Physical leaf defenses – altered by Zea life‐history evolution,domestication, and breeding – mediate oviposition preference of a specialist leafhopper

Plant anti‐herbivore defenses are known to be affected by life‐history evolution, as well as by domestication and breeding in the case of crop species. A suite of plants from the maize genus Zea (Poaceae) and the specialist herbivore Dalbulus maidis (DeLong & Wolcott) (Hemiptera: Cicadellidae) were used to test the hypothesis that anti‐herbivore defenses are affected by plant life‐history evolution and human intervention through domestication and breeding for high yield. The suite of plants included a maize (Zea mays ssp. mays L.) commercial hybrid, a maize landrace, two populations of the annual Balsas teosinte (Z. mays ssp. parviglumis Iltis & Doebley), and perennial teosinte (Z. diploperennis Iltis, Doebley & Guzman). Leaf toughness, pubescence, and oviposition preference were compared among the suite of host plants looking for effects of transitions in life history (i.e., from perennial to annual life cycle), domestication (i.e., from wild annual to domesticated annual), and breeding (i.e., from landrace to hybrid maize) on defense against D. maidis. Results on leaf toughness suggested that the life‐history and domestication transitions weakened the plant's resistance to penetration by the mouthparts and ovipositor of D. maidis, whereas results on pubescence suggested that this putative defense was strengthened with the breeding transition, contrary to expectations. Results on oviposition preference of D. maidis coincided with the expectation that life‐history and domestication transitions would lead to preference for Balsas teosinte over perennial teosinte, and of landrace maize over Balsas teosinte. Also, a negative correlation suggested that oviposition preference is significantly influenced by leaf toughness. Overall, the results suggested that Zea defenses against the specialist herbivore D. maidis were variably affected by plant life‐history evolution, domestication, and breeding, and that chemical defense may play a role in Zea defense against D. maidis because leaf toughness and pubescence only partially explained its host preferences.     

Genetic basis of kernel nutritional traits during maize domestication and improvement

The nutritional traits of maize kernels are important for human and animal nutrition, and these traits have undergone selection to meet the diverse nutritional needs of humans. However, our knowledge of the genetic basis of selecting for kernel nutritional traits is limited. Here, we identified both single and epistatic quantitative trait loci (QTLs) that contributed to the differences of oil and carotenoid traits between maize and teosinte. Over half of teosinte alleles of single QTLs increased the values of the detected oil and carotenoid traits. Based on the pleiotropism or linkage information of the identified single QTLs, we constructed a trait–locus network to help clarify the genetic basis of correlations among oil and carotenoid traits. Furthermore, the selection features and evolutionary trajectories of the genes or loci underlying variations in oil and carotenoid traits revealed that these nutritional traits produced diverse selection events during maize domestication and improvement. To illustrate more, a mutator distance–relative transposable element (TE) in intron 1 of DXS2, which encoded a rate‐limiting enzyme in the methylerythritol phosphate pathway, was identified to increase carotenoid biosynthesis by enhancing DXS2 expression. This TE occurs in the grass teosinte, and has been found to have undergone selection during maize domestication and improvement, and is almost fixed in yellow maize. Our findings not only provide important insights into evolutionary changes in nutritional traits, but also highlight the feasibility of reintroducing back into commercial agricultural germplasm those nutritionally important genes hidden in wild relatives.     

Development and mapping of SSR markers for maize

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.     

Female gametophyte development and double fertilization in Balsas teosinte, Zea mays subsp. parviglumis (Poaceae)

Over the course of maize evolution, domestication played a major role in the structural transition of the vegetative and reproductive characteristics that distinguish it from its closest wild relative, Zea mays subsp. parviglumis (Balsas teosinte). Little is known, however, about impacts of the domestication process on the cellular features of the female gametophyte and the subsequent reproductive events after fertilization, even though they are essential components of plant sexual reproduction. In this study, we investigated the developmental and cellular features of the Balsas teosinte female gametophyte and early developing seed in order to unravel the key structural and evolutionary transitions of the reproductive process associated with the domestication of the ancestor of maize. Our results show that the female gametophyte of Balsas teosinte is a variation of the Polygonum type with proliferative antipodal cells and is similar to that of maize. The fertilization process of Balsas teosinte also is basically similar to domesticated maize. In contrast to maize, many events associated with the development of the embryo and endosperm appear to be initiated earlier in Balsas teosinte. Our study suggests that the pattern of female gametophyte development with antipodal proliferation is common among species and subspecies of Zea and evolved before maize domestication. In addition, we propose that the relatively longer duration of the free nuclear endosperm phase in maize is correlated with the development of a larger fruit (kernel or caryopsis) and with a bigger endosperm compared with Balsas teosinte.     

Maize seedling morphology and defence hormone profiles,but not herbivory tolerance,were mediated by domestication and modern breeding

We addressed whether Zea seedling morphology relevant to performance, defence hormone profiles and tolerance of a phloem‐feeding, specialist herbivore were affected by two processes, plant domestication and modern breeding. Domestication effects were inferred through comparisons between Balsas teosintes (Zea mays parviglumis) and landrace maizes (Z. mays mays), and modern breeding effects through comparisons between landrace maizes and inbred maize lines. Specifically, we compared seedling forms (a composite measure of leaf length, average stem diameter, shoot wet weight, shoot dry weight, total root length, root wet weight, and root dry weight), shapes (forms scaled by seedling dry weight, a proxy for seedling size), and defence hormone profiles among Balsas teosinte and landrace and inbred line maizes, exposed or unexposed to feeding by Dalbulus maidis. Our results suggested that domestication as well as modern breeding strongly mediated both seedling form and shape. Form was more similar between landrace and inbred maize than between Balsas teosinte and landrace maize, suggesting that domestication affected seedling form more than modern breeding. In contrast, shape was more similar between Balsas teosinte and landrace maize than between landrace and inbred maizes, suggesting that modern breeding affected seedling shape more than domestication. Additionally, seedling shoot : root ratios appeared to have been mediated by domestication, but not by modern breeding. In broad terms, individual seedling structures relevant to seedling ecology in wild or managed environments, such as leaf and root lengths, and shoot and root masses, were enlarged with domestication and reduced with modern breeding. Herbivory did not affect seedling shape, but had a weak effect on form so that seedlings were slightly larger in the absence versus presence of D. maidis. Also, both domestication and modern breeding seem to have mediated seedling hormone profiles, with breeding more strongly mediating profiles than domestication. Jasmonic acid isoleucine (JA‐Ile) and salicylic acid (SA) were induced by herbivory in both teosinte and maize. The hormone profiles assays collectively suggested that domestication and modern breeding altered constitutive levels of SA, abscisic acid and JA‐related (JA‐Ile and oxo‐phytodienoic acid) hormone levels in seedlings, particularly by increasing the levels of SA and decreasing those of JA‐related hormones. Altogether, our results suggested that maize domestication and modern breeding significantly altered seedling form, shape, ecologically relevant morphological traits (e.g. leaf and root lengths, and shoot and root masses) and hormonal defences, but not tolerance of D. maidis herbivory.     

Pollination between maize and teosinte: an important determinant of gene flow in Mexico

Gene flow between maize [Zea mays (L.)] and its wild relatives does occur, but at very low frequencies. Experiments were undertaken in Tapachula, Nayarit, Mexico to investigate gene flow between a hybrid maize, landraces of maize and teosinte (Z. mays ssp. mexicana, races Chalco and Central Plateau). Hybridization, flowering synchrony, pollen size and longevity, silk elongation rates, silk and trichome lengths and tassel diameter and morphology were measured. Hybrid and open-pollinated maize ears produced a mean of 8 and 11 seeds per ear, respectively, when hand-pollinated with teosinte pollen, which is approximately 1–2% of the ovules normally produced on a hybrid maize ear. Teosinte ears produced a mean of 0.2–0.3 seeds per ear when pollinated with maize pollen, which is more than one-fold fewer seeds than produced on a maize ear pollinated with teosinte pollen. The pollination rate on a per plant basis was similar in the context of a maize plant with 400–500 seeds and a teosinte plant with 30–40 inflorescences and 9–12 fruitcases per inflorescence. A number of other factors also influenced gene-flow direction: (1) between 90% and 95% of the fruitcases produced on teosinte that was fertilized by maize pollen were sterile; (2) teosinte collections were made in an area where incompatibility systems that limit fertilization are present; (3) silk longevity was much shorter for teosinte than for maize (approx. 4 days vs. approx. 11 days); (4) teosinte produced more pollen on a per plant basis than the landraces and commercial hybrid maize; (5) teosinte frequently produced lateral branches with silks close to a terminal tassel producing pollen. Collectively these factors tend to favor crossing in the direction of teosinte to maize. Our results support the hypothesis that gene flow and the subsequent introgression of maize genes into teosinte populations most probably results from crosses where teosinte first pollinates maize. The resultant hybrids then backcross with teosinte to introgress the maize genes into the teosinte genome. This approach would slow introgression and may help explain why teosinte continues to co-exist as a separate entity even though it normally grows in the vicinity of much larger populations of maize.     

Population genetic structure of a specialist leafhopper on Zea: likely anthropogenic and ecological determinants of gene flow

Corn leafhopper, Dalbulus maidis DeLong & Wolcott (Hemiptera: Cicadellidae), is a specialist herbivore on the genus Zea (Poaceae). The genera Dalbulus and Zea evolved in central Mexico. We sought to determine whether population genetic structuring is prevalent in corn leafhoppers inhabiting three of its host plants: (1) the highland species perennial teosinte (Zea diploperennis Iltis, Doebley & Guzman), (2) the mid‐ to lowland‐species Balsas teosinte (Zea mays ssp. parviglumis Iltis & Doebley), and (3) the ubiquitous domesticated maize (Zea mays ssp. mays L.). We used amplified fragment length polymorphisms to detect population structuring and genetic differentiation among corn leafhoppers on the three host plants in western‐central and ‐northern Mexico. Our results showed that corn leafhopper in Mexico is composed of at least two genetically discrete populations: an ‘Itinerant’ population associated with the annual hosts maize and Balsas teosinte, which appears to be widely distributed in Mexico, and a ‘Las Joyas’ population restricted to perennial teosinte and confined to a small mountain range (Sierra de Manantlán) in western‐central Mexico. Our results further suggested that population structuring is not due to isolation by distance or landscape features: Las Joyas and Itinerant corn leafhopper populations are genetically distinct despite their geographic proximity (ca. 4 km), whereas Itinerant corn leafhoppers separated by hundreds of kilometers (>800 km), mountain ranges, and a maritime corridor (Sea of Cortez) are not genetically distinct. Based on our results and on published ethnohistorical and archaeological data, we propose pre‐Columbian and modern scenarios, including likely ecological and anthropogenic influences, in which the observed genetic population structuring of corn leafhopper could have originated and could be maintained. Also, we hypothesize that after evolving on the lowland Balsas teosinte, corn leafhopper expanded its host range to include maize and then the highland perennial teosinte, following the domestication and spread of maize within the last 9 000 years.     

The Role of cis Regulatory Evolution in Maize Domestication

Gene expression differences between divergent lineages caused by modification of cis regulatory elements are thought to be important in evolution. We assayed genome-wide cis and trans regulatory differences between maize and its wild progenitor, teosinte, using deep RNA sequencing in F₁ hybrid and parent inbred lines for three tissue types (ear, leaf and stem). Pervasive regulatory variation was observed with approximately 70% of ∼17,000 genes showing evidence of regulatory divergence between maize and teosinte. However, many fewer genes (1,079 genes) show consistent cis differences with all sampled maize and teosinte lines. For ∼70% of these 1,079 genes, the cis differences are specific to a single tissue. The number of genes with cis regulatory differences is greatest for ear tissue, which underwent a drastic transformation in form during domestication. As expected from the domestication bottleneck, maize possesses less cis regulatory variation than teosinte with this deficit greatest for genes showing maize-teosinte cis regulatory divergence, suggesting selection on cis regulatory differences during domestication. Consistent with selection on cis regulatory elements, genes with cis effects correlated strongly with genes under positive selection during maize domestication and improvement, while genes with trans regulatory effects did not. We observed a directional bias such that genes with cis differences showed higher expression of the maize allele more often than the teosinte allele, suggesting domestication favored up-regulation of gene expression. Finally, this work documents the cis and trans regulatory changes between maize and teosinte in over 17,000 genes for three tissues.     

Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench]

Simple sequence repeats (SSRs), also known as microsatellites, are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. Three approaches were followed for the development of PCR primers for the amplification of DNA fragments containing SSRs from sorghum [Sorghum bicolor (L.) Moench]: a search for sorghum SSRs in public DNA databases; the use of SSR-specific primers developed in the Poaceae species maize (Zea mays L.) and seashore paspalum grass (Paspalum vaginatum Swartz); and the screening of sorghum genomic libraries by hybridization with SSR oligonucleotides. A total of 49 sorghum SSR-specific PCR primer pairs (two designed from GenBank SSR-containing sequences and 47 from the sequences of genomic clones) were screened on a panel of 17 sorghum and one maize accession. Ten primer pairs from paspalum and 90 from maize were also screened for polymorphism in sorghum. Length polymorphisms among amplification products were detected with 15 of these primer pairs, yielding diversity values ranging from 0.2 to 0.8 with an average diversity of 0.56. These primer pairs are now available for use as markers in crop improvement and conservation efforts.     

Plant defense against fall armyworm in micro‐sympatric maize (Zea mays ssp. mays) and Balsas teosinte (Zea mays ssp. parviglumis)

Maize [Zea mays L. ssp. mays (Poaceae)] was domesticated from Balsas teosinte (Zea mays ssp. parviglumis Iltis & Doebley) in present‐day Mexico. Fall armyworm, Spodoptera frugiperda JE Smith (Lepidoptera: Noctuidae), is among the most important pests of maize in Mexico and Central America. We compared the strength of plant defenses against fall armyworm between micro‐sympatric landrace maize and Balsas teosinte in the field and laboratory. The field comparison, conducted in Mexico, consisted of comparing the frequency of fall armyworm infestation between young maize and Balsas teosinte plants in dryland agricultural fields in which Balsas teosinte grew as a weed. The laboratory comparison contrasted the performance of fall armyworm larvae provided a diet of leaf tissue excised from maize or Balsas teosinte plants that were intact or had been primed by larval feeding. In the field, maize plants were more frequently infested with fall armyworm than Balsas teosinte plants: over 3 years and three fields, maize was infested at a ca. 1.8‐fold greater rate than Balsas teosinte. In the laboratory, larval growth, but not survivorship, was differently affected by feeding on maize vs. Balsas teosinte, and on primed vs. intact plants. Specifically, survivorship was ca. 98%, and did not differ between maize and Balsas teosinte, nor between primed and intact plants. Larvae grew less on intact vs. primed maize, and similarly on intact vs. primed Balsas teosinte; overall, growth was 1.2‐fold greater on maize compared to Balsas teosinte, and on primed compared to intact plants. Parallel observations showed that the differences in growth could not be attributed to the amount of leaf tissue consumed by larvae. We discuss our results in relation to differences in the strength of plant defenses between crops and their ancestors, the relevance of unmanaged Balsas teosinte introgression in the context of fall armyworm defenses in maize, and whether greater growth of larvae on primed vs. intact plants signifies herbivore offense.     

Mining and charactering microsatellites in Cucumis melo expressed sequence tags from sequence database

Simple sequence repeats (SSRs) derived from expressed sequence tags (ESTs) are valuable markers because they represent transcribed regions and often have putative functions. We mined and characterized microsatellites in melon ESTs. Three hundred and eighty‐three SSR loci were identified in 309 of 3188 unigenes assembled by 5747 EST and mRNA sequences in GenBank with occurring frequency of 1/4.7 kb. Twenty‐two polymorphic EST‐SSR markers were developed with the mean allele number of 2.9 per locus and mean expected heterozygosity of 0.442. Amplification products were also detected by 15 pairs of primer in Cucumis sativus. Those informative EST‐SSR markers can be used in melon genetic improvement projects.     

Wide variability in kernel composition, seed characteristics, and zein profiles among diverse maize inbreds, landraces, and teosinte

All crop species have been domesticated from their wild relatives, and geneticists are just now beginning to understand the consequences of artificial (human) selection on agronomic traits that are relevant today. The primary consequence is a basal loss of diversity across the genome, and an additional reduction in diversity for genes underlying traits targeted by selection. An understanding of attributes of the wild relatives may provide insight into target traits and valuable allelic variants for modern agriculture. This is especially true for maize (Zea mays ssp. mays), where its wild ancestor, teosinte (Z. mays ssp. parviglumis), is so strikingly different than modern maize. One obvious target of selection is the size and composition of the kernel. We evaluated kernel characteristics, kernel composition, and zein profiles for a diverse set of modern inbred lines, teosinte accessions, and landraces, the intermediate between inbreds and teosinte. We found that teosinte has very small seeds, but twice the protein content of landraces and inbred lines. Teosinte has a higher average alpha zein content (nearly 89% of total zeins as compared to 72% for inbred lines and 76% for landraces), and there are many novel alcohol-soluble proteins in teosinte relative to the other two germplasm groups. Nearly every zein protein varied in abundance among the germplasm groups, especially the methionine-rich delta zein protein, and the gamma zeins. Teosinte and landraces harbor phenotypic variation that will facilitate genetic dissection of kernel traits and grain quality, ultimately leading to improvement via traditional plant breeding and/or genetic engineering.