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The glossy, green-fleshed fruit of the avocado (Persea americana) has been the object of human selection for thousands of years. Recent interest in healthy nutrition has singled out the avocado as an excellent source of several phytonutrients. Yet as a sizeable, slow-maturing tree crop, it has been largely neglected by genetic studies, owing to a long breeding cycle and costly field trials. We use a small, replicated experimental population of 50 progeny, grown at two locations in two successive years, to explore the feasibility of developing a dense genetic linkage map and to implement quantitative trait locus (QTL) analysis for seven phenotypic traits. Additionally, we test the utility of candidate-gene single-nucleotide polymorphisms developed to genes from biosynthetic pathways of phytonutrients beneficial to human health. The resulting linkage map consisted of 1346 markers (1044.7 cM) distributed across 12 linkage groups. Numerous markers on Linkage Group 10 were associated with a QTL for flowering type. One marker on Linkage Group 1 tracked a QTL for β-sitosterol content of the fruit. A region on Linkage Group 3 tracked vitamin E (α-tocopherol) content of the fruit, and several markers were stable across both locations and study years. We argue that the pursuit of linkage mapping and QTL analysis is worthwhile, even when population size is small.

The Hass cultivar of avocado is the most widely grown commercial cultivar in Mexico. Unfortunately, this cultivar is poorly adapted to the Mexican low-lands with hot dry climates characteristic of northwestern Mexico. Other well-adapted avocado accessions are available for these regions, but their nutritional traits and genetic diversity have yet to be explored. In this study, we analyze oil content, (α-tocopherol) and genetic variation among five local varieties from northwest Mexico that grow in high temperatures regimes in unfertilized soils and without any agronomic management. We report significant phenotypic variability in oil and α-tocopherol components in different accessions of avocado as determined by HPLC. Interestingly, we find higher α-tocopherol content (45.02–50.66 μg/g of fresh pulp) in some local avocados compared to Hass (32.28 μg/g of fresh pulp). The analyzed accessions represent a moderately polymorphic set of genotypes as measured by microsatellite (10 alleles by locus) and SNP (1 SNP every 164.4 bp) analysis of the VTE3 and VTE4 genes, implied in the biosynthesis of tocopherols. SNP data allowed also identifying differences between the local varieties and controls (Hass and the Mexican race accession). The variation observed at the genetic, morphologic and nutritional levels provide significant new information that may be valuable in selecting and developing avocado genotypes adapted to high-temperature environments.

Flower color is an important adaptive trait in many plant species because it determines reproductive success through differential attractiveness to insect pollinators. The genus Ipomoea is a pan tropically distributed plant genus characterized by showy flowers that often differ in color among closely related species. Flower color is determined primarily by products of the anthocyanin biosynthesis pathway. To determine whether flower color evolution among members of the genus is driven by adaptive molecular evolution of the anthocyanin pathway genes, we analyzed data from 10 genes sequenced from 19 species of Ipomoea. Six protein-coding genes from the anthocyanin pathway were drawn for evolutionary analysis in addition to three genes from the unrelated leucine biosynthesis pathway and one MADS box regulatory gene for comparison. The analyses provided: (i) no convincing evidence for positive selection on anthocyanin pathway structural genes, or on the other sampled genes, despite shifts in flower color among species included in the sample; (ii) pathway position correlated weakly with estimates of the intensity of evolutionary constraint on the anthocyanin pathway enzyme coding genes; and (iii) there was substantial gene-specific heterogeneity in the rates of synonymous site evolution. Synonymous rate heterogeneity does not appear to be accounted for by codon bias or local contextual or compositional sequence differences, leading us to implicate heterogeneous rates of mutation among genes as the most probable cause of synonymous rate heterogeneity.

Single-nucleotide polymorphism was used in the construction of an expressed sequence tag map of Aegilops tauschii , the diploid source of the wheat D genome. Comparisons of the map with the rice and sorghum genome sequences revealed 50 inversions and translocations; 2, 8, and 40 were assigned respectively to the rice, sorghum, and Ae. tauschii lineages, showing greatly accelerated genome evolution in the large Triticeae genomes. The reduction of the basic chromosome number from 12 to 7 in the Triticeae has taken place by a process during which an entire chromosome is inserted by its telomeres into a break in the centromeric region of another chromosome. The original centromere–telomere polarity of the chromosome arms is maintained in the new chromosome. An intrachromosomal telomere–telomere fusion resulting in a pericentric translocation of a chromosome segment or an entire arm accompanied or preceded the chromosome insertion in some instances. Insertional dysploidy has been recorded in three grass subfamilies and appears to be the dominant mechanism of basic chromosome number reduction in grasses. A total of 64% and 66% of Ae. tauschii genes were syntenic with sorghum and rice genes, respectively. Synteny was reduced in the vicinity of the termini of modern Ae. tauschii chromosomes but not in the vicinity of the ancient termini embedded in the Ae. tauschii chromosomes, suggesting that the dependence of synteny erosion on gene location along the centromere–telomere axis either evolved recently in the Triticeae phylogenetic lineage or its evolution was recently accelerated.

Hass' avocado (Persea americana Mill.) is characterized by excessive flower and fruit abscission, resulting in extremely low fruit set. Low outcrossing rates might be a factor contributing to low yields. It is hypothesized that self-fertilized flowers and resulting fruit abscise at a much higher rate than fruit that are the product of outcrossing. However, significant relationships between outcrossing rates and yields have only been established in a few avocado studies. The objective of this research was to investigate the importance of outcrossing to yield in a commercial 'Hass' orchard containing 'Bacon', an effective pollinizer of 'Hass'. Microsatellite markers were used to determine the rate of outcrossing of fruit persisting to harvest on 'Hass' trees. Experiments were conducted during sequential on- and off-crop years. During both years, outcrossing rates were not related to yield or alternate bearing. These results indicate that outcrossing was not the primary factor affecting flower and fruit persistence and ultimately yield in this orchard for the two sequential years of this research. Avocado and many other subtropical fruit tree crops are characterized by excessive flower and fruit abscission. Thou- sands of avocado flowers and immature fruit abscise in a single season (Cameron et al., 1952; Garner and Lovatt, 2008; Inoue and Takahashi, 1990; Lahav and Zamet, 1975; Slabbert, 1981), resulting in extremely low fruit set (

It has been difficult to infer the genetic history of avocado breeding, owing to the role of hybridization in the origin of contemporary avocado cultivars. To address this difficulty, we used the model-based clustering program, STRUCTURE, and nucleotide polymorphism in 5960 bp of sequence from 4 nuclear loci to examine population structure in 21 wild avocado accessions. The origins of 33 cultivars were inferred relative to the wild sample. Nucleotide sequence diversity in domesticated avocados ranged between 80% and 90% of that observed for the same loci in wild avocado, depending on the diversity statistic used for comparison. Substantial genetic differentiation among 3 geographic groups of wild germplasm corresponded to the classically defined horticultural races of avocado. Previously undetected genetic differentiation was revealed in wild populations from Central Mexico, where 2 subpopulations were distinguished based on elevation and latitude.

Resequencing studies provide the ultimate resolution of genetic diversity because they identify all mutations in a gene that are present within the sampled individuals. We report a resequencing study of Persea americana, a subtropical tree species native to Meso- and Central America and the progenitor of cultivated avocado. The sample includes 21 wild accessions from Mexico, Costa Rica, Ecuador, and the Dominican Republic. Estimated levels of nucleotide polymorphism and linkage disequilibrium (LD) are obtained from fully resolved haplotype data from 4 nuclear loci that span 5960 nucleotide sites. Results show that, although avocado is a subtropical tree crop and a predominantly outcrossing plant, the overall level of genetic variation is not exceptionally high (nucleotide diversity at silent sites, pi(sil) = 0.0102) compared with available estimates from temperate plant species. Intralocus LD decays rapidly to half the initial value within about 1 kb. Estimates of recombination rate (based on the sequence data) show that the rate is not exceptionally high when compared with annual plants such as wild barley or maize. Interlocus LD is significant owing to substantial population structure induced by mixing of the 3 botanical races of avocado.

ADDITIONAL INDEX WORDS. Persea americana, 'Gwen' progeny, broad-sense heritability, flower abundance, fruit set ABSTRACT. The authors report a quantitative genetic analysis of avocado (Persea americana Mill.) growth rate, flower abundance, and fruit set. The data are based on a total of 204 different genotypes of progeny of 'Gwen' avocado. Each was replicated four times, with two replicates planted in each of two locations in southern California (Irvine and Riverside). Data were collected over 4 years (consecutive) on tree height, canopy diameter, and trunk diameter, representing three distinct measures of growth rate. Growth data were found to fit a linear regression over years, so the slope (growth rate) was used in the analyses. In addition, 2 years of data on flower abundance and 1 year on fruit set were also collected. Quantitative genetic analyses of these data showed that broad-sense heritability (H) was 35.5%, 30.3%, and 26.6% for tree height, canopy diameter, and trunk diameter respectively; and 33.8% and 23.0% for flowering abundance and fruit set respectively. No genotype-by-location effect was noted for growth rate; however, flower abundance and fruit set showed a relatively weak genotype-by-location effect (21.9% and 17.1% respectively). The H estimates are low, probably as a result of sources of uncontrolled environmental error associated with variation in initial planting dates, but fall within the range that should permit quantitative trait locus analyses. The authors also found a moderate positive correlation between tree growth rates and fruit set, but none between growth rates andflower abundance. Different pollen parents havesignificantly different impacts on tree growth rates, flower abundance, and fruit set.

Motivation: Errors in nucleotide sequence and SNP genotyping data are problematic when inferring haplotypes. Previously published methods for error detection in haplotype data make use of pedigree information; however, for many samples, individuals are not related by pedigree. This article describes a method for detecting errors in haplotypes by considering the recombinational history implied by the patterns of variation, three SNPs at a time.Results: Coalescent simulations provide evidence that the method is robust to high levels of recombination as well as homologous gene conversion, indicating that patterns produced by both proximate and distant SNPs may be useful for detecting unlikely three-site haplotypes.Availability: The perl script implementing the described method is called EDUT (Error Detection Using Triplets) and is available on request from the authors.Contact: mclegg@uci.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Cereal agriculture originated with the domestication of barley and early forms of wheat in the Fertile Crescent. There has long been speculation that barley was domesticated more than once. We use differences in haplotype frequency among geographic regions at multiple loci to infer at least two domestications of barley; one within the Fertile Crescent and a second 1,500–3,000 km farther east. The Fertile Crescent domestication contributed the majority of diversity in European and American cultivars, whereas the second domestication contributed most of the diversity in barley from Central Asia to the Far East.

© 2015 Poets et al. Background: The genetic provenance of domesticated plants and the routes along which they were disseminated in prehistory have been a long-standing source of debate. Much of this debate has focused on identifying centers of origins for individual crops. However, many important crops show clear genetic signatures of multiple domestications, inconsistent with geographically circumscribed centers of origin. To better understand the genetic contributions of wild populations to domesticated barley, we compare single nucleotide polymorphism frequencies from 803 barley landraces to 277 accessions from wild populations. Results: We find that the genetic contribution of individual wild populations differs across the genome. Despite extensive human movement and admixture of barley landraces since domestication, individual landrace genomes indicate a pattern of shared ancestry with geographically proximate wild barley populations. This results in landraces with a mosaic of ancestry from multiple source populations rather than discrete centers of origin. We rule out recent introgression, suggesting that these contributions are ancient. The over-representation in landraces of genomic segments from local wild populations suggests that wild populations contributed locally adaptive variation to primitive varieties. Conclusions: This study increases our understanding of the evolutionary process associated with the transition from wild to domesticated barley. Our findings indicate that cultivated barley is comprised of multiple source populations with unequal contributions traceable across the genome. We detect putative adaptive variants and identify the wild progenitor conferring those variants.

High levels of inbreeding cause populations to become composed of homozygous, inbred lines. High levels of homozygosity limit the effectiveness of recombination, and therefore, retard the rate of decay of linkage (gametic phase) disequilibrium (LD) among mutations. Inbreeding and recombination interact to shape the expected pattern of LD. The actual extent of nucleotide sequence level LD within inbreeding species has only been studied in Arabidopsis , a weedy species whose global range has recently expanded. In the present study, we examine the levels of LD within and between 18 nuclear genes in 25 accessions from across the geographic range of wild barley, a species with a selfing rate of ≈98%. In addition to examination of intralocus LD, we employ a resampling method to determine whether interlocus LD exceeds expectations. We demonstrate that, for the majority of wild barley loci, intralocus LD decays rapidly, i.e., at a rate similar to that observed in the outcrossing species, Zea mays (maize). Excess interlocus LD is observed at 15% of two-locus combinations; almost all interlocus LD involves loci with significant geographic structuring of mutational variation.

This paper compares the development of microsatellite markers from two genomic DNA libraries of avocado cultivar Hass enriched for dinucleotide and trinucleotide repeats. Sequencing showed 86 and 31% of clones from the respective libraries to contain microsatellites. However, banding profiles of trinucleotide loci were easier to interpret than those of dinucleotide loci. Of 376 dinucleotide-containing clones, 81% harbored AG repeats and 19% contained AC repeats. A subset of 104 trinucleotide-containing clones consisted of ATG repeats (44%), AGT repeats (30%), and AAG repeats (16%). Array lengths of up to 34 were attained in the dinucleotide repeat microsatellites, whereas trinucleotide arrays never exceeded 11 elements. Typing 37 genotypes at 25 loci (11 dinucleotide and 14 trinucleotide loci) revealed a total of 204 alleles, of which 60% originated from the dinucleotide loci. Average heterozygosity of the di- and trinucleotide loci was 73.4 and 52.6%, respectively. Many loci, especially dinucleotide loci, exhibited allele size differences that were not multiples of the repeat unit, suggesting nonstepwise mutational mechanisms. Several loci were additionally characterized by large gaps in the allele size distribution.

The molecular evolution of the R2R3-MYB gene family is of great interest because it is one of the most important transcription factor gene families in the plant kingdom. Comparative analyses of a gene family may reveal important adaptive changes at the protein level and thereby provide insights that relate structure to function. We have performed a range of comparative and bioinformatics analyses on R2R3-MYB genes identified from the rice (Oryza sativa subsp. japonica and indica) and Arabidopsis genome sequences. The study provides an initial framework to investigate how different evolutionary lineages in a gene family evolve new functions. Our results reveal a remarkable excess of non-synonymous substitutions, an indication of adaptive selection on protein structure that occurred during the evolution of both helix1 and helix2 of rice R2R3-MYB DNA-binding domains. These flexible α-helix regions associated with high frequencies of excess non-synonymous substitutions may play critical roles in the characteristic packing of R2R3-MYB DNA-binding domains and thereby modify the protein-DNA interaction process resulting in the recognition of novel DNA-binding sites. Furthermore, a co-evolutionary pattern is found between the second α-helix of the R2 domain and the second α-helix of the R3 domain by examining all the possible α-helix pairings in both the R2 and R3 domains. This points to the functional importance of pairing interactions between related secondary structures.

Clegg tackles US-Cuba scientific collaboration. The US National Academy of Sciences runs workshops with scientists from Iran, and both governments seem to value this neutral channel of engagement. Cuba has strong capabilities in fields including biotechnology, oceanography, biodiversity, public health and disaster management. Its education system achieved nearly 100% literacy by the 1970s and its higher education and research sectors are impressive for a nation of just over 11 million. Obama had technology-based entrepreneurism and expanded access to the Internet on his wish list for Cuba, along with the chance to work with Cuban biotech efforts, in particular trials for new vaccines for lung cancer and other human and animal diseases.

Analyses of whole-genome data often reveal that some genes have evolutionary histories that diverge from the majority phylogeny estimated for the entire genome. We present a probabilistic model that deals with heterogeneity among gene trees, implement it via the Gibbs sampler, and apply it to the plastid genome. Plastids and their genomes are transmitted as a single block without recombination, hence homogeneity among gene trees within this genome is expected. Nevertheless, previous work has revealed clear heterogeneity among plastid genes (e.g., Delwiche and Palmer 1996). Other studies, using whole plastid genomes of various algae and land plants, found little additional heterogeneity (Martin et al. 1998; Adachi et al. 2000). We augment the earlier studies by using a data set of 14 taxa: 6 land plants, 2 green algae, a diatom, 2 red algae and a cryptophyte, the cyanelle of the glaucocystophyte Cyanophora, and the blue-green alga Synechocystis as an outgroup. Contrary to the earlier analyses, we cannot find even a single, dominant consensus tree. Therefore, we formulate a probabilistic model that divides the genes into two sets: those that follow the consensus tree and those that have independent gene trees. No particular tree is supported by more than three-fourths of the genes. But the set of genes that follows a certain tree is fairly independent of data processing and the method of analysis. With one possible exception, we find no evidence for collinear or functionally related genes to follow similar trees. The phylogenetic pattern also seems independent of bias in amino acid composition. Among possible explanations for the observed phenomenon, the hypothesis that different genes have different covarion structures is difficult to assess. But gene duplication may be possible through the inverted or direct repeat regions, while horizontal gene transfer seems less likely. In contrast to green algae and land plants, inverted repeat regions in red algae and in Cyanophora show abundant differences among the copies. Thus, genes may get duplicated when they are recruited into the inverted repeat region and one of the two copies may be lost after leaving the inverted repeat region.

It has been suggested that evolutionary changes in regulatory genes may be the predominant molecular mechanism governing both physiological and morphological evolution. R2R3-AtMYB is one of the largest transcription factor gene families in Arabidopsis. Using inferred ancestral sequences we show that several lineages in the R2R3-AtMYB phylogeny experienced excess non-synonymous nucleotide substitution upon gene duplication, indicating episodes of positive selection driving adaptive shifts early in the evolution of this gene family. A noise reduction technique was then used to determine individual sites in DNA-binding domains (R2 domain and R3 domain) of R2R3-AtMYB protein sequence that were favored by frequent non-synonymous substitutions. The analyses reveal that the first helix (helix1) and the second helix (helix2) in both R2 and R3 domains are characterized by more frequent non-synonymous substitutions, and thus experienced significantly higher positive selection pressure than the third helix (helix3) in both domains. Previous MYB protein structure studies have suggested that helix1 and helix2 in both R2 and R3 domains are involved in the characteristic packing of R2R3-AtMYB DNA-binding domains. This suggests that excess non-synonymous substitutions in these helices could have resulted in MYB recognition of novel gene target sites.

Patterns of nucleotide sequence diversity are analyzed for three duplicate alcohol dehydrogenase loci (adh1-adh3) within a species-wide sample of 25 accessions of wild barley (Hordeum vulgare ssp. spontaneum). The adh1 and adh2 loci are tightly linked (recombination fraction

Genetic differentiation in natural populations is driven by geographic distance and by ecological or physical features within and between natural habitats that reduce migration. The primary population structure in wild barley differentiates populations east and west of the Zagros Mountains. Genetic differentiation between eastern and western populations is uneven across the genome and is greatest on linkage groups 2H and 5H. Genetic markers in these two regions demonstrate the largest difference in frequency between the primary populations and have the highest informativeness for assignment to each population. Previous cytological and genetic studies suggest there are chromosomal structural rearrangements (inversions or translocations) in these genomic regions. Environmental association analyses identified an association with both temperature and precipitation variables on 2H and with precipitation variables on 5H.

Patterns of nucleotide sequence diversity in the predominantly self-fertilizing species Hordeum vulgare subspecies spontaneum (wild barley) are compared between the putative alcohol dehydrogenase 3 locus (denoted “ adh3 ”) and alcohol dehydrogenase 1 ( adh1 ), two related but unlinked loci. The data consist of a sequence sample of 1,873 bp of “ adh3 ” drawn from 25 accessions that span the species range. There were 104 polymorphic sites in the sequenced region of “ adh3 .” The data reveal a strong geographic pattern of diversity at “ adh3 ” despite geographic uniformity at adh1 . Moreover, levels of nucleotide sequence diversity differ by nearly an order of magnitude between the two loci. Genealogical analysis resolved two distinct clusters of “ adh3 ” alleles (dimorphic sequence types) that coalesce roughly 3 million years ago. One type consists of accessions from the Middle East, and the other consists of accessions predominantly from the Near East. The two “ adh3 ” sequence types are characterized by a high level of differentiation between clusters (≈2.2%), which induces an overall excess of intermediate frequency variants in the pooled sample. Finally, there is evidence of intralocus recombination in the “ adh3 ” data, despite the high level of self-fertilization characteristic of wild barley.

The levels of diversity and extent of linkage disequilibrium in cultivated species are largely determined by diversity in their wild progenitors. We report a comparison of nucleotide sequence diversity in wild and cultivated barley (Hordeum vulgare ssp. spontaneum and ssp. vulgare) at 7 nuclear loci totaling 9296bp, using sequence from Hordeum bulbosum to infer the ancestral state of mutations. The sample includes 36 accessions of cultivated barley, including 23 landraces (cultivated forms not subject to modern breeding) and 13 cultivated lines and genetic stocks compared to either 25 or 45 accessions of wild barley for the same loci. Estimates of nucleotide sequence diversity indicate that landraces retain >80% of the diversity in wild barley. The primary population structure in wild barley, which divides the species into eastern and western populations, is reflected in significant differentiation at all loci in wild accessions and at 3 of 7 loci in landraces. “Oriental” landraces have slightly higher diversity than “Occidental” landraces. Genetic assignment suggests more admixture from Occidental landraces into Oriental landraces than the converse, which may explain this difference. Based on θπ for silent sites, modern western cultivars have ~73% of the diversity found in landraces and ~71% of the diversity in wild barley.

We review the study of flower color polymorphisms in the morning glory as a model for the analysis of adaptation. The pathway involved in the determination of flower color phenotype is traced from the molecular and genetic levels to the phenotypic level. Many of the genes that determine the enzymatic components of flavonoid biosynthesis are redundant, but, despite this complexity, it is possible to associate discrete floral phenotypes with individual genes. An important finding is that almost all of the mutations that determine phenotypic differences are the result of transposon insertions. Thus, the flower color diversity seized on by early human domesticators of this plant is a consequence of the rich variety of mobile elements that reside in the morning glory genome. We then consider a long history of research aimed at uncovering the ecological fate of these various flower phenotypes in the southeastern U.S. A large body of work has shown that insect pollinators discriminate against white phenotypes when white flowers are rare in populations. Because the plant is self-compatible, pollinator bias causes an increase in self-fertilization in white maternal plants, which should lead to an increase in the frequency of white genes, according to modifier gene theory. Studies of geographical distributions indicate other, as yet undiscovered, disadvantages associated with the white phenotype. The ultimate goal of connecting ecology to molecular genetics through the medium of phenotype is yet to be attained, but this approach may represent a model for analyzing the translation between these two levels of biological organization.

Plant genomes appear to exploit the process of gene duplication as a primary means of acquiring biochemical and developmental flexibility. Thus, for example, most of the enzymatic components of plant secondary metabolism are encoded by small families of genes that originated through duplication over evolutionary time. The dynamics of gene family evolution are well illustrated by the genes that encode chalcone synthase (CHS), the first committed step in flavonoid biosynthesis. We review pertinent facts about CHS evolution in flowering plants with special reference to the morning glory genus, Ipomoea. Our review shows that new CHS genes are recruited recurrently in flowering plant evolution. Rates of nucleotide substitution are frequently accelerated in new duplicate genes, and there is clear evidence for repeated shifts in enzymatic function among duplicate copies of CHS genes. In addition, we present new data on expression patterns of CHS genes as a function of tissue and developmental stage in the common morning glory (I. purpurea). These data show extensive differentiation in gene expression among duplicate copies of CHS genes. We also show that a single mutation which blocks anthocyanin biosynthesis in the floral limb is correlated with a loss of expression of one of the six duplicate CHS genes present in the morning glory genome. This suggests that different duplicate copies of CHS have acquired specialized functional roles over the course of evolution. We conclude that recurrent gene duplication and subsequent differentiation is a major adaptive strategy in plant genome evolution.

Lysyl oxidase (EC 1.4.3.13), a cuproenzyme, can account for 10-30% of the copper present in connective tissue. Herein, we assess the extent to which tissue copper concentrations and lysyl oxidase activity are related because the functional activity of lysyl oxidase and the copper content of chick tendon are both related to dietary copper intake. Chicks (1-d old) were fed diets (basal copper concentration, 0.4 microg/g diet) to which copper was added from 0 to 16 microg/g diet. Liver and plasma copper levels tended to normalize in chickens that consumed from 1 to 4 microg copper/g of diet, whereas tendon copper concentrations suggested an unusual accumulation of copper in chickens that consumed 16 microg copper/g diet. The molecular weight of lysyl oxidase was also estimated using matrix-assisted laser desorption ionization/time-of-flight/mass spectrometry (MALDI/TOF/MS). A novel aspect of these measurements was estimation of protein mass directly from the surface of chick tendons and aortae. Whether copper deficiency (0 added copper) or copper supplementation (16 microg copper/g of diet) caused changes in the molecular weight of protein(s) in tendon corresponding to lysyl oxidase was addressed. The average molecular weight of the peak corresponding to lysyl oxidase in tendon and aorta from copper-deficient birds was 28,386 Da +/- 86, whereas the average molecular weight of corresponding protein in tendon from copper-supplemented birds was 28,639 Da +/- 122. We propose that the shift in molecular weight is due in part to copper binding and the formation of lysyl tyrosyl quinone, the cofactor at the active site of lysyl oxidase.

The background selection hypothesis predicts a reduction in nucleotide site diversity and an excess of rare variants, owing to linkage associations with deleterious alleles. This effect is expected to be amplified in species that are predominantly self-fertilizing. To examine the predictions of the background selection hypothesis in self-fertilizing species, we sequenced 1,362 bp of adh1 , a gene for alcohol dehydrogenase (Adh; alcohol:NAD + oxidoreductase, EC 1.1.1.1 ), in a sample of 45 accessions of wild barley, Hordeum vulgare ssp. spontaneum , drawn from throughout the species range. The region sequenced included 786 bp of exon sequence (part of exon 4, all of exons 5–9, and part of exon 10) and 576 bp of intron sequence (all of introns 4–9). There were 19 sites polymorphic for nucleotide substitutions, 8 in introns, and 11 in exons. Of the 11 nucleotide substitutions in codons, 4 were synonymous and 7 were nonsynonymous, occurring uniquely in the sample. There was no evidence of recombination in the region studied, and the estimated effective population size ( N̂ e ) based on synonymous sites was ≈1.8–4.2 × 10 5 . Several tests reveal that the pattern of nonsynonymous substitutions departs significantly from neutral expectations. However, the data do not appear to be consistent with recovery from a population bottleneck, recent population expansion, selective sweep, or strong positive selection. Though several features of the data are consistent with background selection, the distributions of polymorphic synonymous and intron sites are not perturbed toward a significant excess of rare alleles as would be predicted by background selection.

Chalcone synthase (CHS) is a small multigene family with at least four members (CHS-A, B, C and PS) in common morning glory Ipomoea purpurea ROTH. The chalcone synthase enzyme performs the initial condensation reaction that results in the 15-carbon three-ring structure that is the backbone of flavonoid biosynthesis. The biochemical pathway that commences with CHS is important in plant disease defence, pigment biosynthesis and UV protection. Accordingly, it is of substantial interest to characterize levels and patterns of molecular diversity for genes that encode this important enzyme. We report the sequence of 19 CHS-A alleles from Mexican and American populations of common morning glory. American populations of this annual self-compatible vine are believed to have been introduced from Mexico, where the species is native. Individual plants were sampled from populations of common morning glory throughout Mexico and the south-eastern USA. Four American alleles were sequenced and these, together with one allele from Mexico City, were identical in primary nucleotide sequence. These data suggest a restricted origin for the American population, probably as a consequence of selection for domestication by pre-Columbian peoples. Additionally the Mitontic (Chiapas, Mexico) population is significantly more homogeneous than expected by chance indicating that this population may also have experienced a recent population bottleneck. Estimates of nucleotide diversity from the Mexican CHS-A alleles were high. We present evidence that these estimates may, in part, result from low to moderate levels of interlocus recombination/gene conversion. We also present evidence that the ancient duplication of the CHS gene family, preceding the origin of the genus Ipomoea, was associated with heterogeneity in the rate of substitution between the resulting gene family members. The group of gene family members whose sequences possess a signature amino acid of the closely related Stilbene synthase exhibit a significantly faster proportional rate of nonsynonymous substitution.

Ipomoea purpurea (common morning glory) is an annual vine native to Mexico that is well known for its large, showy flowers. Humans have spread morning glories worldwide, owing to the horticultural appeal of morning glory flowers. Ipomoea purpurea is an opportunistic colonizer of disturbed habitats including roadside and agricultural settings, and it is now regarded as a noxious weed in the Southeastern US. Naturalized populations in the Southeastern United States are highly polymorphic for a number of flower color morphs, unlike native Mexican populations that are typically monomorphic for the purple color morph. Although I. purpurea was introduced into the United States from Mexico, little is known about the specific geographic origins of US populations relative to the Mexican source. We use resequencing data from 11 loci and 30 I. purpurea accessions collected from the native range of the species in Central and Southern Mexico and 8 accessions from the Southeastern United States to infer likely geographic origins in Mexico. Based on genetic assignment analysis, haplotype composition, and the degree of shared polymorphism, I. purpurea samples from the Southeastern United States are genetically most similar to samples from the Valley of Mexico and Veracruz State. This supports earlier speculation that I. purpurea in the Southeastern United States was likely to have been introduced by European colonists from sources in Central Mexico. © The American Genetic Association. 2013. All rights reserved.

A number of studies have noted that nucleotide substitution rates at the chloroplast-encoded rbcL locus violate the molecular clock principle. Substitution rate variation at this plastid gene is particularly pronounced between palms and grasses; for example, a previous study estimated that substitution rates in rbcL sequences are approximately 5-fold faster in grasses than in palms. To determine whether a proportionate change in substitution rates also occurs in plant nuclear genes, we characterized nucleotide substitution rates in palm and grass sequences for the nuclear gene Adh. In this article, we report that palm sequences evolve at a rate of 2.61 x 10(-9) substitution per synonymous site per year, a rate which is slower than most plant nuclear genes. Grass Adh sequences evolve approximately 2.5-fold faster than palms at synonymous sites. Thus, synonymous rates in nuclear Adh genes show a marked decrease in palms relative to grasses, paralleling the pattern found at the plastid rbcL locus. This shared pattern indicates that synonymous rates are correlated between a nuclear and a plastid gene. Remarkably, nonsynonymous rates do not show this correlation. Nonsynonymous rates vary between two duplicated grass Adh loci, and nonsynonymous rates at the palm Adh locus are not markedly reduced relative to grasses.

Populations of the common morning glory in the southeastern US are characterized by a striking diversity of flower color polymorphisms. This diversity is probably a consequence of horticultural escapes from cultivation in the 18th and 19th centuries. More than 15 years of research in our laboratory has shown that some color phenotypes are selected by virtue of their differential attraction to insect pollinators. We have studied genetic diversity at isozyme and ribosomal DNA loci and we find reduced diversity in the southeastern US compared to Mexican populations. In an effort to link ecological genetics to molecular evolution, we have cloned and characterized the chalcone synthase (CHS) gene family in morning glory and we have studied the expression of CHS genes in flower development. We have also initiated an investigation of spatial patterns of diversity at CHS genes by sampling and sequencing genes from US and Mexican populations. These investigations reveal (1) that the four CHS genes (CHS A, B, C, and PS) characterized to date evolve rapidly in morning glory and that the gene family in Ipomoea is of relatively recent origin (approximately 21 million years); (2) the duplicate genes in Ipomoea group into two categories (CHS A, C versus CHS B, PS) that may indicate a functional divergence between chalcone synthase and stilbene synthase activities; (3) levels of molecular diversity for CHS A genes sampled from Mexico are much higher than observed in US collections suggesting a major population bottleneck associated with the introduction of morning glory into the southeastern US; and (4) the ratio of amino acid substitution to synonymous substitution between Ipomoea species is remarkably high (about 5.4 synonymous to amino acid substitutions) compared to CHS genes in other plant species. Taken together these data portray a rapidly evolving gene family where functional divergence may arise repeatedly in evolution, despite the central role of chalcone synthase in flavonoid metabolism.

The present diversity of species is the result of a very long and slow process of genetic change and adaptation. The time necessary for the emergence of new species, and even for the accumulation of genetic variants at individual gene loci within species, greatly exceeds the time since the emergence of Homo sapiens. New techniques of molecular biology combined with recent theories in population genetics allow us to assess the time dimension of genetic change; these suggest that some genetic polymorphisms may have originated over a million generations ago. In other words, once lost, any particular genetic adaptation cannot be regained in any realistic time interval. We depend on biological systems for food, fiber, energy and medicinal needs. Continued advances in each of these areas may be compromised by losses of the biological resources — that is the genetic variants — that provide the raw material for innovation.

The common morning glory (Ipomoea purpurea) is an annual vine native to Central and Southern Mexico. The genetics of flower color polymorphisms and interactions with the biotic environment have been extensively studied in I. purpurea and in its sister species I. nil. In this study, we examine nucleotide sequence polymorphism in 11 loci, 9 of which are known to participate in a pathway that produces floral pigments. A sample of 30 I. purpurea accessions from the native range of Central and Southern Mexico comprise the data, along with one accession from each of the two sister species I. alba and I. nil. We observe moderate levels of nucleotide sequence polymorphism of ~1%. The ratio of recombination to mutation parameter estimates (ρ/θ) of ~2.5 appears consistent with a mixed-mating system. Ipomoea resequencing data from these genic regions are noteworthy in providing a good fit to the standard neutral model of molecular evolution. The derived silent site frequency spectrum is very close to that predicted by coalescent simulations of a drift-mutation process, and Tajima's D values are not significantly different from expectations under neutrality.

La naturaleza y los límites de la familia Agavaceae han sido objeto de controversias desde que se propuso en el siglo pasado. En este artículo analizamos las secuencias del gen rbcL del cloroplasto de 9 especies de Agavaceae sensu Hutchinson ( 1934, 1959) junto con 44 secuencias adicionales,. incluyendo 36 secuencias de Lilianae y como grupos externos 8 secuencias de Alismatanae y Arecanae. Presentamos análisis de parsimonia, neighbor-joining (unión de vecinos) y de máxima verosimilitud. Identificamos 3 linajes evolutivos principales en las Agavaceae como fueron definidas por Hutchinson. Estos linajes contienen a la mayoría de las Asparagales tal como fueron definidas por Dahlgren et al. ( 1985) (i) Asparagus (Asparagaceae), Dianella (Phormiaceae), Hemerocallis (Hemerocallidaceae) y las Asphodelacea (Aloe, Haworthia and Kniphofla); (ii) Agavaceae sensu stricto, incluyendo Agave, Manfreda, Beschorneria, Hespera/oe and Yucca, al género asiático Hasta (Funkiaceae) y a la hierba pantropical Chlorophylulll (Anthericaceae); (iii) Sansevieria (Dracaenaceae) , Danae (Ruscaceae) y las Nolinaceae (Nolina, Beaucarnea y Dasylirion). De acuerdo a nuestros análisis otros grupos relacionados a las Agavaceae sensu Hutchinson son: Bowiea (Hyacinthaceae), ledebouria (= Scilla. Hyacinthaceae), Clivia (Amaryllidaceae), Lomandra (Dasypogonaceae). Xanthorrhoea (Xanthorrhoeaceae), Cvanastrum (Cyanastraceae) y las lridaceae (iris. Orthosanthus and Anomatheca). Concluimos que las .Agavaceae, tal como fueron propuestas por Hutchinson ( 1934, 1959) no son un grupo monofílético, aunque la mayoría de sus especies forman el núcleo de un ciado más grande que aproximadamente corresponde (con la adición de las Iridaceae) a las Asparagales tal como fueron propuestas por Dahlgren et al. ( 1985).

The chloroplast genome (cpDNA) of plants has been a focus of research in plant molecular evolution and systematics. Several features of this genome have facilitated molecular evolutionary analyses. First, the genome is small and constitutes an abundant component of cellular DNA. Second, the chloroplast genome has been extensively characterized at the molecular level providing the basic information to support comparative evolutionary research. And third, rates of nucleotide substitution are relatively slow and therefore provide the appropriate window of resolution to study plant phylogeny at deep levels of evolution. Despite a conservative rate of evolution and a relatively stable gene content, comparative molecular analyses reveal complex patterns of mutational changes. Non-coding regions of cpDNA diverge through insertion/deletion changes that are sometimes site dependent. Coding genes exhibit different patterns of codon bias that appear to violate the equilibrium assumptions of some evolutionary models. Rates of molecular change often vary among plant families and orders in a manner that violates the assumption of a simple molecular clock. Finally, protein-coding genes exhibit patterns of amino acid change that appear to depend on protein structure, and these patterns may reveal subtle aspects of structure/function relationships. Only comparative studies of molecular sequences have the resolution to reveal this underlying complexity. A complete description of the complexity of molecular change is essential to a full understanding of the mechanisms of evolutionary change and in the formulation of realistic models of mutational processes.

Resolution of the two haplotypes present in an individual that is heterozygous at a locus has been a difficult problem for nucleotide sequence-based population genetic studies. Here, we demonstrate a method in which allele-specific polymerase chain reaction (AS-PCR) and computational phasing are combined for relatively high-throughput, efficient resolution of phase in resequencing studies. Using data from multiple loci that were fully experimentally phased, we demonstrate that the popular computational tool PHASE can accurately phase heterozygous individuals with common SNPs (single nucleotide polymorphisms) and/or common haplotypes. However, we also demonstrate that experimental phasing with AS-PCR can efficiently supplement computational phasing, providing a rapid means to phase individuals with rare SNPs or haplotypes and with heterozygous insertion/deletion polymorphisms. By following simple stepwise procedures, AS-PCR can result in much more efficient and accurate experimental phasing of haplotypes than is possible with traditional methods such as cloning.

The noncoding DNA region of the chloroplast genome, flanked by the genes rbcL and psaI (ORF36), has been sequenced for seven species of the grass family (Poaceae). This region had previously been observed as a hotspot area for length mutations. Sequence comparison reveals that short duplications, probably resulting from slipped-strand mispairing, account for many small length differences between sequences but that major mutational hotspots are localized in three small areas, two of which show potential secondary structure. Mutation in one of these hotspots appears to be a result of more complex recombination events. All seven species contain a pseudogene for rpl23 and evidence is presented that this pseudogene is being maintained by gene conversion with the functional gene. Different transition/transversion biases and AT contents between the pseudogene and the surrounding noncoding sequences are noted. In the subfamily Panicoideae there is a deletion in which almost 1 kb of ancestral sequence, including the 3' end of the rpl23 pseudogene, has been replaced by a non-homologous 60-base sequence of unknown origin. Two other deletions of almost the same region have occurred in the grass family. The deleted noncoding region has mutational and compositional properties similar to the rbcL coding sequence and the rpl23 pseudogene. The three independent deletions, as well as the pattern of mutation in the localized hotspots, indicate that such noncoding DNA may be misleading for studies of phylogenetic inference.

The rapid development of molecular methods during the 1980s has had a profound effect on the study of plant evolution. Molecular data have accumulated very rapidly, and this abundance of data poses new problems for data analysis. We illustrate three problem areas that arise in plant evolutionary inference. The first problem concerns the use of molecular data to analyse closely related plant species linked through reticulate evolution. The second problem arises from our ability to sample DNA sequences from alleles of a single genetic locus within plant species. We show how cumulative information on selection and random genetic drift can be extracted from such data. The third problem area concerns the limitations of current algorithms for phylogenetic inference when confronted with large sets of DNA sequence data. Based on a consideration of these problem areas, we conclude that: (1) asymmetric transmission of cpDNA markers is useful in resolving the parentage of hybrid plant taxa; (2) simple clustering algorithms can provide useful information on cultivar or variety relation- ships, despite intervarietal hybridisation, if genetic similarities are averaged over sufficient loci; (3) samples of complete DNA sequence data from plant nuclear genes can provide a new dimension of information on historical effective population sizes and on the mechanisms that generate allelic diversity; (4) analyses of the chloroplast gene rbcL, sampled from across the monocotyledon class, reveal large variation in relative rates of nucleotide substitution—these variations, in turn, have im- portant consequences for phylogeny estimation algorithms; and (5) the combined use of algorithms like parsimony and maximum likelihood may represent a more efficient approach to the phylo- genetic analysis of very large (>100) sets of DNA sequences.

The relative rate of occurrence of nucleotide substitutions versus indel (insertion/deletion) events is investigated by comparing complete DNA sequence data from the noncoding portion of the chloroplast genome that maps between the genes rbcL and atp beta. The sequence data are obtained from nine species that represent three tribes of the grass family. Indels could be categorized by those that are deletions or duplications of adjacent or proximal sequences and those that do not appear to be permutations of adjacent sequences. The first category represents 82% of the recorded indels. These indels may also be characterized by being direct duplications of one to several bases usually within runs of As or Ts or by being duplications or deletions of more complex sequences. When viewed from within groups of closely related taxa, indel events appear to occur at an equal or slightly faster rate than do nucleotide substitution events. However, the apparent rate of accumulation of indels in more distantly related species is significantly slower than that of nucleotide substitutions. This difference in apparent accumulation rates between indel events and nucleotide substitutions suggests that the proportion of superimposed changes has been higher among all indel events than among all nucleotide substitution events. Indeed the indels involving more complex sequences were found to be confined across taxa to a number of highly labile sites. Independent, though similar, indel events occur at identical sites in unrelated taxa, yet may not be shared among related taxa, resulting in a type of molecular parallelism. As a result, the phylogenetic tree based on indel events represents an evolutionary hypothesis which is inconsistent with the accepted phylogeny of these grasses. The phylogenetic tree based on nucleotide substitutions is consistent with accepted phylogeny.

A large body of sequence data has accumulated for the chloroplast-encoded gene ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) as the result of a cooperative effort involving many laboratories. The data span all seed plants, including most major lineages from the angiosperms, and as such they provide an unprecedented opportunity to study plant evolutionary history. The full analysis of this large data set poses many problems and opportunities for plant evolutionary biologists and for biostatisticians.

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

We present data on evolution of the Ac/Ds family of transposable elements in select grasses (Poaceae). An Ac-like element was cloned from a DNA library of the grass Pennisetum glaucum (pearl millet) and 2387 bp of it have been sequenced. When the pearl millet Ac-like sequence is aligned with the corresponding region of the maize Ac sequence, it is found that all sequences corresponding to intron II in maize Ac are absent in pearl millet Ac. Kimura's evolutionary distance between maize and pearl millet Ac sequences is estimated to be 0.429 +/- 0.020 nucleotide substitutions per site. This value is not significantly different from the average number of synonymous substitutions for coding regions of the Adh1 gene between maize and pearl millet, which is 0.395 +/- 0.051 nucleotide substitutions per site. If we can assume Ac and Adh1 divergence times are equivalent between maize and pearl millet, then the above calculations suggest Ac-like sequences have probably not been strongly constrained by natural selection. The level of DNA sequence divergence between maize and pearl millet Ac sequences, the estimated date when maize and pearl millet diverged (25-40 million years ago), coupled with their reproductive isolation/lack of current genetic exchange, all support the theory that Ac-like sequences have not been recently introduced into pearl millet from maize. Instead, Ac-like sequences were probably present in the progenitor of maize and pearl millet, and have thus existed in the grasses for at least 25 million years. Ac-like sequences may be widely distributed among the grasses. We also present the first 2 Ds1 controlling element sequences from teosinte species: Zea luxurians and Zea perennis. A total of 10 Ds1 elements had previously been sequenced from maize and a distant maize relative, Tripsacum. When a maximum likelihood network of genetic relationships is constructed for all 12 sequenced Ds1 elements, the 2 teosinte Ds1 elements are as distant from most maize Ds1 elements and from each other, as the maize Ds1 elements are from one another. Our new teosinte sequence data support the previous conclusion that Ds1 elements have been accumulating mutations independently since maize and Tripsacum diverged. We present a scenario for the origin of Ds1 elements.

The molecular evolution of the alcohol dehydrogenase 1 (Adh1; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) locus in members of the grass family is analyzed. We report the complete DNA sequence of a genomic clone of Adh1 from Pennisetum glaucum cv. Tift 23DB (pearl millet). The gene is characterized by ten exons and nine introns. The 5' flanking region of the gene contains sequences corresponding to the anaerobic regulatory element as well as sequences corresponding to the TATA box and the CAAT box identified in the maize Adh1-1S flanking regions. Exon sequences from Pennisetum and maize have been subjected to relative rate tests; the maize and Pennisetum Adh1 lineages evolve at equal rates. These results are compared with similar relative rate studies by using the chloroplast DNA encoding the ribulose-1,5-bisphosphate carboxylase (rbcL) gene. Evolutionary rates of Adh1 are estimated. Nonsynonymous rates are found to be 2.50 x 10(-10) substitutions per site per year, whereas synonymous rates are approximately 7.90 x 10(-9) substitutions per site per year. Molecular phylogenies of the Poaceae based upon Adh1 data are presented.