Perfect Match Genomic Landscape strategy: Refinement and customization of reference genomes.

When addressing a genomic question, having a reliable and adequate reference genome is of utmost importance. This drives the necessity to refine and customize reference genomes (RGs). Our laboratory has recently developed a strategy, the Perfect Match Genomic Landscape (PMGL), to detect variation between genomes [K. Palacios-Flores et al.Genetics 208, 1631-1641 (2018)]. The PMGL is precise and sensitive and, in contrast to most currently used algorithms, is nonstatistical in nature. Here we demonstrate the power of PMGL to refine and customize RGs. As a proof-of-concept, we refined different versions of the Saccharomyces cerevisiae RG. We applied the automatic PMGL pipeline to refine the genomes of microorganisms belonging to the three domains of life: the archaea Methanococcus maripaludis and Pyrococcus furiosus; the bacteria Escherichia coli, Staphylococcus aureus, and Bacillus subtilis; and the eukarya Schizosaccharomyces pombe, Aspergillus oryzae, and several strains of Saccharomyces paradoxus. We analyzed the reference genome of the virus SARS-CoV-2 and previously published viral genomes from patients' samples with COVID-19. We performed a mutation-accumulation experiment in E. coli and show that the PMGL strategy can detect specific mutations generated at any desired step of the whole procedure. We propose that PMGL can be used as a final step for the refinement and customization of any haploid genome, independently of the strategies and algorithms used in its assembly.

Prediction and identification of recurrent genomic rearrangements that generate chimeric chromosomes in Saccharomyces cerevisiae.

Genomes are dynamic structures. Different mechanisms participate in the generation of genomic rearrangements. One of them is nonallelic homologous recombination (NAHR). This rearrangement is generated by recombination between pairs of repeated sequences with high identity. We analyzed rearrangements mediated by repeated sequences located in different chromosomes. Such rearrangements generate chimeric chromosomes. Potential rearrangements were predicted by localizing interchromosomal identical repeated sequences along the nuclear genome of the Saccharomyces cerevisiae S288C strain. Rearrangements were identified by a PCR-based experimental strategy. PCR primers are located in the unique regions bordering each repeated region of interest. When the PCR is performed using forward primers from one chromosome and reverse primers from another chromosome, the break point of the chimeric chromosome structure is revealed. In all cases analyzed, the corresponding chimeric structures were found. Furthermore, the nucleotide sequence of chimeric structures was obtained, and the origin of the unique regions bordering the repeated sequence was located in the expected chromosomes, using the perfect-match genomic landscape strategy (PMGL). Several chimeric structures were searched in colonies derived from single cells. All of the structures were found in DNA isolated from each of the colonies. Our findings indicate that interchromosomal rearrangements that generate chimeric chromosomes are recurrent and occur, at a relatively high frequency, in cell populations of S. cerevisiae.

Precise detection of de novo single nucleotide variants in human genomes.

The precise determination of de novo genetic variants has enormous implications across different fields of biology and medicine, particularly personalized medicine. Currently, de novo variations are identified by mapping sample reads from a parent-offspring trio to a reference genome, allowing for a certain degree of differences. While widely used, this approach often introduces false-positive (FP) results due to misaligned reads and mischaracterized sequencing errors. In a previous study, we developed an alternative approach to accurately identify single nucleotide variants (SNVs) using only perfect matches. However, this approach could be applied only to haploid regions of the genome and was computationally intensive. In this study, we present a unique approach, coverage-based single nucleotide variant identification (COBASI), which allows the exploration of the entire genome using second-generation short sequence reads without extensive computing requirements. COBASI identifies SNVs using changes in coverage of exactly matching unique substrings, and is particularly suited for pinpointing de novo SNVs. Unlike other approaches that require population frequencies across hundreds of samples to filter out any methodological biases, COBASI can be applied to detect de novo SNVs within isolated families. We demonstrate this capability through extensive simulation studies and by studying a parent-offspring trio we sequenced using short reads. Experimental validation of all 58 candidate de novo SNVs and a selection of non-de novo SNVs found in the trio confirmed zero FP calls. COBASI is available as open source at https://github.com/Laura-Gomez/COBASI for any researcher to use.

Genome Wide Association Study of Karnal Bunt Resistance in a Wheat Germplasm Collection from Afghanistan.

Karnal bunt disease of wheat, caused by the fungus Neovossia indica, is one of the most important challenges to the grain industry as it affects the grain quality and also restricts the international movement of infected grain. It is a seed-, soil- and airborne disease with limited effect of chemical control. Currently, this disease is contained through the deployment of host resistance but further improvement is limited as only a few genotypes have been found to carry partial resistance. To identify genomic regions responsible for resistance in a set of 339 wheat accessions, genome-wide association study (GWAS) was undertaken using the DArTSeq® technology, in which 18 genomic regions for Karnal bunt resistance were identified, explaining 5-20% of the phenotypic variation. The identified quantitative trait loci (QTL) on chromosome 2BL showed consistently significant effects across all four experiments, whereas another QTL on 5BL was significant in three experiments. Additional QTLs were mapped on chromosomes 1DL, 2DL, 4AL, 5AS, 6BL, 6BS, 7BS and 7DL that have not been mapped previously, and on chromosomes 4B, 5AL, 5BL and 6BS, which have been reported in previous studies. Germplasm with less than 1% Karnal bunt infection have been identified and can be used for resistance breeding. The SNP markers linked to the genomic regions conferring resistance to Karnal bunt could be used to improve Karnal bunt resistance through marker-assisted selection.

Skene's gland cyst: a simple marsupialization technique.

INTRODUCTION AND HYPOTHESIS: We present a simple approach to the marsupialization of a Skene's gland cyst. METHODS: Our technique facilitates suture placement to exteriorize the cyst wall to efficiently treat a distal Skene's gland cyst and reduce the risk of recurrence. CONCLUSION: Marsupialization is an accepted option for the surgical management of Skene's gland cyst. This technique is an effective and streamlined approach to Skene's gland marsupialization.

RNA-Seq analysis of the multipartite genome of Rhizobium etli CE3 shows different replicon contributions under heat and saline shock.

BACKGROUND: Regulation of transcription is essential for any organism and Rhizobium etli (a multi-replicon, nitrogen-fixing symbiotic bacterium) is no exception. This bacterium is commonly found in the rhizosphere (free-living) or inside of root-nodules of the common bean (Phaseolus vulgaris) in a symbiotic relationship. Abiotic stresses, such as high soil temperatures and salinity, compromise the genetic stability of R. etli and therefore its symbiotic interaction with P. vulgaris. However, it is still unclear which genes are up- or down-regulated to cope with these stress conditions. The aim of this study was to identify the genes and non-coding RNAs (ncRNAs) that are differentially expressed under heat and saline shock, as well as the promoter regions of the up-regulated loci. RESULTS: Analysing the heat and saline shock responses of R. etli CE3 through RNA-Seq, we identified 756 and 392 differentially expressed genes, respectively, and 106 were up-regulated under both conditions. Notably, the set of genes over-expressed under either condition was preferentially encoded on plasmids, although this observation was more significant for the heat shock response. In contrast, during either saline shock or heat shock, the down-regulated genes were principally chromosomally encoded. Our functional analysis shows that genes encoding chaperone proteins were up-regulated during the heat shock response, whereas genes involved in the metabolism of compatible solutes were up-regulated following saline shock. Furthermore, we identified thirteen and nine ncRNAs that were differentially expressed under heat and saline shock, respectively, as well as eleven ncRNAs that had not been previously identified. Finally, using an in silico analysis, we studied the promoter motifs in all of the non-coding regions associated with the genes and ncRNAs up-regulated under both conditions. CONCLUSIONS: Our data suggest that the replicon contribution is different for different stress responses and that the heat shock response is more complex than the saline shock response. In general, this work exemplifies how strategies that not only consider differentially regulated genes but also regulatory elements of the stress response provide a more comprehensive view of bacterial gene regulation.

Narrow-host-range bacteriophages that infect Rhizobium etli associate with distinct genomic types.

In this work, we isolated and characterized 14 bacteriophages that infect Rhizobium etli. They were obtained from rhizosphere soil of bean plants from agricultural lands in Mexico using an enrichment method. The host range of these phages was narrow but variable within a collection of 48 R. etli strains. We obtained the complete genome sequence of nine phages. Four phages were resistant to several restriction enzymes and in vivo cloning, probably due to nucleotide modifications. The genome size of the sequenced phages varied from 43 kb to 115 kb, with a median size of ≈ 45 to 50 kb. A large proportion of open reading frames of these phage genomes (65 to 70%) consisted of hypothetical and orphan genes. The remainder encoded proteins needed for phage morphogenesis and DNA synthesis and processing, among other functions, and a minor percentage represented genes of bacterial origin. We classified these phages into four genomic types on the basis of their genomic similarity, gene content, and host range. Since there are no reports of similar sequences, we propose that these bacteriophages correspond to novel species.

Context-dependent individualization of nucleotides and virtual genomic hybridization allow the precise location of human SNPs.

We have entered the era of individual genomic sequencing, and can already see exponential progress in the field. It is of utmost importance to exclude false-positive variants from reported datasets. However, because of the nature of the used algorithms, this task has not been optimized to the required level of precision. This study presents a unique strategy for identifying SNPs, called COIN-VGH, that largely minimizes the presence of false-positives in the generated data. The algorithm was developed using the X-chromosome-specific regions from the previously sequenced genomes of Craig Venter and James Watson. The algorithm is based on the concept that a nucleotide can be individualized if it is analyzed in the context of its surrounding genomic sequence. COIN-VGH consists of defining the most comprehensive set of nucleotide strings of a defined length that map with 100% identity to a unique position within the human reference genome (HRG). Such set is used to retrieve sequence reads from a query genome (QG), allowing the production of a genomic landscape that represents a draft HRG-guided assembly of the QG. This landscape is analyzed for specific signatures that indicate the presence of SNPs. The fidelity of the variation signature was assessed using simulation experiments by virtually altering the HRG at defined positions. Finally, the signature regions identified in the HRG and in the QG reads are aligned and the precise nature and position of the corresponding SNPs are detected. The advantages of COIN-VGH over previous algorithms are discussed.

Genomic lineages of Rhizobium etli revealed by the extent of nucleotide polymorphisms and low recombination.

BACKGROUND: Most of the DNA variations found in bacterial species are in the form of single nucleotide polymorphisms (SNPs), but there is some debate regarding how much of this variation comes from mutation versus recombination. The nitrogen-fixing symbiotic bacteria Rhizobium etli is highly variable in both genomic structure and gene content. However, no previous report has provided a detailed genomic analysis of this variation at nucleotide level or the role of recombination in generating diversity in this bacterium. Here, we compared draft genomic sequences versus complete genomic sequences to obtain reliable measures of genetic diversity and then estimated the role of recombination in the generation of genomic diversity among Rhizobium etli. RESULTS: We identified high levels of DNA polymorphism in R. etli, and found that there was an average divergence of 4% to 6% among the tested strain pairs. DNA recombination events were estimated to affect 3% to 10% of the genomic sample analyzed. In most instances, the nucleotide diversity (π) was greater in DNA segments with recombinant events than in non-recombinant segments. However, this degree of recombination was not sufficiently large to disrupt the congruence of the phylogenetic trees, and further evaluation of recombination in strains quartets indicated that the recombination levels in this species are proportionally low. CONCLUSION: Our data suggest that R. etli is a species composed of separated lineages with low homologous recombination among the strains. Horizontal gene transfer, particularly via the symbiotic plasmid characteristic of this species, seems to play an important role in diversity but the lineages maintain their evolutionary cohesiveness.

The conjugative plasmid of a bean-nodulating Sinorhizobium fredii strain is assembled from sequences of two Rhizobium plasmids and the chromosome of a Sinorhizobium strain.

BACKGROUND: Bean-nodulating Rhizobium etli originated in Mesoamerica, while soybean-nodulating Sinorhizobium fredii evolved in East Asia. S. fredii strains, such as GR64, have been isolated from bean nodules in Spain, suggesting the occurrence of conjugative transfer events between introduced and native strains. In R. etli CFN42, transfer of the symbiotic plasmid (pRet42d) requires cointegration with the endogenous self-transmissible plasmid pRet42a. Aiming at further understanding the generation of diversity among bean nodulating strains, we analyzed the plasmids of S. fredii GR64: pSfr64a and pSfr64b (symbiotic plasmid). RESULTS: The conjugative transfer of the plasmids of strain GR64 was analyzed. Plasmid pSfr64a was self-transmissible, and required for transfer of the symbiotic plasmid. We sequenced pSfr64a, finding 166 ORFs. pSfr64a showed three large segments of different evolutionary origins; the first one presented 38 ORFs that were highly similar to genes located on the chromosome of Sinorhizobium strain NGR234; the second one harbored 51 ORFs with highest similarity to genes from pRet42d, including the replication, but not the symbiosis genes. Accordingly, pSfr64a was incompatible with the R. etli CFN42 symbiotic plasmid, but did not contribute to symbiosis. The third segment contained 36 ORFs with highest similarity to genes localized on pRet42a, 20 of them involved in conjugative transfer. Plasmid pRet42a was unable to substitute pSfr64a for induction of pSym transfer, and its own transfer was significantly diminished in GR64 background. The symbiotic plasmid pSfr64b was found to differ from typical R. etli symbiotic plasmids. CONCLUSIONS: S. fredii GR64 contains a chimeric transmissible plasmid, with segments from two R. etli plasmids and a S. fredii chromosome, and a symbiotic plasmid different from the one usually found in R. etli bv phaseoli. We infer that these plasmids originated through the transfer of a symbiotic-conjugative-plasmid cointegrate from R. etli to a S. fredii strain, and at least two recombination events among the R. etli plasmids and the S. fredii genome. As in R. etli CFN42, the S. fredii GR64 transmissible plasmid is required for the conjugative transfer of the symbiotic plasmid. In spite of the similarity in the conjugation related genes, the transfer process of these plasmids shows a host-specific behaviour.

Pre-emptive Breeding Against Karnal Bunt Infection in Common Wheat: Combining Genomic and Agronomic Information to Identify Suitable Parents.

Wheat (Triticum aestivum L.) is the most widely grown cereal crop in the world and is staple food to half the world's population. The current world population is expected to reach 9.8 billion people by 2050, but food production is not expected to keep pace with demand in developing countries. Significant opportunities exist for traditional grain exporters to produce and export greater amounts of wheat to fill the gap. Karnal bunt, however, is a major threat, due to its use as a non-tariff trade barrier by several wheat-importing countries. The cultivation of resistant varieties remains the most cost-effective approach to manage the disease, but in countries that are free of the disease, genetic improvement is difficult due to quarantine restrictions. Here we report a study on pre-emptive breeding designed to identify linked molecular markers, evaluate the prospects of genomic selection as a tool, and prioritise wheat genotypes suitable for use as parents. In a genome-wide association (GWAS) study, we identified six DArTseq markers significantly linked to Karnal bunt resistance, which explained between 7.6 and 29.5% of the observed phenotypic variation. The accuracy of genomic prediction was estimated to vary between 0.53 and 0.56, depending on whether it is based solely on the identified Quantitative trait loci (QTL) markers or the use of genome-wide markers. As genotypes used as parents would be required to possess good yield and phenology, further research was conducted to assess the agronomic value of Karnal bunt resistant germplasm from the International Maize and Wheat Improvement Center (CIMMYT). We identified an ideal genotype, ZVS13_385, which possessed similar agronomic attributes to the highly successful Australian wheat variety, Mace. It is phenotypically resistant to Karnal bunt infection (<1% infection) and carried all the favourable alleles detected for resistance in this study. The identification of a genotype combining Karnal bunt resistance with adaptive agronomic traits overcomes the concerns of breeders regarding yield penalty in the absence of the disease.

Evolutionary dynamics of insertion sequences in relation to the evolutionary histories of the chromosome and symbiotic plasmid genes of Rhizobium etli populations.

Insertion sequences (IS) are mobile genetic elements that are distributed in many prokaryotes. In particular, in the genomes of the symbiotic nitrogen-fixing bacteria collectively known as rhizobia, IS are fairly abundant in plasmids or chromosomal islands that carry the genes needed for symbiosis. Here, we report an analysis of the distribution and genetic conservation of the IS found in the genome of Rhizobium etli CFN42 in a collection of 87 Rhizobium strains belonging to populations with different geographical origins. We used PCR to generate presence/absence profiles of the 39 IS found in R. etli CFN42 and evaluated whether the IS were located in consistent genomic contexts. We found that the IS from the symbiotic plasmid were frequently present in the analyzed strains, whereas the chromosomal IS were observed less frequently. We then examined the evolutionary dynamics of these strains based on a population genetic analysis of two chromosomal housekeeping genes (glyA and dnaB) and three symbiotic sequences (nodC and the two IS elements). Our results indicate that the IS contained within the symbiotic plasmid have a higher degree of genomic context conservation, lower nucleotide diversity and genetic differentiation, and fewer recombination events than the chromosomal housekeeping genes. These results suggest that the R. etli populations diverged recently in Mexico, that the symbiotic plasmid also had a recent origin, and that the IS elements have undergone a process of cyclic infection and expansion.

Conserved symbiotic plasmid DNA sequences in the multireplicon pangenomic structure of Rhizobium etli.

Strains of the same bacterial species often show considerable genomic variation. To examine the extent of such variation in Rhizobium etli, the complete genome sequence of R. etli CIAT652 and the partial genomic sequences of six additional R. etli strains having different geographical origins were determined. The sequences were compared with each other and with the previously reported genome sequence of R. etli CFN42. DNA sequences common to all strains constituted the greater part of these genomes and were localized in both the chromosome and large plasmids. About 700 to 1,000 kb of DNA that did not match sequences of the complete genomes of strains CIAT652 and CFN42 was unique to each R. etli strain. These sequences were distributed throughout the chromosome as individual genes or chromosomal islands and in plasmids, and they encoded accessory functions, such as transport of sugars and amino acids, or secondary metabolism; they also included mobile elements and hypothetical genes. Sequences corresponding to symbiotic plasmids showed high levels of nucleotide identity (about 98 to 99%), whereas chromosomal sequences and the sequences with matches to other plasmids showed lower levels of identity (on average, about 90 to 95%). We concluded that R. etli has a pangenomic structure with a core genome composed of both chromosomal and plasmid sequences, including a highly conserved symbiotic plasmid, despite the overall genomic divergence.

Unravelling the Complex Genetics of Karnal Bunt (Tilletia indica) Resistance in Common Wheat (Triticum aestivum) by Genetic Linkage and Genome-Wide Association Analyses.

Karnal bunt caused by Tilletia indica Mitra [syn. Neovossia indica (Mitra) Mundkur] is a significant biosecurity concern for wheat-exporting countries that are free of the disease. It is a seed-, soil-and air-borne disease with no effective chemical control measures. The current study used data from multi-year field experiments of two bi-parental populations and a genome-wide association (GWA) mapping panel to unravel the genetic basis for resistance in common wheat. Broad-sense heritability for Karnal bunt resistance in the populations varied from 0.52 in the WH542×HD29 population, to 0.61 in the WH542×W485 cross and 0.71 in a GWAS panel. Quantitative trait locus (QTL) analysis with seven years of phenotypic data identified a major locus on chromosome 3B (R2 = 27.8%) and a minor locus on chromosome 1A (R2 = 12.2%), in the WH542×HD29 population, with both parents contributing the high-value alleles. A major locus (R2 = 27.8%) and seven minor loci (R2 = 4.4-15.8%) were detected in the WH542×W485 population. GWA mapping validated QTL regions in the bi-parent populations, but also identified novel loci not previously associated with Karnal bunt resistance. Meta-QTL analysis aligned the results from this study with those reported in wheat over the last two decades. Two major clusters were detected, the first on chromosome 4B, which clustered with Qkb.ksu-4B, QKb.cimmyt-4BL, Qkb.cim-4BL, and the second on chromosome 3B, which clustered with Qkb.cnl-3B, QKb.cimmyt-3BS and Qkb.cim-3BS1 The results provide definitive chromosomal assignments for QTL/genes controlling Karnal bunt resistance in common wheat, and will be useful in pre-emptive breeding against the pathogen in wheat-producing areas that are free of the disease.

The Rhizobium etli sigma70 (SigA) factor recognizes a lax consensus promoter.

A collection of Rhizobium etli promoters was isolated from a genomic DNA library constructed in the promoter-trap vector pBBMCS53, by their ability to drive the expression of a gusA reporter gene. Thirty-seven clones were selected, and their transcriptional start-sites were determined. The upstream sequence of these 37 start-sites, and the sequences of seven previously identified promoters were compared. On the basis of sequence conservation and mutational analysis, a consensus sequence CTTGACN16-23TATNNT was obtained. In this consensus sequence, nine on of twelve bases are identical to the canonical Escherichia coli sigma70 promoter, however the R.etli promoters only contain 6.4 conserved bases on average. We show that the R.etli sigma factor SigA recognizes all R.etli promoters studied in this work, and that E.coli RpoD is incapable of recognizing them. The comparison of the predicted structure of SigA with the known structure of RpoD indicated that regions 2.4 and 4.2, responsible for promoter recognition, are different only by a single amino acid, whereas the region 1 of SigA contains 72 extra residues, suggesting that the differences contained in this region could be related to the lax promoter recognition of SigA.

A Perfect Match Genomic Landscape Provides a Unified Framework for the Precise Detection of Variation in Natural and Synthetic Haploid Genomes.

We present a conceptually simple, sensitive, precise, and essentially nonstatistical solution for the analysis of genome variation in haploid organisms. The generation of a Perfect Match Genomic Landscape (PMGL), which computes intergenome identity with single nucleotide resolution, reveals signatures of variation wherever a query genome differs from a reference genome. Such signatures encode the precise location of different types of variants, including single nucleotide variants, deletions, insertions, and amplifications, effectively introducing the concept of a general signature of variation. The precise nature of variants is then resolved through the generation of targeted alignments between specific sets of sequence reads and known regions of the reference genome. Thus, the perfect match logic decouples the identification of the location of variants from the characterization of their nature, providing a unified framework for the detection of genome variation. We assessed the performance of the PMGL strategy via simulation experiments. We determined the variation profiles of natural genomes and of a synthetic chromosome, both in the context of haploid yeast strains. Our approach uncovered variants that have previously escaped detection. Moreover, our strategy is ideally suited for further refining high-quality reference genomes. The source codes for the automated PMGL pipeline have been deposited in a public repository.

Genetic Mapping of Resistance in Hexaploid Wheat for a Quarantine Disease: Karnal Bunt.

Karnal bunt (KB) of wheat, caused by Tilletia indica, is one of the greatest challenges to grain industry, not because of yield loss, but quarantine regulations that restrict international movement and trade of affected stocks. Genetic resistance is the best way to manage this disease. Although several different sources of resistance have been identified to date, very few of those have been subjected to genetic analyses. Understanding the genetics of resistance, characterization and mapping of new resistance loci can help in development of improved germplasm. The objective of this study was to identify and characterize resistance loci (QTL) in two independent recombinant inbred lines (RILs) populations utilizing different wheat lines as resistance donors. Elite CIMMYT wheat lines Blouk#1 and Huirivis#1 were used as susceptible female parents and WHEAR/KUKUNA/3/C80.1/3∗BATAVIA//2∗WBLL1 (WKCBW) and Mutus as moderately resistant male parents in Pop1 and Pop2 populations, respectively. Populations were evaluated for KB resistance in 2015-16 and 2016-17 cropping seasons at two seeding dates (total four environments) in Cd. Obregon, Mexico. Two stable QTL from each population were identified in each environment: QKb.cim-2B and QKb.cim-3D (Pop1), QKb.cim-3B1 and QKb.cim-5B2 (Pop2). Other than those four QTL, other QTL were detected in each population which were specific to environments: QKb.cim-5B1, QKb.cim-6A, and QKb.cim-7A (Pop1), QKb.cim-3B2, QKb.cim-4A1, QKb.cim-4A2, QKb.cim-4B, QKb.cim-5A1, QKb.cim-5A2, and QKb.cim-7A2 (Pop2). Among the four stable QTL, all but QKb.cim-3B1 were derived from the resistant parent. QKb.cim-2B and QKb.cim-3D in Pop1 and QKb.cim-3B1 and QKb.cim-5B2 in Pop2 explained 5.0-11.4% and 3.3-7.1% phenotypic variance, respectively. A combination of two stable QTL in each population reduced KB infection by 24-33%, respectively. Transgressive resistant segregants lines derived with resistance alleles from both parents in each population were identified. Single nucleotide polymorphism (SNP) markers flanking these QTL regions may be amenable to marker-assisted selection. The best lines from both populations (in agronomy, end-use quality and KB resistance) carrying resistance alleles at all identified loci, may be used for inter-crossing and selection of improved germplasm in future. Markers flanking these QTL may assist in selection of such lines.

Harnessing genetic potential of wheat germplasm banks through impact-oriented-prebreeding for future food and nutritional security.

The value of exotic wheat genetic resources for accelerating grain yield gains is largely unproven and unrealized. We used next-generation sequencing, together with multi-environment phenotyping, to study the contribution of exotic genomes to 984 three-way-cross-derived (exotic/elite1//elite2) pre-breeding lines (PBLs). Genomic characterization of these lines with haplotype map-based and SNP marker approaches revealed exotic specific imprints of 16.1 to 25.1%, which compares to theoretical expectation of 25%. A rare and favorable haplotype (GT) with 0.4% frequency in gene bank identified on chromosome 6D minimized grain yield (GY) loss under heat stress without GY penalty under irrigated conditions. More specifically, the 'T' allele of the haplotype GT originated in Aegilops tauschii and was absent in all elite lines used in study. In silico analysis of the SNP showed hits with a candidate gene coding for isoflavone reductase IRL-like protein in Ae. tauschii. Rare haplotypes were also identified on chromosomes 1A, 6A and 2B effective against abiotic/biotic stresses. Results demonstrate positive contributions of exotic germplasm to PBLs derived from crosses of exotics with CIMMYT's best elite lines. This is a major impact-oriented pre-breeding effort at CIMMYT, resulting in large-scale development of PBLs for deployment in breeding programs addressing food security under climate change scenarios.

Rapid evolutionary change of common bean (Phaseolus vulgaris L) plastome, and the genomic diversification of legume chloroplasts.

BACKGROUND: Fabaceae (legumes) is one of the largest families of flowering plants, and some members are important crops. In contrast to what we know about their great diversity or economic importance, our knowledge at the genomic level of chloroplast genomes (cpDNAs or plastomes) for these crops is limited. RESULTS: We sequenced the complete genome of the common bean (Phaseolus vulgaris cv. Negro Jamapa) chloroplast. The plastome of P. vulgaris is a 150,285 bp circular molecule. It has gene content similar to that of other legume plastomes, but contains two pseudogenes, rpl33 and rps16. A distinct inversion occurred at the junction points of trnH-GUG/rpl14 and rps19/rps8, as in adzuki bean 1. These two pseudogenes and the inversion were confirmed in 10 varieties representing the two domestication centers of the bean. Genomic comparative analysis indicated that inversions generally occur in legume plastomes and the magnitude and localization of insertions/deletions (indels) also vary. The analysis of repeat sequences demonstrated that patterns and sequences of tandem repeats had an important impact on sequence diversification between legume plastomes and tandem repeats did not belong to dispersed repeats. Interestingly, P. vulgaris plastome had higher evolutionary rates of change on both genomic and gene levels than G. max, which could be the consequence of pressure from both mutation and natural selection. CONCLUSION: Legume chloroplast genomes are widely diversified in gene content, gene order, indel structure, abundance and localization of repetitive sequences, intracellular sequence exchange and evolutionary rates. The P. vulgaris plastome is a rapidly evolving genome.

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.