Background: Triticum monococcum L., an A genome diploid einkorn wheat, was the first domesticated... more Background: 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 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. 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.
Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. EccDNApowered co... more Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. EccDNApowered copy number variation plays diverse roles, from oncogenesis in humans to herbicide resistance in crop weeds. Here, we report interspecific eccDNA flow and its dynamic behavior in soma cells of natural populations and F 1 hybrids of Amaranthus sp. The glyphosate resistance (GR) trait is controlled by eccDNA-based amplification harboring the 5-enolpyruvylshikimate-3phosphate synthase (EPSPS) gene (eccDNA replicon), the molecular target of glyphosate. We documented pollen-mediated transfer of eccDNA in experimental hybrids between glyphosate susceptible A. tuberculatus and GR A. palmeri. Experimental hybridization and fluorescence in situ hybridization (FISH) analysis revealed that the eccDNA replicon in A. spinosus derived from GR A. palmeri by natural hybridization. FISH analysis also revealed random chromosome anchoring and massive eccDNA replicon copy number variation in soma cells of weedy hybrids. The results suggest that eccDNAs are inheritable across compatible species, contributing to genome plasticity and rapid adaptive evolution.
In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an i... more In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an important herbicide invariably used in the cultivation of glyphosate-resistant crops to control weeds. Overuse of glyphosate results in the evolution of glyphosate-resistant weeds. Evolution of glyphosate resistance (GR) in Amaranthus palmeri (AP) is a serious concern in the USA. Investigation of the mechanism of GR in AP identified different resistance mechanisms of which 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification is predominant. Molecular analysis of GR AP identified the presence of a 5-to >160-fold increase in copies of the EPSPS gene than in a glyphosate-susceptible (GS) population. This increased copy number of the EPSPS gene increased the genome size ranging from 3.5 to 11.8%, depending on the copy number compared to the genome size of GS AP. FISH analysis using a 399-kb EPSPS cassette derived from bacterial artificial chromosomes (BACs) as probes identified that amplified EPSPS copies in GR AP exist in extra-chromosomal circular DNA (eccDNA) in addition to the native copy in the chromosome. The EPSPS gene-containing eccDNA having a size of ∼400 kb is termed EPSPS-eccDNA and showed somatic mosacism in size and copy number. EP-SPS-eccDNA has a genetic mechanism to tether randomly to mitotic or meiotic chromosomes during cell division or gamete formation and is inherited to daughter cells or progeny generating copy number variation. These eccDNAs are stable genetic elements that can replicate and exist independently. The genomic characterization of the EPSPS locus, along with the flanking regions, identified the presence of a complex array of repeats and mobile genetic elements. The cytogenomics approach in understanding the biology of EPSPS-eccDNA sheds light on various characteristics of EPSPS-eccDNA that favor GR in AP.
Centromeres, because of their repeat structure and lack of sequence conservation, are difficult t... more Centromeres, because of their repeat structure and lack of sequence conservation, are difficult to assemble and compare across organisms. It was recently discovered that rice centromeres often contain genes. This suggested a method for studying centromere homologies between wheat and rice chromosomes by mapping rice centromeric genes onto wheat aneuploid stocks. Three of the seven cDNA clones of centromeric genes from rice centromere 8 (Cen8), 6729.t09, 6729.t10, and 6730.t11 which lie in the Cen8 kinetochore region, and three wheat ESTs, BJ301191, BJ305475, and BJ280500, with similarity to sequences of rice centromeric genes, were mapped to the centromeric regions of the wheat group-7 (W7) chromosomes. A possible pericentric inversion in chromosome 7D was detected. Genomewide comparison of wheat ESTs that mapped to centromeric regions against rice genome sequences revealed high conservation and a one-to-one correspondence of centromeric regions between wheat and rice chromosome pairs W1-R5, W2-R7, W3-R1, W5-R12, W6-R2, and W7-R8. The W4 centromere may share homology with R3 only or with R3 1 R11. Wheat ESTs that mapped to the pericentromeric region of the group-5 long arm anchored to the rice BACs located in the recently duplicated region at the distal ends of the short arms of rice chromosomes 11 and 12. A pericentric inversion specific to the rice lineage was detected. The depicted framework provides a working model for further studies on the structure and evolution of cereal chromosome centromeres.
Proceedings of the National Academy of Sciences of the United States of America, Dec 1, 1992
Bread wheat (Triticum aestivum L. em Thell) is well suited for cytogenetic analysis because the g... more Bread wheat (Triticum aestivum L. em Thell) is well suited for cytogenetic analysis because the genome, buffered by polyploidy, can tolerate structurally and numeri- cally engineered chromosomes for analysis over infinite gen- erations. This feature of polyploidy can be used in developing a high-resolution, cytogenetically based physical map of the wheat genome. We show that numerous deletions, observed in the progeny of a monosomic addition of a chromosome from Trtcum cylinduicum in wheat, result from single breakpoints and a concomitant loss ofdistal fragments. Breakages occurred in euchromatic and heterochromatic regions. Forty-one dele- tions for chromosomes 7A, 7B, and 7D, and a set of genetically mapped DNA probes, were used to construct physical maps. Recombination was low in proximal chromosomal regions and very high toward the distal ends. Deletion mapping was more efficient than genetic mapping in resolving the order of proximal loci. Despite variation in size and arm ratio, relative gene position was largely conserved among chromosomes 7A, 7B, and 7D and a consensus group 7 physical map was constructed. Several molecularly tagged chromosome regions (MTCRs) of approximately one to a few million base pafrs were identified that may be resolved by long-range mapping of DNA fragments. Thus, a cytogenetically based physical map may be used to integrate chromosome and DNA-based maps. The MTCRs may simplify strategies for cloning of agronomically useful genes despite the genetic complexity and the large genome size of wheat.
bioRxiv (Cold Spring Harbor Laboratory), Mar 2, 2023
Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. The eccDNA-power... more Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. The eccDNA-powered copy number variation plays diverse roles from oncogenesis in humans to herbicide resistance in crop weeds. Here we report interspecific eccDNA flow and its dynamic behavior in soma cells of natural populations and F 1 hybrids of Amaranthus sp. The glyphosate resistance (GR) trait is controlled by eccDNA-based amplification harboring the 5enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (EPSPS-eccDNA), the molecular target of glyphosate. We documented pollen-mediated transfer of eccDNA in experimental hybrids between GS A. tuberculatus x GR A. palmeri. Experimental hybridization and fluorescence in situ hybridization (FISH) analysis revealed that the EPSPS-eccDNA present in A. spinosus was derived from GR A. palmeri by natural hybridization. FISH analysis also revealed random chromosome anchoring and massive EPSPS-eccDNA copy number variation in soma cells of weedy hybrids. The results suggest that eccDNAs are inheritable across compatible species contributing to genome plasticity and rapid adaptive evolution.
With this rather cursory look at worldwide small grain cereal genetics efforts, I want to make a ... more With this rather cursory look at worldwide small grain cereal genetics efforts, I want to make a few comments about the ITMI workshop program in Manhattan. We focused the program on genome mapping (Imkage and physical mapping), mapping using nonconventional approaches, such as chemiluminescence and PCR, and on a survey of national genome initiatives, cytogenetic stocks, data management software demonstration, and poster sessions. About 100 scientists from eleven countries participated. Based on the comments from the participants, the program was quite successhl. The intent of the program was to present exciting science and separate the business sessions from scientific presentations. I hope this strategy will be followed in future meetings. Of course, the organization of such a meeting entails a lot of hard work. One single individual who did more than anyone else to make this meeting such a success is John Raupp. John deserves our heartfelt thanks. I want to thank Duane Wilson and the rest of my laboratory staff for their tremendous efforts. We received considerable help from the ITMI office in Davis, CA, particularly from Cal Qualset, Pat McGuire, and Harold Corke. I express my appreciation to them. I shall close by offering a few suggestions for future meetings. First and foremost, the invited speakers must present cutting-edge science, new data, new protocols, innovative research. It is imperative that laboratory leaders encourage participation from their colleagues, bright postdoctoral fellows, and graduate students; the hands-on researchers who have much to contribute. We must encourage participation from all over the world. We must constantly encourage comparative mapping in wheat, rye, barley, oats, and beyond. We need communal library resources such as YAC libraries and cDNA libraries that are beyond the resources of individual laboratories. We need techniques for transformation, chromosome walking, and so on, so that small grain and polyploid genetics research remains an exciting and competitive enterprise.
The wheat wild relative Aegilops tauschii was previously used to transfer the Lr42 leaf rust resi... more The wheat wild relative Aegilops tauschii was previously used to transfer the Lr42 leaf rust resistance gene into bread wheat. Lr42 confers resistance at both seedling and adult stages, and it is broadly effective against all leaf rust races tested to date. Lr42 has been used extensively in the CIMMYT international wheat breeding program with resulting cultivars deployed in several countries. Here, using a bulked segregant RNA-Seq (BSR-Seq) mapping strategy, we identify three candidate genes for Lr42. Overexpression of a nucleotide-binding site leucine-rich repeat (NLR) gene AET1Gv20040300 induces strong resistance to leaf rust in wheat and a mutation of the gene disrupted the resistance. The Lr42 resistance allele is rare in Ae. tauschii and likely arose from ectopic recombination. Cloning of Lr42 provides diagnostic markers and over 1000 CIMMYT wheat lines carrying Lr42 have been developed documenting its widespread use and impact in crop improvement.
Aegilops tauschii, the D-genome donor of bread wheat, Triticum aestivum, is a storehouse of genet... more Aegilops tauschii, the D-genome donor of bread wheat, Triticum aestivum, is a storehouse of genetic diversity, and an important resource for future wheat improvement. Genomic and population analysis of 549 Ae. tauschii and 103 wheat accessions was performed by using 13,135 high quality SNPs. Population structure, principal component, and cluster analysis confirmed the differentiation of Ae. tauschii into two lineages; lineage 1 (L1) and lineage 2 (L2), the latter being the wheat D-genome donor. Lineage L1 contributes only 2.7% of the total introgression from Ae. tauschii for a set of United States winter wheat lines, confirming the great amount of untapped genetic diversity in L1. Lineage L2 accessions had overall greater allelic diversity and wheat accessions had the least allelic diversity. Both lineages also showed intra-lineage differentiation with L1 being driven by longitudinal gradient and L2 differentiated by altitude. There has previously been little reported on natural hybridization between L1 and L2. We found nine putative inter-lineage hybrids in the population structure analysis, each containing numerous lineage-specific private alleles from both lineages. One hybrid was confirmed as a recombinant inbred between the two lineages, likely artificially post collection. Of the remaining eight putative hybrids, a group of seven from Georgia carry 713 SNPs with private alleles, which points to the possibility of a novel L1-L2 hybrid lineage. To facilitate the use of Ae. tauschii in wheat improvement, a MiniCore consisting of 29 L1 and 11 L2 accessions, has been developed based on genotypic, phenotypic and geographical data. MiniCore reduces the collection size by over 10-fold and captures 84% of the total allelic diversity in the whole collection.
During evolutionary history many grasses from the tribe Triticeae have undergone interspecific hy... more During evolutionary history many grasses from the tribe Triticeae have undergone interspecific hybridization, resulting in allopolyploidy; whereas homoploid hybrid speciation was found only in rye. Homoeologous chromosomes within the Triticeae preserved cross-species macrocolinearity, except for a few species with rearranged genomes. Aegilops markgrafii, a diploid wild relative of wheat (2n = 2x = 14), has a highly asymmetrical karyotype that is indicative of chromosome rearrangements. Molecular cytogenetics and next-generation sequencing were used to explore the genome organization. Fluorescence in situ hybridization with a set of wheat cDNAs allowed the macrostructure and cross-genome homoeology of the Ae. markgrafii chromosomes to be established. Two chromosomes maintained colinearity, whereas the remaining were highly rearranged as a result of inversions and inter-and intrachromosomal translocations. We used sets of barley and wheat orthologous gene sequences to compare discrete parts of the Ae. markgrafii genome involved in the rearrangements. Analysis of sequence identity profiles and phylogenic relationships grouped chromosome blocks into two distinct clusters. Chromosome painting revealed the distribution of transposable elements and differentiated chromosome blocks into two groups consistent with the sequence analyses. These data suggest that introgressive hybridization accompanied by gross chromosome rearrangements might have had an impact on karyotype evolution and homoploid speciation in Ae. markgrafii.
Gene transfer from wild wheat relatives to bread wheat is restricted to homologous recombination.... more Gene transfer from wild wheat relatives to bread wheat is restricted to homologous recombination. The presence of the Pairing homoeologous 1 (Ph1) gene in the long arm of wheat chromosome 5B allows only homologous chromosomes to pair and recombine, resulting in diploid inheritance of polyploid wheat. Previously, we identified a potent homoeologous pairing promotor gene(s) (Hpp-5M g ); its carrier chromosome 5M g derived from Aegilops geniculata and its wheat homoeologous chromosome 5D freely recombined in the presence of the Ph1 gene. In this study, we investigated the effect of Hpp-5M g on homoeologous recombination in the absence of Ph1. In Hpp-5M g /ph1bph1b plants, we observed a vast genome-wide increase in homoeologous recombination and multiple crossovers (CO), including CO breakpoints in proximal regions of the chromosomes where recombination is known to be suppressed. We tested the efficacy of Hpp-5M g /ph1bph1b-induced homoeologous recombination by producing new recombinants for the wheat streak mosaic virus resistance gene, Wsm3, present in the wheat-Thinopyrum intermedium Robertsonian translocation (RobT T7BS.7S#3L). A recombination frequency of 6.5% was detected by screening the progenies double monosomic for T7BS.7S#3L and 7B by genomic in situ hybridization. This recombination frequency was about 100-fold higher compared with the recombinant frequency of 0.06% observed by using ph1b-induced homoeologous recombination alone. Our results indicate that chromosome 5M g promotes homoeologous recombination between wheat and wild wheat relative chromosomes, which helps in the generation of pre-breeding materials thereby accelerating wheat crop improvement.
In most eukaryotes, centromeres assemble at a single location per chromosome. Naturally occurring... more In most eukaryotes, centromeres assemble at a single location per chromosome. Naturally occurring telocentric chromosomes (telosomes) with a terminal centromere are rare but do exist. Telosomes arise through misdivision of centromeres in normal chromosomes, and their cytological stability depends on the structure of their kinetochores. The instability of telosomes may be attributed to the relative centromere size and the degree of completeness of their kinetochore. Here we test this hypothesis by analyzing the cytogenetic structure of wheat telosomes. We used a population of 80 telosomes arising from the misdivision of the 21 chromosomes of wheat that have shown stable inheritance over many generations. We analyzed centromere size by probing with the centromere-specific histone H3 variant, CENH3. Comparing the signal intensity for CENH3 between the intact chromosome and derived telosomes showed that the telosomes had approximately half the signal intensity compared to that of normal chromosomes. Immunofluorescence of CENH3 in a wheat stock with 28 telosomes revealed that none of the telosomes received a complete CENH3 domain. Some of the telosomes lacked centromere specific retrotransposons of wheat in the CENH3 domain, indicating that the stability of telosomes depends on the presence of CENH3 chromatin and not on the presence of CRW repeats. In addition to providing evidence for centromere shift, we also observed chromosomal aberrations including inversions and deletions in the short arm telosomes of double ditelosomic 1D and 6D stocks. The role of centromere-flanking, pericentromeric heterochromatin in mitosis is discussed with respect to genome/chromosome integrity.
We report the map-based cloning of the leaf rust resistance gene Lr21, previously mapped to a gen... more We report the map-based cloning of the leaf rust resistance gene Lr21, previously mapped to a generich region at the distal end of chromosome arm 1DS of bread wheat (Triticum aestivum L.). Molecular cloning of Lr21 was facilitated by diploid/polyploid shuttle mapping strategy. Cloning of Lr21 was confirmed by genetic transformation and by a stably inherited resistance phenotype in transgenic plants. Lr21 spans 4318 bp and encodes a 1080-amino-acid protein containing a conserved nucleotide-binding site (NBS) domain, 13 imperfect leucine-rich repeats (LRRs), and a unique 151-amino-acid sequence missing from known NBS-LRR proteins at the N terminus. Fine-structure genetic analysis at the Lr21 locus detected a noncrossover (recombination without exchange of flanking markers) within a 1415-bp region resulting from either a gene conversion tract of at least 191 bp or a double crossover. The successful map-based cloning approach as demonstrated here now opens the door for cloning of many crop-specific agronomic traits located in the gene-rich regions of bread wheat.
These WCM-resistance sources have not been utilized mosaic virus which is vectored by the wheat c... more These WCM-resistance sources have not been utilized mosaic virus which is vectored by the wheat curl mite (WCM) Aceria tosicheilla (Keifer). Host resistance to WCM has reduced losses. This commercially, but some are being advanced in wheat study was conducted to evaluate the effectiveness of resistance in breeding programs. As different collections of WCM wheat to WCM collected from various locations in the Great Plains. vary in degree of virulence to resistant wheat (Harvey Collections of WCM from Montana, Nebraska, South Dakota, Texas, et al., 1995a), it is important to evaluate the range in Alberta, Canada, and eight locations in Kansas were compared for response among WCM strains collected from areas for their ability to survive and reproduce in the greenhouse on seven which resistant cultivars are targeted. The objective of lines of wheat and wheat relatives previously identified as resistant. this research was to expand on a previous report (Har-The lines and their sources of resistance were: AC PGR16635 (Aegivey et al., 1995a) by using a wider range of WCM cullops tauschii Coss., Cmc1 ), PI 525452 (Thinopyrum ponticum (Podp.) tures to evaluate the effectiveness of resistance provided Liu and Wang, Cmc2 ), KS96WGRC40 (Ae. tauschii and Secale cerby the previously tested resistant sources and two reeale L.), TA920 (Triticum timopheevii (Zhuk.) Zhuk spp. armenidcum ), PI 475772 (S. cereale ), 'TAM 107' (S. cereale ), PI 222655 (T. cently identified sources of resistance. aestivum ). KS96WGRC40 and TA920 were the only entries that were resistant to all WCM collections. Other sources of resistance were MATERIALS AND METHODS effective against WCMs from some but not all locations. PI 222655 Sources of WCM-resistant germplasm are given in Table was resistant to WCMs from Nebraska and central Kansas but not 1. The wheat cultivar Larned, (CI 17650) was used in all tests 2). This relationship held for collections from other
Leaf rust resistance gene Lr58 derived from Aegilops triuncialis L. was transferred to the hard r... more Leaf rust resistance gene Lr58 derived from Aegilops triuncialis L. was transferred to the hard red winter wheat (HRWW) cultivars Jagger and Overley by standard backcrossing and markerassisted selection (MAS). A co-dominant PCR-based sequence tagged site (STS) marker was developed based on the sequence information of the RFLP marker (XksuH16) diagnostically detecting the alien segment in T2BSÁ2BL-2 t L(0.95). STS marker Xncw-Lr58-1 was used to select backcross F 1 plants with rust resistance. The co-dominant marker polymorphism detected by primer pair NCW-Lr58-1 efficiently identified the homozygous BC 3 F 2 plants with rust resistance gene Lr58. The STS marker Xncw-Lr58-1 showed consistent diagnostic polymorphism between the resistant source and the wheat cultivars selected by the US Wheat Coordinated Agricultural Project. The utility and compatibility of the STS marker in MAS programs involving robust genotyping platforms was demonstrated in both agarosebased and capillary-based platforms. Screening backcross derivatives carrying Lr58 with various rust races at seedling stage suggested the transferred rust resistance in adapted winter wheats is stable in both cultivar backgrounds. Lr58 in adapted winter wheat backgrounds could be used in combination with other resistance genes in wheat rust resistance breeding.
The distribution of genes and recombination in the wheat genome was studied by comparing physical... more The distribution of genes and recombination in the wheat genome was studied by comparing physical maps with the genetic linkage maps. The physical maps were generated by mapping 80 DNA and two phenotypic markers on an array of 65 deletion lines for homoeologous group 5 chromosomes. The genetic maps were constructed for chromosome 5B in wheat and 50 in Triticum tauschii. No marker mapped in the proximal 20% chromosome region surrounding the centromere. More than 60% of the long arm markers were present in three major clusters that physically encompassed <18% of the arm. Because 48% of the markers were cDNA clones and the distributions of the cDNA and genomic clones were similar, the marker distribution may represent the distribution of genes. The gene clusters were identified and allocated to very small chromosome regions because of a higher number of deletions in their surrounding regions. The recombination was suppressed in the centromeric regions and mainly occurred in the gene-rich regions. The bp/cM estimates varied from 118 kb for gene-rich regions to 22 Mb for gene-poor regions. The wheat genes present in these clusters are, therefore, amenable to molecular manipulations parallel to the plants with smaller genomes like rice.
Brachypodium distachyon is a wild annual grass belonging to the Pooideae, more closely related to... more Brachypodium distachyon is a wild annual grass belonging to the Pooideae, more closely related to wheat, barley, and forage grasses than rice and maize. As an experimental model, the completed genome sequence of B. distachyon provides a unique opportunity to study centromere evolution during the speciation of grasses. Centromeric satellite sequences have been identified in B. distachyon, but little is known about centromeric retrotransposons in this species. In the present study, BAC-fluorescence in situ hybridization was conducted in maize, rice, barley, wheat, and rye using B. distachyon (Bd) centromere-specific BAC clones. Eight Bd centromeric BAC clones gave no detectable FISH signals on the chromosomes of rice and maize, and three of them also did not yield any FISH signals in barley, wheat, and rye. In addition, four of five Triticeae centromeric BAC clones did not hybridize to the B. distachyon centromeres, implying certain unique features of Brachypodium centromeres. Analysis of Brachypodium centromeric BAC sequences identified a long terminal repeat (LTR)centromere retrotransposon of B. distachyon (CRBd1). This element was found in high copy number accounting for 1.6% of the B. distachyon genome, and is enriched in Brachypodium centromeric regions. CRBd1 accumulated in active centromeres, but was lost from inactive ones. The LTR of CRBd1 appears to be specific to B. distachyon centromeres. These results reveal different evolutionary events of this retrotransposon family across grass species.
Background: Lack of nutritionally appropriate foods is one of the leading causes of obesity in th... more Background: 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. Results: 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-ΔABD. The Jag-ssiia-ΔABD had an amylose content of 35.70% compared to 31.15% in Jagger, leading to ~118% increase in resistant starch in the Jag-ssiia-ΔABD genotype of Jagger wheat. The single individual genome mutations also had various effects on starch composition. Conclusions: Our full null Jag-ssiia-ΔABD 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.
The International Weed Genomics Consortium is a collaborative group of researchers focused on dev... more The International Weed Genomics Consortium is a collaborative group of researchers focused on developing genomic resources for the study of weedy plants. Weeds are attractive systems for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Our goal is to use genomic information to develop sustainable and effective weed control methods and to provide insights about biotic and abiotic stress tolerance to assist crop breeding. Here, we outline resources under development by the consortium and highlight areas of research that will be impacted by these enabling resources.
International audienceBecause of its 17 Gb hexaploid genome, wheat genomics has been lagging behi... more International audienceBecause of its 17 Gb hexaploid genome, wheat genomics has been lagging behind the one of the other major crops. Two years after the establishment of the first physical map of the biggest wheat chromosome, the 3B, which represents 1 Gb, its complete sequencing is now underway (ANR project 3BSEQ). In order to prepare for its complete sequencing and analysis, we performed a pilot project on 18 Mb of large BAC contigs which allowed us to improve our understanding of the wheat genome composition and dynamics. Comparative and evolutionary analyses revealed that increase of the genome size, mainly mediated by transposable element amplification, was uneven and has led to the formation of small gene islands. In addition, it was accompanied by rearrangement of the ancestral gene content. Indeed, a significant amount of nonsyntenic genes are interspersed into a conserved ancestral grass gene backbone, revealing that, in our sample, about 50% of the annotated genes was une...
Background: Triticum monococcum L., an A genome diploid einkorn wheat, was the first domesticated... more Background: 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 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. 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.
Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. EccDNApowered co... more Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. EccDNApowered copy number variation plays diverse roles, from oncogenesis in humans to herbicide resistance in crop weeds. Here, we report interspecific eccDNA flow and its dynamic behavior in soma cells of natural populations and F 1 hybrids of Amaranthus sp. The glyphosate resistance (GR) trait is controlled by eccDNA-based amplification harboring the 5-enolpyruvylshikimate-3phosphate synthase (EPSPS) gene (eccDNA replicon), the molecular target of glyphosate. We documented pollen-mediated transfer of eccDNA in experimental hybrids between glyphosate susceptible A. tuberculatus and GR A. palmeri. Experimental hybridization and fluorescence in situ hybridization (FISH) analysis revealed that the eccDNA replicon in A. spinosus derived from GR A. palmeri by natural hybridization. FISH analysis also revealed random chromosome anchoring and massive eccDNA replicon copy number variation in soma cells of weedy hybrids. The results suggest that eccDNAs are inheritable across compatible species, contributing to genome plasticity and rapid adaptive evolution.
In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an i... more In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an important herbicide invariably used in the cultivation of glyphosate-resistant crops to control weeds. Overuse of glyphosate results in the evolution of glyphosate-resistant weeds. Evolution of glyphosate resistance (GR) in Amaranthus palmeri (AP) is a serious concern in the USA. Investigation of the mechanism of GR in AP identified different resistance mechanisms of which 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification is predominant. Molecular analysis of GR AP identified the presence of a 5-to >160-fold increase in copies of the EPSPS gene than in a glyphosate-susceptible (GS) population. This increased copy number of the EPSPS gene increased the genome size ranging from 3.5 to 11.8%, depending on the copy number compared to the genome size of GS AP. FISH analysis using a 399-kb EPSPS cassette derived from bacterial artificial chromosomes (BACs) as probes identified that amplified EPSPS copies in GR AP exist in extra-chromosomal circular DNA (eccDNA) in addition to the native copy in the chromosome. The EPSPS gene-containing eccDNA having a size of ∼400 kb is termed EPSPS-eccDNA and showed somatic mosacism in size and copy number. EP-SPS-eccDNA has a genetic mechanism to tether randomly to mitotic or meiotic chromosomes during cell division or gamete formation and is inherited to daughter cells or progeny generating copy number variation. These eccDNAs are stable genetic elements that can replicate and exist independently. The genomic characterization of the EPSPS locus, along with the flanking regions, identified the presence of a complex array of repeats and mobile genetic elements. The cytogenomics approach in understanding the biology of EPSPS-eccDNA sheds light on various characteristics of EPSPS-eccDNA that favor GR in AP.
Centromeres, because of their repeat structure and lack of sequence conservation, are difficult t... more Centromeres, because of their repeat structure and lack of sequence conservation, are difficult to assemble and compare across organisms. It was recently discovered that rice centromeres often contain genes. This suggested a method for studying centromere homologies between wheat and rice chromosomes by mapping rice centromeric genes onto wheat aneuploid stocks. Three of the seven cDNA clones of centromeric genes from rice centromere 8 (Cen8), 6729.t09, 6729.t10, and 6730.t11 which lie in the Cen8 kinetochore region, and three wheat ESTs, BJ301191, BJ305475, and BJ280500, with similarity to sequences of rice centromeric genes, were mapped to the centromeric regions of the wheat group-7 (W7) chromosomes. A possible pericentric inversion in chromosome 7D was detected. Genomewide comparison of wheat ESTs that mapped to centromeric regions against rice genome sequences revealed high conservation and a one-to-one correspondence of centromeric regions between wheat and rice chromosome pairs W1-R5, W2-R7, W3-R1, W5-R12, W6-R2, and W7-R8. The W4 centromere may share homology with R3 only or with R3 1 R11. Wheat ESTs that mapped to the pericentromeric region of the group-5 long arm anchored to the rice BACs located in the recently duplicated region at the distal ends of the short arms of rice chromosomes 11 and 12. A pericentric inversion specific to the rice lineage was detected. The depicted framework provides a working model for further studies on the structure and evolution of cereal chromosome centromeres.
Proceedings of the National Academy of Sciences of the United States of America, Dec 1, 1992
Bread wheat (Triticum aestivum L. em Thell) is well suited for cytogenetic analysis because the g... more Bread wheat (Triticum aestivum L. em Thell) is well suited for cytogenetic analysis because the genome, buffered by polyploidy, can tolerate structurally and numeri- cally engineered chromosomes for analysis over infinite gen- erations. This feature of polyploidy can be used in developing a high-resolution, cytogenetically based physical map of the wheat genome. We show that numerous deletions, observed in the progeny of a monosomic addition of a chromosome from Trtcum cylinduicum in wheat, result from single breakpoints and a concomitant loss ofdistal fragments. Breakages occurred in euchromatic and heterochromatic regions. Forty-one dele- tions for chromosomes 7A, 7B, and 7D, and a set of genetically mapped DNA probes, were used to construct physical maps. Recombination was low in proximal chromosomal regions and very high toward the distal ends. Deletion mapping was more efficient than genetic mapping in resolving the order of proximal loci. Despite variation in size and arm ratio, relative gene position was largely conserved among chromosomes 7A, 7B, and 7D and a consensus group 7 physical map was constructed. Several molecularly tagged chromosome regions (MTCRs) of approximately one to a few million base pafrs were identified that may be resolved by long-range mapping of DNA fragments. Thus, a cytogenetically based physical map may be used to integrate chromosome and DNA-based maps. The MTCRs may simplify strategies for cloning of agronomically useful genes despite the genetic complexity and the large genome size of wheat.
bioRxiv (Cold Spring Harbor Laboratory), Mar 2, 2023
Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. The eccDNA-power... more Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. The eccDNA-powered copy number variation plays diverse roles from oncogenesis in humans to herbicide resistance in crop weeds. Here we report interspecific eccDNA flow and its dynamic behavior in soma cells of natural populations and F 1 hybrids of Amaranthus sp. The glyphosate resistance (GR) trait is controlled by eccDNA-based amplification harboring the 5enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (EPSPS-eccDNA), the molecular target of glyphosate. We documented pollen-mediated transfer of eccDNA in experimental hybrids between GS A. tuberculatus x GR A. palmeri. Experimental hybridization and fluorescence in situ hybridization (FISH) analysis revealed that the EPSPS-eccDNA present in A. spinosus was derived from GR A. palmeri by natural hybridization. FISH analysis also revealed random chromosome anchoring and massive EPSPS-eccDNA copy number variation in soma cells of weedy hybrids. The results suggest that eccDNAs are inheritable across compatible species contributing to genome plasticity and rapid adaptive evolution.
With this rather cursory look at worldwide small grain cereal genetics efforts, I want to make a ... more With this rather cursory look at worldwide small grain cereal genetics efforts, I want to make a few comments about the ITMI workshop program in Manhattan. We focused the program on genome mapping (Imkage and physical mapping), mapping using nonconventional approaches, such as chemiluminescence and PCR, and on a survey of national genome initiatives, cytogenetic stocks, data management software demonstration, and poster sessions. About 100 scientists from eleven countries participated. Based on the comments from the participants, the program was quite successhl. The intent of the program was to present exciting science and separate the business sessions from scientific presentations. I hope this strategy will be followed in future meetings. Of course, the organization of such a meeting entails a lot of hard work. One single individual who did more than anyone else to make this meeting such a success is John Raupp. John deserves our heartfelt thanks. I want to thank Duane Wilson and the rest of my laboratory staff for their tremendous efforts. We received considerable help from the ITMI office in Davis, CA, particularly from Cal Qualset, Pat McGuire, and Harold Corke. I express my appreciation to them. I shall close by offering a few suggestions for future meetings. First and foremost, the invited speakers must present cutting-edge science, new data, new protocols, innovative research. It is imperative that laboratory leaders encourage participation from their colleagues, bright postdoctoral fellows, and graduate students; the hands-on researchers who have much to contribute. We must encourage participation from all over the world. We must constantly encourage comparative mapping in wheat, rye, barley, oats, and beyond. We need communal library resources such as YAC libraries and cDNA libraries that are beyond the resources of individual laboratories. We need techniques for transformation, chromosome walking, and so on, so that small grain and polyploid genetics research remains an exciting and competitive enterprise.
The wheat wild relative Aegilops tauschii was previously used to transfer the Lr42 leaf rust resi... more The wheat wild relative Aegilops tauschii was previously used to transfer the Lr42 leaf rust resistance gene into bread wheat. Lr42 confers resistance at both seedling and adult stages, and it is broadly effective against all leaf rust races tested to date. Lr42 has been used extensively in the CIMMYT international wheat breeding program with resulting cultivars deployed in several countries. Here, using a bulked segregant RNA-Seq (BSR-Seq) mapping strategy, we identify three candidate genes for Lr42. Overexpression of a nucleotide-binding site leucine-rich repeat (NLR) gene AET1Gv20040300 induces strong resistance to leaf rust in wheat and a mutation of the gene disrupted the resistance. The Lr42 resistance allele is rare in Ae. tauschii and likely arose from ectopic recombination. Cloning of Lr42 provides diagnostic markers and over 1000 CIMMYT wheat lines carrying Lr42 have been developed documenting its widespread use and impact in crop improvement.
Aegilops tauschii, the D-genome donor of bread wheat, Triticum aestivum, is a storehouse of genet... more Aegilops tauschii, the D-genome donor of bread wheat, Triticum aestivum, is a storehouse of genetic diversity, and an important resource for future wheat improvement. Genomic and population analysis of 549 Ae. tauschii and 103 wheat accessions was performed by using 13,135 high quality SNPs. Population structure, principal component, and cluster analysis confirmed the differentiation of Ae. tauschii into two lineages; lineage 1 (L1) and lineage 2 (L2), the latter being the wheat D-genome donor. Lineage L1 contributes only 2.7% of the total introgression from Ae. tauschii for a set of United States winter wheat lines, confirming the great amount of untapped genetic diversity in L1. Lineage L2 accessions had overall greater allelic diversity and wheat accessions had the least allelic diversity. Both lineages also showed intra-lineage differentiation with L1 being driven by longitudinal gradient and L2 differentiated by altitude. There has previously been little reported on natural hybridization between L1 and L2. We found nine putative inter-lineage hybrids in the population structure analysis, each containing numerous lineage-specific private alleles from both lineages. One hybrid was confirmed as a recombinant inbred between the two lineages, likely artificially post collection. Of the remaining eight putative hybrids, a group of seven from Georgia carry 713 SNPs with private alleles, which points to the possibility of a novel L1-L2 hybrid lineage. To facilitate the use of Ae. tauschii in wheat improvement, a MiniCore consisting of 29 L1 and 11 L2 accessions, has been developed based on genotypic, phenotypic and geographical data. MiniCore reduces the collection size by over 10-fold and captures 84% of the total allelic diversity in the whole collection.
During evolutionary history many grasses from the tribe Triticeae have undergone interspecific hy... more During evolutionary history many grasses from the tribe Triticeae have undergone interspecific hybridization, resulting in allopolyploidy; whereas homoploid hybrid speciation was found only in rye. Homoeologous chromosomes within the Triticeae preserved cross-species macrocolinearity, except for a few species with rearranged genomes. Aegilops markgrafii, a diploid wild relative of wheat (2n = 2x = 14), has a highly asymmetrical karyotype that is indicative of chromosome rearrangements. Molecular cytogenetics and next-generation sequencing were used to explore the genome organization. Fluorescence in situ hybridization with a set of wheat cDNAs allowed the macrostructure and cross-genome homoeology of the Ae. markgrafii chromosomes to be established. Two chromosomes maintained colinearity, whereas the remaining were highly rearranged as a result of inversions and inter-and intrachromosomal translocations. We used sets of barley and wheat orthologous gene sequences to compare discrete parts of the Ae. markgrafii genome involved in the rearrangements. Analysis of sequence identity profiles and phylogenic relationships grouped chromosome blocks into two distinct clusters. Chromosome painting revealed the distribution of transposable elements and differentiated chromosome blocks into two groups consistent with the sequence analyses. These data suggest that introgressive hybridization accompanied by gross chromosome rearrangements might have had an impact on karyotype evolution and homoploid speciation in Ae. markgrafii.
Gene transfer from wild wheat relatives to bread wheat is restricted to homologous recombination.... more Gene transfer from wild wheat relatives to bread wheat is restricted to homologous recombination. The presence of the Pairing homoeologous 1 (Ph1) gene in the long arm of wheat chromosome 5B allows only homologous chromosomes to pair and recombine, resulting in diploid inheritance of polyploid wheat. Previously, we identified a potent homoeologous pairing promotor gene(s) (Hpp-5M g ); its carrier chromosome 5M g derived from Aegilops geniculata and its wheat homoeologous chromosome 5D freely recombined in the presence of the Ph1 gene. In this study, we investigated the effect of Hpp-5M g on homoeologous recombination in the absence of Ph1. In Hpp-5M g /ph1bph1b plants, we observed a vast genome-wide increase in homoeologous recombination and multiple crossovers (CO), including CO breakpoints in proximal regions of the chromosomes where recombination is known to be suppressed. We tested the efficacy of Hpp-5M g /ph1bph1b-induced homoeologous recombination by producing new recombinants for the wheat streak mosaic virus resistance gene, Wsm3, present in the wheat-Thinopyrum intermedium Robertsonian translocation (RobT T7BS.7S#3L). A recombination frequency of 6.5% was detected by screening the progenies double monosomic for T7BS.7S#3L and 7B by genomic in situ hybridization. This recombination frequency was about 100-fold higher compared with the recombinant frequency of 0.06% observed by using ph1b-induced homoeologous recombination alone. Our results indicate that chromosome 5M g promotes homoeologous recombination between wheat and wild wheat relative chromosomes, which helps in the generation of pre-breeding materials thereby accelerating wheat crop improvement.
In most eukaryotes, centromeres assemble at a single location per chromosome. Naturally occurring... more In most eukaryotes, centromeres assemble at a single location per chromosome. Naturally occurring telocentric chromosomes (telosomes) with a terminal centromere are rare but do exist. Telosomes arise through misdivision of centromeres in normal chromosomes, and their cytological stability depends on the structure of their kinetochores. The instability of telosomes may be attributed to the relative centromere size and the degree of completeness of their kinetochore. Here we test this hypothesis by analyzing the cytogenetic structure of wheat telosomes. We used a population of 80 telosomes arising from the misdivision of the 21 chromosomes of wheat that have shown stable inheritance over many generations. We analyzed centromere size by probing with the centromere-specific histone H3 variant, CENH3. Comparing the signal intensity for CENH3 between the intact chromosome and derived telosomes showed that the telosomes had approximately half the signal intensity compared to that of normal chromosomes. Immunofluorescence of CENH3 in a wheat stock with 28 telosomes revealed that none of the telosomes received a complete CENH3 domain. Some of the telosomes lacked centromere specific retrotransposons of wheat in the CENH3 domain, indicating that the stability of telosomes depends on the presence of CENH3 chromatin and not on the presence of CRW repeats. In addition to providing evidence for centromere shift, we also observed chromosomal aberrations including inversions and deletions in the short arm telosomes of double ditelosomic 1D and 6D stocks. The role of centromere-flanking, pericentromeric heterochromatin in mitosis is discussed with respect to genome/chromosome integrity.
We report the map-based cloning of the leaf rust resistance gene Lr21, previously mapped to a gen... more We report the map-based cloning of the leaf rust resistance gene Lr21, previously mapped to a generich region at the distal end of chromosome arm 1DS of bread wheat (Triticum aestivum L.). Molecular cloning of Lr21 was facilitated by diploid/polyploid shuttle mapping strategy. Cloning of Lr21 was confirmed by genetic transformation and by a stably inherited resistance phenotype in transgenic plants. Lr21 spans 4318 bp and encodes a 1080-amino-acid protein containing a conserved nucleotide-binding site (NBS) domain, 13 imperfect leucine-rich repeats (LRRs), and a unique 151-amino-acid sequence missing from known NBS-LRR proteins at the N terminus. Fine-structure genetic analysis at the Lr21 locus detected a noncrossover (recombination without exchange of flanking markers) within a 1415-bp region resulting from either a gene conversion tract of at least 191 bp or a double crossover. The successful map-based cloning approach as demonstrated here now opens the door for cloning of many crop-specific agronomic traits located in the gene-rich regions of bread wheat.
These WCM-resistance sources have not been utilized mosaic virus which is vectored by the wheat c... more These WCM-resistance sources have not been utilized mosaic virus which is vectored by the wheat curl mite (WCM) Aceria tosicheilla (Keifer). Host resistance to WCM has reduced losses. This commercially, but some are being advanced in wheat study was conducted to evaluate the effectiveness of resistance in breeding programs. As different collections of WCM wheat to WCM collected from various locations in the Great Plains. vary in degree of virulence to resistant wheat (Harvey Collections of WCM from Montana, Nebraska, South Dakota, Texas, et al., 1995a), it is important to evaluate the range in Alberta, Canada, and eight locations in Kansas were compared for response among WCM strains collected from areas for their ability to survive and reproduce in the greenhouse on seven which resistant cultivars are targeted. The objective of lines of wheat and wheat relatives previously identified as resistant. this research was to expand on a previous report (Har-The lines and their sources of resistance were: AC PGR16635 (Aegivey et al., 1995a) by using a wider range of WCM cullops tauschii Coss., Cmc1 ), PI 525452 (Thinopyrum ponticum (Podp.) tures to evaluate the effectiveness of resistance provided Liu and Wang, Cmc2 ), KS96WGRC40 (Ae. tauschii and Secale cerby the previously tested resistant sources and two reeale L.), TA920 (Triticum timopheevii (Zhuk.) Zhuk spp. armenidcum ), PI 475772 (S. cereale ), 'TAM 107' (S. cereale ), PI 222655 (T. cently identified sources of resistance. aestivum ). KS96WGRC40 and TA920 were the only entries that were resistant to all WCM collections. Other sources of resistance were MATERIALS AND METHODS effective against WCMs from some but not all locations. PI 222655 Sources of WCM-resistant germplasm are given in Table was resistant to WCMs from Nebraska and central Kansas but not 1. The wheat cultivar Larned, (CI 17650) was used in all tests 2). This relationship held for collections from other
Leaf rust resistance gene Lr58 derived from Aegilops triuncialis L. was transferred to the hard r... more Leaf rust resistance gene Lr58 derived from Aegilops triuncialis L. was transferred to the hard red winter wheat (HRWW) cultivars Jagger and Overley by standard backcrossing and markerassisted selection (MAS). A co-dominant PCR-based sequence tagged site (STS) marker was developed based on the sequence information of the RFLP marker (XksuH16) diagnostically detecting the alien segment in T2BSÁ2BL-2 t L(0.95). STS marker Xncw-Lr58-1 was used to select backcross F 1 plants with rust resistance. The co-dominant marker polymorphism detected by primer pair NCW-Lr58-1 efficiently identified the homozygous BC 3 F 2 plants with rust resistance gene Lr58. The STS marker Xncw-Lr58-1 showed consistent diagnostic polymorphism between the resistant source and the wheat cultivars selected by the US Wheat Coordinated Agricultural Project. The utility and compatibility of the STS marker in MAS programs involving robust genotyping platforms was demonstrated in both agarosebased and capillary-based platforms. Screening backcross derivatives carrying Lr58 with various rust races at seedling stage suggested the transferred rust resistance in adapted winter wheats is stable in both cultivar backgrounds. Lr58 in adapted winter wheat backgrounds could be used in combination with other resistance genes in wheat rust resistance breeding.
The distribution of genes and recombination in the wheat genome was studied by comparing physical... more The distribution of genes and recombination in the wheat genome was studied by comparing physical maps with the genetic linkage maps. The physical maps were generated by mapping 80 DNA and two phenotypic markers on an array of 65 deletion lines for homoeologous group 5 chromosomes. The genetic maps were constructed for chromosome 5B in wheat and 50 in Triticum tauschii. No marker mapped in the proximal 20% chromosome region surrounding the centromere. More than 60% of the long arm markers were present in three major clusters that physically encompassed <18% of the arm. Because 48% of the markers were cDNA clones and the distributions of the cDNA and genomic clones were similar, the marker distribution may represent the distribution of genes. The gene clusters were identified and allocated to very small chromosome regions because of a higher number of deletions in their surrounding regions. The recombination was suppressed in the centromeric regions and mainly occurred in the gene-rich regions. The bp/cM estimates varied from 118 kb for gene-rich regions to 22 Mb for gene-poor regions. The wheat genes present in these clusters are, therefore, amenable to molecular manipulations parallel to the plants with smaller genomes like rice.
Brachypodium distachyon is a wild annual grass belonging to the Pooideae, more closely related to... more Brachypodium distachyon is a wild annual grass belonging to the Pooideae, more closely related to wheat, barley, and forage grasses than rice and maize. As an experimental model, the completed genome sequence of B. distachyon provides a unique opportunity to study centromere evolution during the speciation of grasses. Centromeric satellite sequences have been identified in B. distachyon, but little is known about centromeric retrotransposons in this species. In the present study, BAC-fluorescence in situ hybridization was conducted in maize, rice, barley, wheat, and rye using B. distachyon (Bd) centromere-specific BAC clones. Eight Bd centromeric BAC clones gave no detectable FISH signals on the chromosomes of rice and maize, and three of them also did not yield any FISH signals in barley, wheat, and rye. In addition, four of five Triticeae centromeric BAC clones did not hybridize to the B. distachyon centromeres, implying certain unique features of Brachypodium centromeres. Analysis of Brachypodium centromeric BAC sequences identified a long terminal repeat (LTR)centromere retrotransposon of B. distachyon (CRBd1). This element was found in high copy number accounting for 1.6% of the B. distachyon genome, and is enriched in Brachypodium centromeric regions. CRBd1 accumulated in active centromeres, but was lost from inactive ones. The LTR of CRBd1 appears to be specific to B. distachyon centromeres. These results reveal different evolutionary events of this retrotransposon family across grass species.
Background: Lack of nutritionally appropriate foods is one of the leading causes of obesity in th... more Background: 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. Results: 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-ΔABD. The Jag-ssiia-ΔABD had an amylose content of 35.70% compared to 31.15% in Jagger, leading to ~118% increase in resistant starch in the Jag-ssiia-ΔABD genotype of Jagger wheat. The single individual genome mutations also had various effects on starch composition. Conclusions: Our full null Jag-ssiia-ΔABD 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.
The International Weed Genomics Consortium is a collaborative group of researchers focused on dev... more The International Weed Genomics Consortium is a collaborative group of researchers focused on developing genomic resources for the study of weedy plants. Weeds are attractive systems for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Our goal is to use genomic information to develop sustainable and effective weed control methods and to provide insights about biotic and abiotic stress tolerance to assist crop breeding. Here, we outline resources under development by the consortium and highlight areas of research that will be impacted by these enabling resources.
International audienceBecause of its 17 Gb hexaploid genome, wheat genomics has been lagging behi... more International audienceBecause of its 17 Gb hexaploid genome, wheat genomics has been lagging behind the one of the other major crops. Two years after the establishment of the first physical map of the biggest wheat chromosome, the 3B, which represents 1 Gb, its complete sequencing is now underway (ANR project 3BSEQ). In order to prepare for its complete sequencing and analysis, we performed a pilot project on 18 Mb of large BAC contigs which allowed us to improve our understanding of the wheat genome composition and dynamics. Comparative and evolutionary analyses revealed that increase of the genome size, mainly mediated by transposable element amplification, was uneven and has led to the formation of small gene islands. In addition, it was accompanied by rearrangement of the ancestral gene content. Indeed, a significant amount of nonsyntenic genes are interspersed into a conserved ancestral grass gene backbone, revealing that, in our sample, about 50% of the annotated genes was une...
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Papers by Bikram Gill