to have similar functions. Interactions between the RAD50 wheat proteins and their MRE11 counterparts in the MRN complex were observed. The level of expression of Rad50 in each of the species examined was determined and compared with those previously reported for the Mre11 genes. In some cases similar levels of expression were seen, as expected. The expression of the RAD50 homoeologous genes was assessed in two polyploid wheat species using quantitative PCR. In both cases, an overexpression of the Rad50B gene was detected. Although the results indicate the maintenance of function of these species?? three homoeologous Rad50 genes, the biased expression of Rad50B might indicate ongoing silencing of one or both other homoeologues in polyploid wheat. To assess the consequences of such silencing on the formation of the MRN complex, the interactions between individual homoeologues of Rad50 and their genomic counterpart Mre11 genes were examined. The results indicate the inexistence of genomic specificity in the interactions between these genes. This would guarantee the formation of an MRN complex in wheat.     

Improving the yellow pigment content of bread wheat flour by selecting the three homoeologous copies of Psy1

The yellow pigment content (YPC) of endosperm affects the quality and nutritional value of wheat grain products. Major quantitative trait loci (QTL) for endosperm YPC have been repeatedly mapped on chromosomes 7A and 7B in durum and bread wheats. The genes coding for phytoene synthase (PSY1), which is involved in the biosynthesis of carotenoids, generally co-segregate with these QTL, indicating their role in determining YPC. Here, to study the genetic factors underlying endosperm YPC in bread wheat, the sequence polymorphism of the homoeologous A, B and D copies of genes coding for PSY1, Psy-A1, Psy-B1, and Psy-D1, was studied in a worldwide core collection, which was also phenotyped for flour YPC. Seven novel alleles of Psy-A1 and two novel alleles of Psy-B1 were detected, which confirms the high level of polymorphism of these genes. Two major QTL with respective candidate genes Psy-A1 and Psy-B1 were identified in the distal region of chromosomes 7A and 7B using progeny of a cross between Apache and Ornicar, high and low YPC cultivars, respectively. Association mapping confirms the role of these genes in YPC and shows that the D copy also significantly influences this trait. These results indicate that breeders need to consider all three Psy1 copies when seeking to improve the YPC of wheat endosperm.     

Exploiting the <Emphasis Type="Italic">ZIP4</Emphasis> homologue within the wheat <Emphasis Type="Italic">Ph1</Emphasis> locus has identified two lines exhibiting homoeologous crossover in wheat-wild relative hybrids

Despite possessing related ancestral genomes, hexaploid wheat behaves as a diploid during meiosis. The wheat Ph1 locus promotes accurate synapsis and crossover of homologous chromosomes. Interspecific hybrids between wheat and wild relatives are exploited by breeders to introgress important traits from wild relatives into wheat, although in hybrids between hexaploid wheat and wild relatives, which possess only homoeologues, crossovers do not take place during meiosis at metaphase I. However, in hybrids between Ph1 deletion mutants and wild relatives, crossovers do take place. A single Ph1 deletion (ph1b) mutant has been exploited for the last 40 years for this activity. We show here that chemically induced mutant lines, selected for a mutation in TaZIP4-B2 within the Ph1 locus, exhibit high levels of homoeologous crossovers when crossed with wild relatives. Tazip4-B2 mutant lines may be more stable over multiple generations, as multivalents causing accumulation of chromosome translocations are less frequent. Exploitation of such Tazip4-B2 mutants, rather than mutants with whole Ph1 locus deletions, may therefore improve introgression of wild relative chromosome segments into wheat.     

Localization of Structural and Regulatory Genes for Phosphodiesterase in Wheat (TRITICUM AESTIVUM)

Genes (Pde-A3; Pde-B3; Pde-D3) for phosphodiesterase (PDE; E.C. 3.1.4.1.) isoenzymes in hexaploid wheat were located on the three homoeologous chromosomes of group 3 by testing the electrophoretic banding pattern of monosomic, nullisomic and nullisomic/tetrasomic compensation lines of "Chinese Spring" variety. In plants nullisomic for chromosome 5B, the 3D structural gene is not expressed and this lack of expression can be overcome by four doses of either homoeologous chromosome 5A or 5D. Our data conclusively indicate that there are genes on group 5 chromosomes which positively control the expression of the 3D structural gene. In addition, the expression of the "regulatory genes" is dosage dependent. Thus, our study reveals a complex interaction of the three genomes of wheat for regulation of PDE gene expression.     

Mapping of homoeologous chromosome exchanges influencing quantitative trait variation in Brassica napus

Genomic rearrangements arising during polyploidization are an important source of genetic and phenotypic variation in the recent allopolyploid crop Brassica napus. Exchanges among homoeologous chromosomes, due to interhomoeologue pairing, and deletions without compensating homoeologous duplications are observed in both natural B. napus and synthetic B. napus. Rearrangements of large or small chromosome segments induce gene copy number variation (CNV) and can potentially cause phenotypic changes. Unfortunately, complex genome restructuring is difficult to deal with in linkage mapping studies. Here, we demonstrate how high‐density genetic mapping with codominant, physically anchored SNP markers can detect segmental homoeologous exchanges (HE) as well as deletions and accurately link these to QTL. We validated rearrangements detected in genetic mapping data by whole‐genome resequencing of parental lines along with cytogenetic analysis using fluorescence in situ hybridization with bacterial artificial chromosome probes (BAC‐FISH) coupled with PCR using primers specific to the rearranged region. Using a well‐known QTL region influencing seed quality traits as an example, we confirmed that HE underlies the trait variation in a DH population involving a synthetic B. napus trait donor, and succeeded in narrowing the QTL to a small defined interval that enables delineation of key candidate genes.