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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.

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.

© 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.

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.

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

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.

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.

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.

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.

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.

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.

The avocado (Persea americana) is a major crop commodity worldwide. Moreover, avocado, a paleopolyploid, is an evolutionary "outpost" among flowering plants, representing a basal lineage (the magnoliid clade) near the origin of the flowering plants themselves. Following centuries of selective breeding, avocado germplasm has been characterized at the level of microsatellite and RFLP markers. Nonetheless, little is known beyond these general diversity estimates, and much work remains to be done to develop avocado as a major subtropical-zone crop. Among the goals of avocado improvement are to develop varieties with fruit that will "store" better on the tree, show uniform ripening and have better post-harvest storage. Avocado transcriptome sequencing, genome mapping and partial genomic sequencing will represent a major step toward the goal of sequencing the entire avocado genome, which is expected to aid in improving avocado varieties and production, as well as understanding the evolution of flowers from non-flowering seed plants (gymnosperms). Additionally, continued evolutionary and other comparative studies of flower and fruit development in different avocado strains can be accomplished at the gene expression level, including in comparison with avocado relatives, and these should provide important insights into the genetic regulation of fruit development in basal angiosperms.

A central goal of evolutionary genetics is to trace the causal pathway between mutations at particular genes and adaptation at the phenotypic level. The proximate objective is to identify adaptations through the analysis of molecular sequence data from specific candidate genes or their regulatory elements. In this paper, we consider the molecular evolution of floral color in the morning glory genus (Ipomoea) as a model for relating molecular and phenotypic evolution. To begin, flower color variation usually conforms to simple Mendelian transmission, thus facilitating genetic and molecular analyses. Population genetic studies of flower color polymorphisms in the common morning glory (Ipomoea purpurea) have shown that some morphs are subject to complex patterns of selection. Striking differences in floral color and morphology are also associated with speciation in the genus Ipomoea. The molecular bases for these adaptive shifts can be dissected because the biosynthetic pathways that determine floral pigmentation are well understood and many of the genes of flavonoid biosynthesis have been isolated and extensively studied. We present a comparative analysis of the level of gene expression in Ipomoea for several key genes in flavonoid biosynthesis. Specifically we ask: how frequently are adaptive shifts in flower color phenotypes associated with changes in regulation of gene expression versus mutations in structural genes? The results of this study show that most species differences in this crucial phenotype are associated with changes in the regulation of gene expression.

Mutations arise in a single individual and at a single point in time and space. The geographic distribution of mutations reflects both historical population size and frequency of migration. We employ coalescence-based methods to coestimate effective population size, frequency of migration, and level of recombination compatible with observed genealogical relationships in sequence data from nine nuclear genes in wild barley ( Hordeum vulgare ssp. spontaneum ), a highly self-fertilizing grass species. In self-fertilizing plants, gamete dispersal is severely limited; dissemination occurs primarily through seed dispersal. Also, heterozygosity is greatly reduced, which renders recombination less effective at randomizing genetic variation and causes larger portions of the genome to trace a similar history. Despite these predicted effects of this mating system, the majority of loci show evidence of recombination. Levels of nucleotide variation and the patterns of geographic distribution of mutations in wild barley are highly heterogeneous across loci. Two of the nine sampled loci maintain highly diverged, geographic region-specific suites of mutations. Two additional loci include region-specific haplotypes with a much shallower coalescence. Despite inbreeding, sessile growth habit, and the observation of geographic structure at almost half of sampled loci, parametric estimates of migration suggest that seed dispersal is sufficient for migration across the ≈3,500-km range of the species. Recurrent migration is also evident based on the geographic distribution of mutational variation at some loci. At one locus a single haplotype has spread rapidly enough to occur, unmodified by mutation, across the range of the species.

Flowers have long fascinated humans. The scientific study of floral biology unifies many diverse areas of research, ranging from systematics to ecology, and from genetics to molecular biology. Despite this unity, few plant species offer the experimental versatility to encompass all levels of biological investigation in a single system. An exception is the morning glory genus Ipomoea, in which a broad picture of floral evolution, ranging from ecology to molecular biology, is emerging.

We investigated the evolutionary dynamics of the Adh gene family within the grasses (Poaceae), with the goal of using molecular evolutionary tools to understand the process of gene family diversification. We analyzed 21 Adh sequences representing a broad array of grasses. Phylogenetic analyses suggested that Adh duplicated into Adh1 and Adh2 before the radiation of the grasses roughly 65 MYA. Gene structure, including intron length, has varied little over this period. Conservation of intron length prompted investigation into the dynamics of intron evolution, particularly the ability of intron sequences to form secondary structures. Intron sequences did not have an extremely high or low minimum free energy of folding relative to permuted sequences, suggesting that individual Adh introns do not evolve under secondary structural constraints. For coding sequences, the diversification of Adh1 and Adh2 was marked by a shift in third-position G + C content. This shift may reflect differential selection for codon use. Diversification between Adh1 and Adh2 was also typified by a shift in nonsynonymous nucleotide substitution rates, but there was no evidence that relatively fast nonsynonymous nucleotide substitution rates in the Adh2 clade were a product of diversifying selection. Gene conversion may have played a role in retarding diversification of Adh1 and Adh2 in rice, but there is no evidence of gene conversion between paralogs in other taxa. Although the reasons for retention of two functional Adh genes remain obscure, we propose that a shift in gene expression was important for the retention of the two Adh gene copies within the grasses.

This chapter explores the role of recombination, broadly defined, as a major agency in the elaboration of adaptive novelties in plant genomes. We consider the horizontal transfer of genes from the plastid genome into the nuclear genome, the integration of foreign DNA within the plant mitochondrial genome and the evidence for gene duplication within plant genomes as evidence of recombinational influences on plant genomes. We argue that each of these processes has lead to biochemical and metabolic adaptations that have shaped plant evolution. We also discuss the role of ectopic exchange and the effect of linkage on gene evolution in plant species. There is clear evidence for the significance of intragenic recombination as a major factor in the origin of novel alleles. Finally, we consider the interaction between linkage and selection in the structuring of plant genetic diversity.

The alcohol dehydrogenase (Adh; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) gene family has two or three loci in a broad array of angiosperm species. The relative stability in the number of Adh loci led Gottlieb [Gottlieb, L. D. (1982) Science 216, 373-380] to propose that the Adh gene family arose from an ancient gene duplication. In this study, the isolation of three loci from the California fan palm (Washingtonia robusta) is reported. The three loci from palm are highly diverged. One palm Adh gene, referred to here as adhB, has been completely sequenced, including 950 nucleotides of the upstream regulatory region. For the second locus, adhA, 81% of the exon sequence is complete. Both show the same basic structure as grass Adh genes in terms of intron number and intron location. The third locus, adhC, for which only a small amount of sequence is available (12% of exon sequence) appears to be more highly diverged. Comparison of the Adh gene families from palms and grasses shows that the adh1 and adh2 genes of grasses, and the adhA and adhB genes of palms, arose by duplication following the divergence of the two families. This finding suggests that the multiple Adh loci in different monocot lineages are not the result of a single ancestral duplication but, rather, of multiple duplication events.

We investigated nucleotide polymorphism in the Adh1 locus of pearl millet (Pennisetum glaucum) (Poaceae) by determining the DNA sequence of 20 alleles from 10 individuals. The individuals were sampled from throughout pearl millet's indigenous range and represent both wild and cultivated accessions. Our results indicated that there is little nucleotide polymorphism in the Adh1 locus. Estimates of per site nucleotide polymorphism did not differ significantly between cultivated and wild millet accessions. We compared nucleotide polymorphism in pearl millet Adh1 with nucleotide polymorphism in maize (Zea mays) Adh1 and conclude that the maize Adh1 sample is more polymorphic. Increased polymorphism in maize Adh1 may be attributable, in part, to faster substitution rates in the maize lineage. Analysis suggests that substitution rates in the maize Adh1 lineage are approximately 1.7 times faster than substitution rates in the millet Adh1 lineage.

Coding sequences from maize, rice, tobacco, and liverwort chloroplasts are aligned and subjected to relative rate tests. Results of rate tests suggest that coding sequences from maize and rice are evolving with homogeneous rates of nucleotide substitution while coding sequences from the grass lineages (i.e., maize and rice) are evolving at a faster rate than coding sequences from the tobacco chloroplast. Rate tests also suggest that particular loci evolve at significantly faster rates in grass chloroplast genomes than the tobacco chloroplast genome. These loci encode proteins important to RNA polymerase, the H(+)-ATPase complex, and the ribosomal proteins. Much of the variation at these loci can be attributed to differences in nonsynonymous substitution rates. Taken together, these studies suggest that the chloroplast DNA molecular clock varies both between evolutionary lineages and between protein coding loci.

We subjected 35 rbcL nucleotide sequences from monocotyledonous taxa to maximum likelihood relative rate tests and estimated relative differences in rates of nucleotide substitution between groups of sequences without relying on knowledge of divergence times between taxa. Rate tests revealed that there is a hierarchy of substitution rate at the rbcL locus within the monocots. Among the taxa analyzed the grasses have the most rapid substitution rate; they are followed in rate by the Orchidales, the Liliales, the Bromeliales, and the Arecales. The overall substitution rate for the rbcL locus of grasses is over 5 times the substitution rate in the rbcL of the palms. The substitution rate at the third codon positions in the rbcL of the grasses is over 8 times the third position rate in the palms. The pattern of rate variation is consistent with the generation-time-effect hypothesis. Heterogenous rates of substitution have important implications for phylogenetic reconstruction.

Analysis of a 2175-base pair (bp) SmaI-HindIII fragment of barley chloroplast DNA revealed that rbcL (the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase) and atpB (the gene for the β subunit of ATPase) are transcribed divergently and are separated by an untranscribed region of 155-166 bp. The rbcL mRNA has a 320-residue untranslated leader region, whereas the atpB mRNA has a 296- to 309-residue leader region. The sequence of these regions, together with the initial 113 bp of the atpB-coding region and the initial 1279 bp of the rbcL-coding region, is compared with the analogous maize chloroplast DNA sequences. Two classes of nucleotide differences are present, substitutions and insertions/deletions. Nucleotide substitutions show a 1.9-fold bias toward transitions in the rbcL-coding region and a 1.5-fold bias toward transitions in the noncoding region. The level of nucleotide substitutions between the barley and maize sequences is about 0.065/bp. Seventy-one percent of the substitutions in the rbcL-coding region are at the third codon position, and 95% of these are synonymous changes. Insertion/deletion events, which are confined to the noncoding regions, are not randomly distributed in these regions and are often associated with short repeated sequences. The extent of change for the noncoding regions (about 0.093 events/bp) is less than the extent of change at the third codon positions in the rbcL-coding region (about 0.135 events/bp), including insertion/delection events. Limited sequence analysis of the analogous DNA from a wild line (Hordeum spontaneum) and a primitive Iranian barley (H. vulgare) suggested a low rate of chloroplast DNA evolution. Compared to spinach chloroplast DNA, the barley rbcL-atpB untranslated region is extremely diverged, with only the putative rbcL promoters and ribosome-binding site being extensively conserved.

The nucleotide sequence of a barley chloroplast DNA 3.7 kb SmaI-HindIII fragment is presented. This fragment contains atpBE, the genes for the beta and epsilon subunits of ATPase; trnM2, the gene for tRNA2met; and trnV1, the gene for tRNA1va1. The atpE-trnM2 interval is 126 bp and trnM2 is transcribed towards atpBE. The trnM2-trnV1 interval is 203 bp and trnV1 is transcribed away from trnM2. The trnV1 locus has a 597 bp intervening sequence. the organization and sequences of these genes are compared to the analogous genes from maize and tobacco chloroplast DNA. Using the latter comparisons the nature of sequence divergence between chloroplast DNAs is discussed.

A procedure is outlined for purifying DNA from a single plant. A crude organelle pellet consisting of nuclei, chromatin, chloroplasts, and mitochondria is prepared, suspended, and immediately lysed with detergents. The DNA is separated from RNA, protein, and polysaccharides by banding it in CsCl density equilibrium gradients. Ethidium bromide is included in all buffers to act as an inhibitor of DNAase activity. The DNA prepared in this manner can be digested with restriction endonucleases, separated by gel electrophoresis, and used to identify specific genes by hybridization of cloned DNA sequences.

In plants possessing a mixed selfing and random mating system, outcrossing rate t can be estimated from progeny genotype arrays. Computer simulation studies show that if there is heterogeneity of gene frequency between subpopulations, these estimates of t are severely biased downwards, the degree of bias increasing as the true value of t increases. This effect has been demonstrated in the field using experimental populations of Ipomoea purpurea. These field studies also provide evidence for pollen “carryover” in I. purpurea.

In a series of previous studies, it has been shown that populations of the common morning glory Ipomoea purpurea are typically polymorphic for flower-color genes that bias pollinator service and, hence, the rate of outcrossing. In this study, we show that the rate of outcrossing for the white flower-color morph depends on its frequency in experimental populations of the morning glory. White flowers are visited less often by the primary pollinator, bumblebees, and have lower outcrossing rates than colored flowers when they are in the minority. In contrast, blue or pink flower morphs are not undervisited and do not have lowered outcrossing rates when they are rare. In plant populations where genes that increase selling are selectively favored due to their transmission bias, undervisitation of rare morphs may act to stabilize genetic variation for outcrossing rates.

Comparative data on the evolution of chloroplast genes are reviewed. The chloroplast genome has maintained a similar structural organization over most plant taxa so far examined. Comparisons of nucleotide sequence divergence among chloroplast genes reveals marked similarity across the plant kingdom and beyond to the cyanobacteria (blue-green algae). Estimates of rates of nucleotide substitution indicate a synonymous rate of 1.1 x 10(-9) substitutions per site per year. Noncoding regions also appear to be constrained in their evolution, although addition/deletion events are common. There have also been evolutionary changes in the distribution of introns in chloroplast encoded genes. Relative to mammalian mitochondrial DNA, the chloroplast genome evolves at a conservative rate.

The chloroplast rDNA genes of pearl millet (Pennisetum americanum) have been cloned and physically mapped. The chloroplast genome of the pearl millet contains two identical rRNA genes located on DNA sequences that are inverted with respect to one another and separated by 12 kb of single-copy DNA. The rRNA genes were positioned on a restriction endonuclease map by using as hybridization probes specific cloned rDNA sequences from the chloroplast DNA of the alga Euglena gracilis. The 16S and 23S rRNA genes were shown to be approx. 2 kb from one another, and the 5S RNA gene is immediately adjacent to the 23S tRNA gene.

A cDNA library was prepared from ripe avocado fruit (Persea americana Mill. cv. Hass) and screened for clones hybridizing to a 600 bp cDNA clone (pAV5) coding for avocado fruit cellulase. This screening led to the isolation of a clone (pAV363) containing a 2021 nucleotide transcribed sequence and an approximately 150 nucleotide poly(A) tail. Hybridization of pAV363 to a northern blot shows that the length of the homologous message is approximately 2.2 kb. The nucleotide sequence of this putative full-length mRNA clone contains an open reading frame of 1482 nucleotides which codes for a polypeptide of 54.1 kD. The deduced amino acid composition compares favorably with the amino acid composition of native avocado cellulase determined by amino acid analysis. Southern blot analysis of Hind III and Eco RI endonuclease digested genomic DNA indicates a small family of cellulase genes.

Avocado (Persea americana) is a subtropical tree prized for its large and nutritious fruit. Although native to Mesoamerica, avocado is now grown in tropical and subtropical regions around the world, and consumer demand for avocado continues to grow at a considerable rate. Despite the appeal of avocado, its genetic improvement has been slow owing to substantial land and labor requirements combined with the fact that young trees do not produce fruit for several years and a pollination system that makes it difficult to produce genetic crosses. Molecular markers promise to accelerate the rate of breeding progress, especially for simple traits of high heritability. One of the distinguishing features of the avocado fruit is the presence of a number of compounds that have been linked to human health. As a prelude to the use of molecular markers for the improvement of nutritional traits, this article reports estimates of the heritability of carotenoids, β-sitosterol, and α-tocopherol content (the most biologically active form of vitamin E) in ripe avocado fruit. Each of these three compounds has been linked to beneficial health outcomes, and each is shown to have a sufficiently high heritability to predict successful marker-assisted selection.

Experimental populations of the annual plant, Ipomoea purpurea, composed of individuals belonging to two flower color morphs were studied to determine the effect of flower color on outcrossing rate and reproductive success as a male parent. Analyses of parent and offspring genotypes show that the pigmented and white morphs outcross at similar rates, but that the white morph is favored as a pollen donor. The result suggests that the dynamics of selection occurring at the locus coding for white versus pigmented flowers are more complex than previously believed. Factors such as frequency-dependent outcrossing rates and epistatic effects of the white allele may be operating. The results also suggest that pollinator observations are unreliable indicators of the actual mating system.