Triple null mutations in starch synthase SSIIa gene homoeologs lead to high amylose and resistant starch in hexaploid wheat

Lack of nutritionally appropriate foods is one of the leading causes of obesity in the US and worldwide. Wheat (Triticum aestivum) provides 20% of the calories consumed daily across the globe. The nutrients in the wheat grain come primarily from the starch composed of amylose and amylopectin. Resistant starch content, which is known to have significant human health benefits, can be increased by modifying starch synthesis pathways. Starch synthase enzyme SSIIa, also known as starch granule protein isoform-1 (SGP-1), is integral to the biosynthesis of the branched and readily digestible glucose polymer amylopectin. The goal of this work was to develop a triple null mutant genotype for SSIIa locus in the elite hard red winter wheat variety ‘Jagger’ and evaluate the effect of the knock-out mutations on resistant starch content in grains with respect to wild type. Knock-out mutations in SSIIa in the three genomes of wheat variety ‘Jagger’ were identified using TILLING. Subsequently, these loss-of function mutations on A, B, and D genomes were combined by crossing to generate a triple knockout mutant genotype Jag-ssiia-∆ABD. The Jag-ssiia-∆ABD had an amylose content of 35.70% compared to 31.15% in Jagger, leading to ~ 118% increase in resistant starch in the Jag-ssiia-∆ABD genotype of Jagger wheat. The single individual genome mutations also had various effects on starch composition. Our full null Jag-ssiia-∆ABD mutant showed a significant increase in RS without the shriveled grain phenotype seen in other ssiia knockouts in elite wheat cultivars. Moreover, this study shows the potential for developing nutritionally improved foods in a non-GM approach. Since all the mutants have been developed in an elite wheat cultivar, their adoption in production and supply will be feasible in future.https://doi.org/10.1186/s12870-020-02822-

Reassessment of the evolution of wheat chromosomes 4A, 5A, and 7B.

Key messageComparison of genome sequences of wild emmer wheat and Aegilops tauschii suggests a novel scenario of the evolution of rearranged wheat chromosomes 4A, 5A, and 7B. Past research suggested that wheat chromosome 4A was subjected to a reciprocal translocation T(4AL;5AL)1 that occurred in the diploid progenitor of the wheat A subgenome and to three major rearrangements that occurred in polyploid wheat: pericentric inversion Inv(4AS;4AL)1, paracentric inversion Inv(4AL;4AL)1, and reciprocal translocation T(4AL;7BS)1. Gene collinearity along the pseudomolecules of tetraploid wild emmer wheat (Triticum turgidum ssp. dicoccoides, subgenomes AABB) and diploid Aegilops tauschii (genomes DD) was employed to confirm these rearrangements and to analyze the breakpoints. The exchange of distal regions of chromosome arms 4AS and 4AL due to pericentric inversion Inv(4AS;4AL)1 was detected, and breakpoints were validated with an optical Bionano genome map. Both breakpoints contained satellite DNA. The breakpoints of reciprocal translocation T(4AL;7BS)1 were also found. However, the breakpoints that generated paracentric inversion Inv(4AL;4AL)1 appeared to be collocated with the 4AL breakpoints that had produced Inv(4AS;4AL)1 and T(4AL;7BS)1. Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 either originated sequentially, and Inv(4AL;4AL)1 was produced by recurrent chromosome breaks at the same breakpoints that generated Inv(4AS;4AL)1 and T(4AL;7BS)1, or Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 originated simultaneously. We prefer the latter hypothesis since it makes fewer assumptions about the sequence of events that produced these chromosome rearrangements

A diploid wheat TILLING resource for wheat functional genomics

Citation: Rawat, N., . . . & Gill, B. (2012). A diploid wheat TILLING resource for wheat functional genomics. BMC Plant Biology, 12(1), 205. https://doi.org/10.1186/1471-2229-12-205Background: Triticum monococcum L., an A genome diploid einkorn wheat, was the first domesticated crop. As a diploid, it is attractive genetic model for the study of gene structure and function of wheat-specific traits. Diploid wheat is currently not amenable to reverse genetics approaches such as insertion mutagenesis and post-transcriptional gene silencing strategies. However, TILLING offers a powerful functional genetics approach for wheat gene analysis. Results: We developed a TILLING population of 1,532 M[subscript 2] families using EMS as a mutagen. A total of 67 mutants were obtained for the four genes studied. Waxy gene mutation frequencies are known to be 1/17.6 - 34.4 kb DNA in polyploid wheat TILLING populations. The T. monococcum diploid wheat TILLING population had a mutation frequency of 1/90 kb for the same gene. Lignin biosynthesis pathway genes- COMT1, HCT2, and 4CL1 had mutation frequencies of 1/86 kb, 1/92 kb and 1/100 kb, respectively. The overall mutation frequency of the diploid wheat TILLING population was 1/92 kb. Conclusion: The mutation frequency of a diploid wheat TILLING population was found to be higher than that reported for other diploid grasses. The rate, however, is lower than tetraploid and hexaploid wheat TILLING populations because of the higher tolerance of polyploids to mutations. Unlike polyploid wheat, most mutants in diploid wheat have a phenotype amenable to forward and reverse genetic analysis and establish diploid wheat as an attractive model to study gene function in wheat. We estimate that a TILLING population of 5, 520 will be needed to get a non-sense mutation for every wheat gene of interest with 95% probability

Physical mapping of a large plant genome using global high-information-content-fingerprinting: the distal region of the wheat ancestor Aegilops tauschii chromosome 3DS

Extent: 10p.Background: Physical maps employing libraries of bacterial artificial chromosome (BAC) clones are essential for comparative genomics and sequencing of large and repetitive genomes such as those of the hexaploid bread wheat. The diploid ancestor of the D-genome of hexaploid wheat (Triticum aestivum), Aegilops tauschii, is used as a resource for wheat genomics. The barley diploid genome also provides a good model for the Triticeae and T. aestivum since it is only slightly larger than the ancestor wheat D genome. Gene co-linearity between the grasses can be exploited by extrapolating from rice and Brachypodium distachyon to Ae. tauschii or barley, and then to wheat. Results: We report the use of Ae. tauschii for the construction of the physical map of a large distal region of chromosome arm 3DS. A physical map of 25.4 Mb was constructed by anchoring BAC clones of Ae. tauschii with 85 EST on the Ae. tauschii and barley genetic maps. The 24 contigs were aligned to the rice and B. distachyon genomic sequences and a high density SNP genetic map of barley. As expected, the mapped region is highly collinear to the orthologous chromosome 1 in rice, chromosome 2 in B. distachyon and chromosome 3H in barley. However, the chromosome scale of the comparative maps presented provides new insights into grass genome organization. The disruptions of the Ae. tauschii-rice and Ae. tauschii-Brachypodium syntenies were identical. We observed chromosomal rearrangements between Ae. tauschii and barley. The comparison of Ae. tauschii physical and genetic maps showed that the recombination rate across the region dropped from 2.19 cM/Mb in the distal region to 0.09 cM/Mb in the proximal region. The size of the gaps between contigs was evaluated by comparing the recombination rate along the map with the local recombination rates calculated on single contigs. Conclusions: The physical map reported here is the first physical map using fingerprinting of a complete Triticeae genome. This study demonstrates that global fingerprinting of the large plant genomes is a viable strategy for generating physical maps. Physical maps allow the description of the co-linearity between wheat and grass genomes and provide a powerful tool for positional cloning of new genes.Delphine Fleury, Ming-Cheng Luo, Jan Dvorak, Luke Ramsay, Bikram S Gill, Olin D Anderson, Frank M You, Zahra Shoaei, Karin R Deal and Peter Langridg

Development of a D genome specific marker resource for diploid and hexaploid wheat

Citation: Wang, Y., Drader, T., Tiwari, V. K., Dong, L. L., Kumar, A., Huo, N. X., . . . Gu, Y. Q. (2015). Development of a D genome specific marker resource for diploid and hexaploid wheat. Bmc Genomics, 16, 12. https://doi.org/10.1186/s12864-015-1852-2Background: Mapping and map-based cloning of genes that control agriculturally and economically important traits remain great challenges for plants with complex highly repetitive genomes such as those within the grass tribe, Triticeae. Mapping limitations in the Triticeae are primarily due to low frequencies of polymorphic gene markers and poor genetic recombination in certain genetic regions. Although the abundance of repetitive sequence may pose common problems in genome analysis and sequence assembly of large and complex genomes, they provide repeat junction markers with random and unbiased distribution throughout chromosomes. Hence, development of a high-throughput mapping technology that combine both gene-based and repeat junction-based markers is needed to generate maps that have better coverage of the entire genome. Results: In this study, the available genomics resource of the diploid Aegilop tauschii, the D genome donor of bread wheat, were used to develop genome specific markers that can be applied for mapping in modern hexaploid wheat. A NimbleGen array containing both gene-based and repeat junction probe sequences derived from Ae. tauschii was developed and used to map the Chinese Spring nullisomic-tetrasomic lines and deletion bin lines of the D genome chromosomes. Based on these mapping data, we have now anchored 5,171 repeat junction probes and 10,892 gene probes, corresponding to 5,070 gene markers, to the delineated deletion bins of the D genome. The order of the gene-based markers within the deletion bins of the Chinese Spring can be inferred based on their positions on the Ae. tauschii genetic map. Analysis of the probe sequences against the Chinese Spring chromosome sequence assembly database facilitated mapping of the NimbleGen probes to the sequence contigs and allowed assignment or ordering of these sequence contigs within the deletion bins. The accumulated length of anchored sequence contigs is about 155 Mb, representing similar to 3.2 % of the D genome. A specific database was developed to allow user to search or BLAST against the probe sequence information and to directly download PCR primers for mapping specific genetic loci. Conclusions: In bread wheat, aneuploid stocks have been extensively used to assign markers linked with genes/traits to chromosomes, chromosome arms, and their specific bins. Through this study, we added thousands of markers to the existing wheat chromosome bin map, representing a significant step forward in providing a resource to navigate the wheat genome. The database website (http://probes.pw.usda.gov/ATRJM/) provides easy access and efficient utilization of the data. The resources developed herein can aid map-based cloning of traits of interest and the sequencing of the D genome of hexaploid wheat

TILL-D: An Aegilops tauschii TILLING Resource for Wheat Improvement

Aegilops tauschii (2n = 2x = 14, genome DD), also known as Tausch’s goatgrass, is the D genome donor of bread or hexaploid wheat Triticum aestivum (2n = 2x = 42, AABBDD genome). It is a rich reservoir of useful genes for biotic and abiotic stress tolerance for wheat improvement. We developed a TILLING (Targeting Induced Local Lesions In Genomes) resource for Ae. tauschii for discovery and validation of useful genes in the D genome of wheat. The population, referred to as TILL-D, was developed with ethyl methanesulfonate (EMS) mutagen. The survival rate in M1 generation was 73%, out of which 22% plants were sterile. In the M2 generation 25% of the planted seeds showed phenotypic mutations such as albinos, chlorinas, no germination, variegated, sterile and partially fertile events, and 2,656 produced fertile M2 plants. The waxy gene was used to calculate the mutation frequency (1/70 kb) of the developed population, which was found to be higher than known mutation frequencies for diploid plants (1/89–1/1000 kb), but lower than that for a polyploid species (1/24–1/51 kb). The TILL-D resource, together with the newly published Ae. tauschii reference genome sequence, will facilitate gene discoveries and validations of agronomically important traits and their eventual fine transfer in bread wheat

Development of a D genome specific marker resource for diploid and hexaploid wheat

Background: Mapping and map-based cloning of genes that control agriculturally and economically important traits remain great challenges for plants with complex highly repetitive genomes such as those within the grass tribe, Triticeae. Mapping limitations in the Triticeae are primarily due to low frequencies of polymorphic gene markers and poor genetic recombination in certain genetic regions. Although the abundance of repetitive sequence may pose common problems in genome analysis and sequence assembly of large and complex genomes, they provide repeat junction markers with random and unbiased distribution throughout chromosomes. Hence, development of a high-throughput mapping technology that combine both gene-based and repeat junction-based markers is needed to generate maps that have better coverage of the entire genome. Results: In this study, the available genomics resource of the diploid Aegilop tauschii, the D genome donor of bread wheat, were used to develop genome specific markers that can be applied for mapping in modern hexaploid wheat. A NimbleGen array containing both gene-based and repeat junction probe sequences derived from Ae. tauschii was developed and used to map the Chinese Spring nullisomic-tetrasomic lines and deletion bin lines of the D genome chromosomes. Based on these mapping data, we have now anchored 5,171 repeat junction probes and 10,892 gene probes, corresponding to 5,070 gene markers, to the delineated deletion bins of the D genome. The order of the gene-based markers within the deletion bins of the Chinese Spring can be inferred based on their positions on the Ae. tauschii genetic map. Analysis of the probe sequences against the Chinese Spring chromosome sequence assembly database facilitated mapping of the NimbleGen probes to the sequence contigs and allowed assignment or ordering of these sequence contigs within the deletion bins. The accumulated length of anchored sequence contigs is about 155 Mb, representing ~ 3.2 % of the D genome. A specific database was developed to allow user to search or BLAST against the probe sequence information and to directly download PCR primers for mapping specific genetic loci. Conclusions: In bread wheat, aneuploid stocks have been extensively used to assign markers linked with genes/traits to chromosomes, chromosome arms, and their specific bins. Through this study, we added thousands of markers to the existing wheat chromosome bin map, representing a significant step forward in providing a resource to navigate the wheat genome. The database website (http://probes.pw.usda.gov/ATRJM/) provides easy access and efficient utilization of the data. The resources developed herein can aid map-based cloning of traits of interest and the sequencing of the D genome of hexaploid wheat.Keywords: Recombination, Repeat junction markers, Genetic map, NimbleGen array, Molecular markers, Wheat deletion bin

Nucleotide diversity maps reveal variation in diversity among wheat genomes and chromosomes

<p>Abstract</p> <p>Background</p> <p>A genome-wide assessment of nucleotide diversity in a polyploid species must minimize the inclusion of homoeologous sequences into diversity estimates and reliably allocate individual haplotypes into their respective genomes. The same requirements complicate the development and deployment of single nucleotide polymorphism (SNP) markers in polyploid species. We report here a strategy that satisfies these requirements and deploy it in the sequencing of genes in cultivated hexaploid wheat (<it>Triticum aestivum</it>, genomes AABBDD) and wild tetraploid wheat (<it>Triticum turgidum </it>ssp. <it>dicoccoides</it>, genomes AABB) from the putative site of wheat domestication in Turkey. Data are used to assess the distribution of diversity among and within wheat genomes and to develop a panel of SNP markers for polyploid wheat.</p> <p>Results</p> <p>Nucleotide diversity was estimated in 2114 wheat genes and was similar between the A and B genomes and reduced in the D genome. Within a genome, diversity was diminished on some chromosomes. Low diversity was always accompanied by an excess of rare alleles. A total of 5,471 SNPs was discovered in 1791 wheat genes. Totals of 1,271, 1,218, and 2,203 SNPs were discovered in 488, 463, and 641 genes of wheat putative diploid ancestors, <it>T. urartu</it>, <it>Aegilops speltoides</it>, and <it>Ae. tauschii</it>, respectively. A public database containing genome-specific primers, SNPs, and other information was constructed. A total of 987 genes with nucleotide diversity estimated in one or more of the wheat genomes was placed on an <it>Ae. tauschii </it>genetic map, and the map was superimposed on wheat deletion-bin maps. The agreement between the maps was assessed.</p> <p>Conclusions</p> <p>In a young polyploid, exemplified by <it>T. aestivum</it>, ancestral species are the primary source of genetic diversity. Low effective recombination due to self-pollination and a genetic mechanism precluding homoeologous chromosome pairing during polyploid meiosis can lead to the loss of diversity from large chromosomal regions. The net effect of these factors in <it>T. aestivum </it>is large variation in diversity among genomes and chromosomes, which impacts the development of SNP markers and their practical utility. Accumulation of new mutations in older polyploid species, such as wild emmer, results in increased diversity and its more uniform distribution across the genome.</p

A whole-genome, radiation hybrid mapping resource of hexaploid wheat

Generating a contiguous, ordered reference sequence of a complex genome such as hexaploid wheat (2n = 6x = 42; approximately 17 GB) is a challenging task due to its large, highly repetitive, and allopolyploid genome. In wheat, ordering of whole-genome or hierarchical shotgun sequencing contigs is primarily based on recombination and comparative genomics-based approaches. However, comparative genomics approaches are limited to syntenic inference and recombination is suppressed within the pericentromeric regions of wheat chromosomes, thus, precise ordering of physical maps and sequenced contigs across the whole-genome using these approaches is nearly impossible. We developed a whole-genome radiation hybrid (WGRH) resource and tested it by genotyping a set of 115 randomly selected lines on a high-density single nucleotide polymorphism (SNP) array. At the whole-genome level, 26 299 SNP markers were mapped on the RH panel and provided an average mapping resolution of approximately 248 Kb/cR₁₅₀₀ with a total map length of 6866 cR₁₅₀₀. The 7296 unique mapping bins provided a five- to eight-fold higher resolution than genetic maps used in similar studies. Most strikingly, the RH map had uniform bin resolution across the entire chromosome(s), including pericentromeric regions. Our research provides a valuable and low-cost resource for anchoring and ordering sequenced BAC and next generation sequencing (NGS) contigs. The WGRH developed for reference wheat line Chinese Spring (CS-WGRH), will be useful for anchoring and ordering sequenced BAC and NGS based contigs for assembling a high-quality, reference sequence of hexaploid wheat. Additionally, this study provides an excellent model for developing similar resources for other polyploid species.Keywords: gamma radiation, physical mapping, Radiation hybrid mapping, genome assembly, wheat resource, Triticum aestivu