Peter J. Maughan

Amplified fragment length polymorphism (AFLP) in soybean: species diversity, inheritance, and near-isogenic line analysis

Amplified fragment length polymorphism (AFLP) analysis is a PCR-based technique capable of detecting more than 50 independent loci in a single PCR reaction. The objectives of the present study were to: (1) assess the extent of AFLP variation in cultivated (Gycine max L. Merr.) and wild soybean (G. soja Siebold & Zucc.), (2) determine genetic relationships among soybean accessions using AFLP data, and (3) evaluate the usefulness of AFLPs as genetic markers. Fifteen AFLP primer pairs detected a total of 759 AFLP fragments in a sample of 23 accessions of wild and cultivated soybean, with an average of 51 fragments produced per primer pair per accession. Two-hundred and seventy four fragments (36% of the total observed) were polymorphic, among which 127 (17%) were polymorphic in G. max and 237 (31%) were polymorphic in G. soja. F2 segregation analysis of six AFLP fragments indicated that they segregate as stable Mendelian loci. The number of polymorphic loci detected per AFLP primer pair in a sample of 23 accessions ranged from 9 to 27. The AFLP phenotypic diversity values were greater in wild than in cultivated soybean. Cluster and principal component analyses using AFLP data clearly separated G. max and G. soja accessions. Within the G. max group, adapted soybean cultivars were tightly clustered, illustrating the relatively low genetic diversity present in cultivated soybean. AFLP analysis of four soybean near-isogenic lines (NILs) identified three AFLP markers putatively linked to a virus resistance gene from two sources. The capacity of AFLP analysis to detect thousands of independent genetic loci with minimal cost and time requirements makes them an ideal marker for a wide array of genetic investigations.

Molecular marker analysis of seed-weight : genomic locations, gene action, and evidence for orthologous evolution among three legume species

The objectives of this study were to use molecular markers to: (1) identify quantitative trait loci (QTL) controlling seed-weight in soybean, (2) characterize the genetic basis of seed-weight expression, and (3) determine whether soybean shares orthologous seed-weight genes with cowpea and/or mung bean. An F2 population was developed between a large-seeded Glycine max breeding line and a small-seeded G. soja plant introduction. DNA samples from 150 F2 individuals were analyzed with 91 polymorphic genetic markers, including RFLPs, RAPDs and SSRs. Seed-weight was analyzed by randomly sampling 100 seeds from each of 150 greenhouse-grown F2 individuals, and their 150 F2∶3 lines, from a replicated field trial. Markers associated with seed-weight were identified using the computer program MapMaker-QTL and a one-way analysis of variance. Three and five markers were significantly associated with seed-weight variation (P<0.01) in the F2 and F2∶3 generations, respectively. Tests for digenic epistasis revealed three significant interactions in both generations. In a combined analysis, these markers and interactions explained 50 and 60% of the phenotypic variation for seed-weight in the F2 and F2∶3 generations, respectively. Comparison of our results in soybean (Glycine) with those previously reported in cowpea and mung bean (Vigna) indicated that soybean and cowpea share an orthologous seed-weight gene. In both species, a genomic region significantly associated with seed-weight spanned the same RFLP markers in the same linkage order. A significant digenic interaction involving this genomic region was conserved in all three species. These results suggest that the exploitation of “comparative QTL mapping” is an invaluable tool for quantitative geneticists working with poorly characterized plant systems.

Targeted enrichment strategies for next-generation plant biology

PREMISE OF THE STUDY The dramatic advances offered by modern DNA sequencers continue to redefine the limits of what can be accomplished in comparative plant biology. Even with recent achievements, however, plant genomes present obstacles that can make it difficult to execute large-scale population and phylogenetic studies on next-generation sequencing platforms. Factors like large genome size, extensive variation in the proportion of organellar DNA in total DNA, polyploidy, and gene number/redundancy contribute to these challenges, and they demand flexible targeted enrichment strategies to achieve the desired goals. METHODS In this article, we summarize the many available targeted enrichment strategies that can be used to target partial-to-complete organellar genomes, as well as known and anonymous nuclear targets. These methods fall under four categories: PCR-based enrichment, hybridization-based enrichment, restriction enzyme-based enrichment, and enrichment of expressed gene sequences. KEY RESULTS Examples of plant-specific applications exist for nearly all methods described. While some methods are well established (e.g., transcriptome sequencing), other promising methods are in their infancy (hybridization enrichment). A direct comparison of methods shows that PCR-based enrichment may be a reasonable strategy for accessing small genomic targets (e.g., ≤50 kbp), but that hybridization and transcriptome sequencing scale more efficiently if larger targets are desired. CONCLUSIONS While the benefits of targeted sequencing are greatest in plants with large genomes, nearly all comparative projects can benefit from the improved throughput offered by targeted multiplex DNA sequencing, particularly as the amount of data produced from a single instrument approaches a trillion bases per run.

Noncontact dipole effects on channel permeation. I. Experiments with (5F-indole)Trp13 gramicidin A channels.

Gramicidin A (gA), with four Trp residues per monomer, has an increased conductance compared to its Phe replacement analogs. When the dipole moment of the Trp13 side chain is increased by fluorination at indole position 5 (FgA), the conductance is expected to increase further. gA and FgA conductances to Na+, K+, and H+ were measured in planar diphytanoylphosphatidylcholine (DPhPC) or glycerylmonoolein (GMO) bilayers. In DPhPC bilayers, Na+ and K+ conductances increased upon fluorination, whereas in GMO they decreased. The low ratio in the monoglyceride bilayer was not reversed in GMO-ether bilayers, solvent-inflated or -deflated bilayers, or variable fatty acid chain monoglyceride bilayers. In both GMO and DPhPC bilayers, fluorination decreased conductance to H+ but increased conductance in the mixed solution, 1 M KCl at pH 2.0, where K+ dominates conduction. Eadie-Hofstee plot slopes suggest similar destabilization of K+ binding in both lipids. Channel lifetimes were not affected by fluorination in either lipid. These observations indicate that fluorination does not change the rotameric conformation of the side chain. The expected difference in the rate-limiting step for transport through channels in the two bilayers qualitatively explains all of the above trends.

Analysis of the barley and rice genomes by comparative RFLP linkage mapping

Comparative genetic mapping of rice and barley, both major crop species with extensive genetic resources, offers the possibility of uniting two well-established and characterized genetic systems. In the present study, we screened 229 molecular markers and utilized 110 polymorphic orthologous loci to construct comparative maps of the rice and barley genomes. While extensive chromosomal rearrangements, including inversions and intrachromosomal translocations, differentiate the rice and barley genomes, several syntenous chromosomes are evident. Indeed, several chromosomes and chromosome arms appear to share nearly identical gene content and gene order. Seventeen regions of conserved organization were detected, spanning 287 cM (24%) and 321 cM (31%) of the rice and barley genomes, respectively. The results also indicate that most (72%) of the single-copy sequences in barley are also single copy in rice, suggesting that the large barley genome arose by unequal crossing over and amplification of repetitive DNA sequences and not by the duplication of single-copy sequences. Combining these results with those previously reported for comparative analyses of rice and wheat identified nine putatively syntenous chromosomes among barley, wheat and rice. The high degree of gene-order conservation as detected by comparative mapping has astonishing implications for interpreting genetic information among species and for elucidating chromosome evolution and speciation.

The genome of Chenopodium quinoa

Chenopodium quinoa (quinoa) is a highly nutritious grain identified as an important crop to improve world food security. Unfortunately, few resources are available to facilitate its genetic improvement. Here we report the assembly of a high-quality, chromosome-scale reference genome sequence for quinoa, which was produced using single-molecule real-time sequencing in combination with optical, chromosome-contact and genetic maps. We also report the sequencing of two diploids from the ancestral gene pools of quinoa, which enables the identification of sub-genomes in quinoa, and reduced-coverage genome sequences for 22 other samples of the allotetraploid goosefoot complex. The genome sequence facilitated the identification of the transcription factor likely to control the production of anti-nutritional triterpenoid saponins found in quinoa seeds, including a mutation that appears to cause alternative splicing and a premature stop codon in sweet quinoa strains. These genomic resources are an important first step towards the genetic improvement of quinoa.

SNP Discovery via Genomic Reduction, Barcoding, and 454-Pyrosequencing in Amaranth

The grain amaranths are important pseudo‐cereals native to the New World. During the last decade they have garnered increased international attention for their nutritional quality, tolerance to abiotic stress, and importance as a symbol of indigenous cultures. We report the development of a novel genomic reduction protocol based on restriction‐site conservation and multiplex identifiers (MID) barcodes to discover single nucleotide polymorphisms (SNPs) from a pooled amaranth library of four mapping parents. The incorporation of MID barcodes allowed for DNA sample pooling, sequence deconvolution, and the identification of SNPs for specific mapping populations without additional genotyping. Approximately 1.3 million sequence‐reads with an average read length of 440 bp were collected from a single 454‐pyrosequencing run. Contigs specific to each of the four mapping populations were assembled. The assembled contigs had an average read‐length of 464 bp, and an average read‐depth of 16X. A total of 27,658 SNPs were observed across all populations. The average base coverage at all SNPs was 20X. Thirty‐four of 35 (97%) predicted SNPs were verified via resequencing and the random genomic distribution of the SNPs generated using this approach was shown in Arabidopsis. The method does not require a priori sequence information and should be readily adaptable to other species.

Development, Characterization, and Linkage Mapping of Single Nucleotide Polymorphisms in the Grain Amaranths (Amaranthus sp.)

The grain amaranths (Amaranthus sp.) are important pseudo‐cereals native to the New World. During the last decade they have garnered increased international attention for their nutritional quality, tolerance to abiotic stress, and importance as a symbol of indigenous cultures. We describe the development of the first single nucleotide polymorphism (SNP) assays for amaranth. In addition, we report the characterization of the first complete genetic linkage map in the genus. The SNP assays are based on KASPar genotyping chemistry and were detected using the Fluidigm dynamic array platform. A diversity screen of 41 accessions of the cultivated amaranth species and their putative ancestor species (Amaranth hybridus L.) showed that the minor allele frequency (MAF) of these markers ranged from 0.05 to 0.5 with an average MAF of 0.27 per SNP locus. One hundred and forty‐one of the SNP loci were considered highly polymorphic (MAF ≥ 0.3). Linkage mapping placed all 411 markers into 16 linkage groups, presumably corresponding to each of the 16 amaranth haploid chromosomes. The map spans 1288 cM with an average marker density of 3.1 cM per marker. The work reported here represents the initial first steps toward the genetic dissection of agronomically important characteristics in amaranth.

SNP Discovery and Chromosome Anchoring Provide the First Physically-Anchored Hexaploid Oat Map and Reveal Synteny with Model Species

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.

Evolution of the NANOG pseudogene family in the human and chimpanzee genomes

BackgroundThe NANOG gene is expressed in mammalian embryonic stem cells where it maintains cellular pluripotency. An unusually large family of pseudogenes arose from it with one unprocessed and ten processed pseudogenes in the human genome. This article compares the NANOG gene and its pseudogenes in the human and chimpanzee genomes and derives an evolutionary history of this pseudogene family.ResultsThe NANOG gene and all pseudogenes except NANOGP8 are present at their expected orthologous chromosomal positions in the chimpanzee genome when compared to the human genome, indicating that their origins predate the human-chimpanzee divergence. Analysis of flanking DNA sequences demonstrates that NANOGP8 is absent from the chimpanzee genome.ConclusionBased on the most parsimonious ordering of inferred source-gene mutations, the deduced evolutionary origins for the NANOG pseudogene family in the human and chimpanzee genomes, in order of most ancient to most recent, are NANOGP6, NANOGP5, NANOGP3, NANOGP10, NANOGP2, NANOGP9, NANOGP7, NANOGP1, and NANOGP4. All of these pseudogenes were fixed in the genome of the human-chimpanzee common ancestor. NANOGP8 is the most recent pseudogene and it originated exclusively in the human lineage after the human-chimpanzee divergence. NANOGP1 is apparently an unprocessed pseudogene. Comparison of its sequence to the functional NANOG genes reading frame suggests that this apparent pseudogene remained functional after duplication and, therefore, was subject to selection-driven conservation of its reading frame, and that it may retain some functionality or that its loss of function may be evolutionarily recent.