Isolation and characterization of 14 microsatellite markers in <Emphasis Type="Italic">Macrodasyceras</Emphasis><Emphasis Type="Italic">hirsutum</Emphasis> (Hymenoptera: Torymidae)

Macrodasyceras hirsutum Kamijo is the seed parasitoid wasp of the bird-dispersed, dioecious tree, Ilex integra Thunb. The wasp reduces the level of dispersal mutualism between the Ilex tree and its frugivorous birds by manipulating the color of mature berries. The female trees do not blossom every year and sometimes change sex. Thus, the reproduction biology of I. integra affects the population size and structure of M. hirsutum in a forest and consequently influences the seed dispersal mutualism between the tree and birds, because of limited ability of adult locomotion. To investigate the wasp population structure with reference to the dispersal mutualism between trees and birds, we isolated 14 microsatellite loci of M. hirsutum wasps. Every locus was polymorphic among 20 females, with 3–13 alleles per locus, without linkage disequilibrium. The observed and expected heterozygosities ranged from 0.100 to 0.900 and 0.099 to 0.818, respectively, indicating their utility in molecular analyses of the wasp population.     

Temporal variations in seed dispersal patterns of a bird-dispersed tree, <Emphasis Type="Italic">Swida controversa</Emphasis> (Cornaceae), in a temperate forest

The seed dispersal patterns of bird-dispersed trees often show substantial seasonal and annual variation due to temporal changes in frugivorous bird and bird-dispersed fruit distributions. Elucidating such variation and how it affects plant regeneration is important for understanding the evolution and seed dispersal maintenance strategies of these plants. In this study, we investigated the seed dispersal quantity and distance of a bird-dispersed plant, Swida controversa, for 2 years and detected large seasonal variations in dispersal pattern. Early in the fruiting season, short seed dispersal distance and large amounts of fruit consumption by birds (seed dispersal quantity) were observed. In contrast, late in the fruiting season, a long seed dispersal distance and small seed dispersal quantity were observed. This relationship between seed dispersal distance and quantity may help to maintain constant seed dispersal effectiveness during the long S. controversa fruiting season. Annual variation was also detected for both seed dispersal quantity and distance. More effective seed dispersal was achieved in the masting year, because both seed dispersal quantity and distance were greater than that in the non-masting year. These seed dispersal dynamics may contribute to the evolution and maintenance of S. controversa masting behavior. Thus, we identified substantial temporal variation on both seasonal and annual scales in the seed dispersal pattern of a bird-dispersed plant. The temporal variation in seed dispersal pattern revealed in this study probably plays a substantial role in the life history and population dynamics of S. controversa.     

Dispersal of remnant endangered trees in a fragmented and disturbed forest by frugivorous birds

Most endangered plant species in a fragmented forest behave as a unique source population, with a high dependence on frugivorous birds for recruitment and persistence. In this study, we combined field data of dispersal behavior of birds and GIS information of patch attributes to estimate how frugivorous birds could affect the effective dispersal pattern of Chinese yew (Taxus chinensis) in a fragmented and disturbed forest. Nine bird species were observed to visit T. chinensis trees, with Urocissa erythrorhyncha, Zoothera dauma and Picus canus being the most common dispersers. After foraging, six disperser species exhibited different perching patterns. Three specialist species, P. canus, Turdus hortulorum, and Z. dauma stayed in the source patch, while three generalist species, U. erythrorhyncha, Hypsipetes mcclellandii, and H. castanonotus, could perch in bamboo patches and varied in movement ability due to body size. As a consequence of perching, dispersers significantly contributed to the seed bank, but indirectly affected seedling recruitment. Moreover, the recruitment of T. chinensis was also affected by patch attributes in a fragmented forest (distances to source patch, patch type, size). Our results highlighted the ability of unique source population regeneration of T. chinensis in a fragmented forest, with high dependence on both frugivorous birds and patch attributes, which should be considered in future planning for forest management and conservation.     

The <Emphasis Type="Italic">APX4</Emphasis> locus regulates seed vigor and seedling growth in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The amino acid sequence of APX4 is similar to other ascorbate peroxidases (APXs), a group of proteins that protect plants from oxidative damage by transferring electrons from ascorbate to detoxify peroxides. In this study, we characterized two apx4 mutant alleles. Translational fusions with GFP indicated APX4 localizes to chloroplasts. Both apx4 mutant alleles formed chlorotic cotyledons with significantly reduced chlorophyll a, chlorophyll b and lutein. Given the homology of APX to ROS-scavenging proteins, this result is consistent with APX4 protecting seedling photosystems from oxidation. The growth of apx4 seedlings was stunted early in seedling development. In addition, APX4 altered seed quality by affecting seed coat formation. While apx4 seed development appeared normal, the seed coat was darker and more permeable than the wild type. In addition, accelerated aging tests showed that apx4 seeds were more sensitive to environmental stress than the wild-type seeds. If APX4 affects seed pigment biosynthesis or reduction, the seed coat color and permeability phenotypes are explained. apx4 mutants had cotyledon chlorosis, increased H₂O₂ accumulation, and reduced soluble APX activity in seedlings. These results indicate that APX4 is involved in the ROS-scavenging process in chloroplasts.     

An Ecohydrological Cellular Automata Model Investigation of Juniper Tree Encroachment in a Western North American Landscape

Woody plant encroachment over the past 140 years has substantially changed grasslands in western North American. We studied encroachment of western juniper (Juniperus occidentalis var. occidentalis) into a previously mixed shrub–grassland site in central Oregon (USA) using a modified version of Cellular Automata Tree–Grass–Shrub Simulator (CATGraSS) ecohydrological model. We developed simple algorithms to simulate three encroachment factors (grazing, fire frequency reduction, and seed dispersal by herbivores) in CATGraSS. Local ecohydrological dynamics represented by the model were first evaluated using satellite-derived leaf area index and measured evapotranspiration data. Reconstructed pre-encroachment vegetation cover percentages and the National Land Cover Database (NLCD 2006) vegetation map were used to estimate parameters for encroachment factors to represent juniper encroachment in CATGraSS. Model sensitivity experiments examined the influence of each encroachment factor and their combinations on trajectories of modeled percent cover of each plant functional type and emergent spatial vegetation patterns in the modeled domain. Simulation results identified grazing as the key factor leading to juniper encroachment, by reducing shrub and grass cover and promoting the formation of juniper tree clusters. Reduced fire frequency and increased seed dispersal by grazers further amplified juniper encroachment into grassland patches between clusters of juniper trees. Each encroachment factor showed different consequences on modeled vegetation patterns. Time series of modeled plant cover and spatial patterns of plant functional types were found to be consistent with an existing conceptual model described in the literature. The proposed model provides a tool that can be used to improve our understanding of the drivers and processes of woody plant encroachment and vegetation response to global change.     

Screening for potential <Emphasis Type="Italic">Striga hermonthica</Emphasis> fungal and bacterial biocontrol agents from suppressive soils in Western Kenya

Striga hermonthica is a hemiparasitic weed that causes huge grain yield losses to small-scale farmers in Africa. Effective biocontrol agents against S. hermonthica can sustainably mitigate these losses. This study characterized the biocontrol potential of culturable fungal and bacterial isolates from S. hermonthica suppressive soils of western Kenya. These isolates were screened for their ability to produce antibiotic compounds and extra cellular enzymes and also their ability to cause S. hermonthica seed decay. Genomic DNA of the selected bacterial and fungal isolates was extracted and partial characterization of 16S rRNA and 18S rRNA genes performed respectively. Analysis show that antibiosis and enzymatic properties of potential biocontrol isolates correlated positively. Isolate KY041696 recorded high antibiosis, enzymatic and seed decay values. This study also revealed that bioactive bacterial isolates belonged to Bacillus, Streptomyces and Rhizobium genera. In this study, no fungal isolate caused S. hermonthica seed decay. This study therefore provides baseline information on the potential biocontrol microbes against S. hermonthica in Western Kenya that could be exploited further in the management of the weed.     

Exploiting the <Emphasis Type="Italic">Rht</Emphasis> portfolio for hybrid wheat breeding

Key message

The portfolio of available Reduced height loci (Rht-B1, Rht-D1, and Rht24) can be exploited for hybrid wheat breeding to achieve the desired heights in the female and male parents, as well as in the hybrids, without adverse effects on other traits relevant for hybrid seed production.

Abstract

Plant height is an important trait in wheat line breeding, but is of even greater importance in hybrid wheat breeding. Here, the height of the female and male parental lines must be controlled and adjusted relative to each other to maximize hybrid seed production. In addition, the height of the resulting hybrids must be fine-tuned to meet the specific requirements of the farmers in the target regions. Moreover, this must be achieved without adversely impacting traits relevant for hybrid seed production. In this study, we explored Reduced height (Rht) loci effective in elite wheat and exploited their utilization for hybrid wheat breeding. We performed association mapping in a panel of 1705 wheat hybrids and their 225 parental lines, which besides the Rht-B1 and Rht-D1 loci revealed Rht24 as a major QTL for plant height. Furthermore, we found that the Rht-1 loci also reduce anther extrusion and thus cross-pollination ability, whereas Rht24 appeared to have no adverse effect on this trait. Our results suggest different haplotypes of the three Rht loci to be used in the female or male pool of a hybrid breeding program, but also show that in general, plant height is a quantitative trait controlled by numerous small-effect QTL. Consequently, marker-assisted selection for the major Rht loci must be complemented by phenotypic selection to achieve the desired height in the female and male parents as well as in the wheat hybrids.

     

Coffee volatiles induced after mechanical injury and beetle herbivory attract the coffee berry borer and two of its parasitoids

The coffee berry borer (CBB), Hypothenemus hampei (Ferrari), is the most important insect pest of coffee worldwide. In this study, we used headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry to sample and identify volatile compounds from Robusta coffee berries, Coffea canephora Pierre ex Froehner, infested with CBB and with mechanical damage. Furthermore, we evaluated the behavioral responses of the CBB and two of its parasitoids, Prorops nasuta Waterstone and Phymastichus coffea LaSalle, to three selected coffee volatile compounds in a Y-tube olfactometer. We found in the effluvia of red coffee berry compounds not previously reported for this coffee species. Our results show that Robusta coffee berries release induced volatiles either by insect herbivory or by mechanical damage. Small amount of butyl acetate, unknown compound 2, α-longipinene, longiborneol and longiborneol acetate are produced only in infested coffee berries fruits. Quantitatively, nine compounds account for the difference between healthy berries, infested, or mechanically damaged berries. Trans-ocimene, 4,8-dimethyl-3,7-nonadien-2-ol, α-copaene and kaurene increased amount levels in infested berries, while amount of methyl salicylate and linalool increased in mechanically damaged coffee berries. The olfactometric bioassays showed that CBB females and its two parasitoids were attracted to methyl salicylate. In addition, H. hampei and P. nasuta were attracted to linalool, and P. nasuta and P. coffea were attracted to trans-ocimene.     

Map-based cloning of the dominant genic male sterile <Emphasis Type="Italic">Ms</Emphasis>-<Emphasis Type="Italic">cd1</Emphasis> gene in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

Key message

Using map-based cloning, we delimited the Ms - cd1 gene responsible for the male sterile phenotype in B. oleracea to an approximately 39-kb fragment. Expression analysis suggests that a new predicted gene, a homolog of the Arabidopsis SIED1 gene, is a potential candidate gene.

Abstract

A dominant genic male sterile (DGMS) mutant 79-399-3 in Brassica oleracea (B. oleracea) is controlled by a single gene named Ms-cd1, which was genetically mapped on chromosome C09. The derived DGMS lines of 79-399-3 have been successfully applied in hybrid cabbage breeding and commercial hybrid seed production of several B. oleracea cultivars in China. However, the Ms-cd1 gene responsible for the DGMS has not been identified, and the molecular basis of the DGMS is unclear, which then limits its widespread application in hybrid cabbage seed production. In the present study, a large BC₉ population with 12,269 individuals was developed for map-based cloning of the Ms-cd1 gene, and Ms-cd1 was mapped to a 39.4-kb DNA fragment between two InDel markers, InDel14 and InDel24. Four genes were identified in this region, including two annotated genes based on the available B. oleracea annotation database and two new predicted open reading frames (ORFs). Finally, a newly predicted ORF designated Bol357N3 was identified as the candidate of the Ms-cd1 gene. These results will be useful to reveal the molecular mechanism of the DGMS and develop more practical DGMS lines with stable male sterility for hybrid seed production in cabbage.

     

Seed protein fraction electrophoresis in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) accessions and wild species

Total seed storage proteins were studied in 50 accessions of A. hypogaea (11 A. hypogaea ssp. hypogaea var hypogaea, 13 A. hypogaea ssp. hypogaea var hirsuta, 11 A. hypogaea ssp. fastigiata var fastigiata and 15 A. hypogaea ssp. fastigiata var. vulgaris accessions) in SDS PAGE. These accessions were also analysed for albumin and globulin seed protein fractions. Among the six seed protein markers presently used, it was found that globulin fraction showed maximum diversity (77.2%) in A. hypogaea accessions followed by albumin (52.3%), denatured total soluble protein fraction in embryo (33.3%) and cotyledon (28.5%). The cluster analysis based on combined data of cotyledons, embryos, albumins and globulins seed protein fractions demarcated the accessions of two subspecies hypogaea and fastigiata into two separate clusters supported by 51% bootstrap value, with few exceptions, suggesting the genotypes to be moderately diverse. Native and denatured total soluble seed storage proteins were also electrophoretically analysed in 27 wild Arachis species belonging to six sections of the genus. Cluster analysis using different methods were performed for different seed proteins data alone and also in combination. Section Caulorrhizae (C genome) and Triseminatae (T genome) formed one, distantly related group to A. hypogaea and other section Arachis species in the dendrogram based on denatured seed storage proteins data. The present analysis has maintained that the section Arachis species belong to primary and secondary genepools and, sections Procumbenetes and Erectoides belong to tertiary gene pools.     

Seed specific expression and analysis of recombinant human adenosine deaminase (<Emphasis Type="Italic">hADA</Emphasis>) in three host plant species

The plant seed is a leading platform amongst plant-based storage systems for the production of recombinant proteins. In this study, we compared the activity of human adenosine deaminase (hADA) expressed in transgenic seeds of three different plant species: pea (Pisum sativum L.), Nicotiana benthamiana L. and tarwi (Lupinus mutabilis Sweet). All three species were transformed with the same expression vector containing the hADA gene driven by the seed-specific promoter LegA2 with an apoplast targeting pinII signal peptide. During the study, several independent transgenic lines were generated and screened from each plant species and only lines with a single copy of the gene of interest were used for hADA expression analysis. A stable transgenic canola line expressing the ADA protein, under the control of 35S constitutive promoter was used as both as a positive control and for comparative study with the seed specific promoter. Significant differences were detected in the expression of hADA. The highest activity of the hADA enzyme (Units/g seed) was reported in tarwi (4.26 U/g) followed by pea (3.23 U/g) and Nicotiana benthamiana (1.69 U/g). The expression of mouse ADA in canola was very low in both seed and leaf tissue compared to other host plants, confirming higher activity of seed specific promoter. Altogether, these results suggest that tarwi could be an excellent candidate for the production of valuable recombinant proteins.     

Influences of the invasive tamarisk leaf beetle (<Emphasis Type="Italic">Diorhabda carinulata</Emphasis>) on avian diets along the Dolores River in Southwestern Colorado USA

The tamarisk leaf beetle (Diorhabda carinulata), introduced from Eurasia in 2001 as a biological control agent for the invasive plant Tamarix ramosissima, has spread widely throughout the western USA. With D. carinulata now very abundant, scientists and restoration managers have questioned what influence this introduced arthropod might have upon the avian component of riparian ecosystems. From 2009 through 2012 we studied the consequences of biological invasions of the introduced tamarisk shrub and tamarisk leaf beetles on the diets of native birds along the Dolores River in southwestern Colorado, USA. We examined avian foraging behavior, sampled the arthropod community, documented bird diets and the use of invasive tamarisk shrubs and tamarisk leaf beetles by birds. We documented D. carinulata abundance, on what plants the beetles occurred, and to what degree they were consumed by birds as compared to other arthropods. We hypothesized that if D. carinulata is an important new avian food source, birds should consume beetles at least in proportion to their abundance. We also hypothesized that birds should forage more in tamarisk in the late summer when tamarisk leaf beetle larvae are more abundant than in early summer, and that birds should select beetle-damaged tamarisk shrubs. We found that D. carinulata composed 24.0 percent (±?19.9–27.4%) and 35.4% biomass of all collected arthropods. From the gut contents of 188 birds (25 passerine species), only four species (n?=?11 birds) contained tamarisk leaf beetle parts. Although D. carinulata comprised one-quarter of total insect abundance, frequency of occurrence in bird gut contents was only 2.1% by abundance and 3.4% biomass. Birds used tamarisk shrubs for foraging in proportion to their availability, but foraging frequency did not increase during the late summer when more tamarisk leaf beetles were present and birds avoided beetle-damaged tamarisk shrubs. Despite D. carinulata being the most abundant arthropod in the environment, these invasive beetles were not frequently consumed by birds and seem not to provide a significant contribution to avian diets.     

Non-redundancy in seed dispersal and germination by native and introduced frugivorous birds: implications of invasive bird impact on native plant communities

Seed dispersal by vertebrate animals is important for the establishment of many fleshy-fruited plant species. Different frugivorous species can provide different seed dispersal services according to their specific dietary preferences as well as behaviour and body traits (e.g. body size and beak size of birds). Our aim was to study redundancies and complementarities in seed dispersal and germination between the two main native seed disperser birds and the introduced silver pheasant Lophura nycthemera in the temperate Patagonian forests. For this, we collected fresh droppings from the studied species and analyzed seed content. We conducted germination trials for four plant species common in bird droppings; two native species (Aristotelia chilensis and Rhaphithamnus spinosus) and two invasive non-native species (Rubus ulmifolius and Rosa rubiginosa). Both native frugivorous birds and the silver pheasant dispersed fruits of non- native fleshy-fruited plants, but their roles were non-redundant in terms of species dispersed and effect on seed germination. The silver pheasant dispersed a proportionally high number of non-native seeds, while native birds dispersed a high number of native seeds. In addition, the effect of gut treatment in seed germination differed between seed dispersers. Native birds promoted the germination for the two native plant species studied, while the silver pheasant promoted the germination of one non-native plant. This suggests that seed dispersal by the silver pheasant may contribute to the spread of some invasive fleshy-fruited plants in the ecosystems that otherwise would not be dispersed by any other bird. The understanding of redundancies and complementarities on seed dispersal and germination between native and introduced birds will allow improving the management of fleshy-fruited non-native plants.     

Map-based cloning and characterization of <Emphasis Type="Italic">Zea mays male sterility33</Emphasis> (<Emphasis Type="Italic">ZmMs33</Emphasis>) gene,encoding a glycerol-3-phosphate acyltransferase

Key message

Map-based cloning of maize ms33 gene showed that ZmMs33 encodes a sn-2 glycerol-3-phosphate acyltransferase, the ortholog of rice OsGPAT3, and it is essential for male fertility in maize.

Abstract

Genetic male sterility has been widely studied for its biological significance and commercial value in hybrid seed production. Although many male-sterile mutants have been identified in maize (Zea mays L.), it is likely that most genes that cause male sterility are unknown. Here, we report a recessive genetic male-sterile mutant, male sterility33 (ms33), which displays small, pale yellow anthers, and complete male sterility. Using a map-based cloning approach, maize GRMZM2G070304 was identified as the ms33 gene (ZmMs33). ZmMs33 encodes a novel sn-2 glycerol-3-phosphate acyltransferase (GPAT) in maize. A functional complementation experiment showed that GRMZM2G070304 can rescue the male-sterile phenotype of the ms33-6029 mutant. GRMZM2G070304 was further confirmed to be the ms33 gene via targeted knockouts induced by the clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system. ZmMs33 is preferentially expressed in the immature anther from the quartet to early-vacuolate microspore stages and in root tissues at the fifth leaf growth stage. Phylogenetic analysis indicated that ZmMs33 and OsGPAT3 are evolutionarily conserved for anther and pollen development in monocot species. This study reveals that the monocot-specific GPAT3 protein plays an important role in male fertility in maize, and ZmMs33 and mutants in this gene may have value in maize male-sterile line breeding and hybrid seed production.

     

Genetic dissection of seed weight by QTL analysis and detection of allelic variation in Indian and east European gene pool lines of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Key message

Seed weight QTL identified in different populations were synthesized into consensus QTL which were shown to harbor candidate genes by in silico mapping. Allelic variation inferred would be useful in breeding B. juncea lines with high seed weight.

Abstract

Seed weight is an important yield influencing trait in oilseed Brassicas and is a multigenic trait. Among the oilseed Brassicas, Brassica juncea harbors the maximum phenotypic variation wherein thousand seed weight varies from around 2.0 g to more than 7.0 g. In this study, we have undertaken quantitative trait locus/quantitative trait loci (QTL) analysis of seed weight in B. juncea using four bi-parental doubled-haploid populations. These four populations were derived from six lines (three Indian and three east European lines) with parental phenotypic values for thousand seed weight ranging from 2.0 to 7.6 g in different environments. Multi-environment QTL analysis of the four populations identified a total of 65 QTL ranging from 10 to 25 in each population. Meta-analysis of these component QTL of the four populations identified six ‘consensus’ QTL (C-QTL) in A3, A7, A10 and B3 by merging 33 of the 65 component Tsw QTL from different bi-parental populations. Allelic diversity analysis of these six C-QTL showed that Indian lines, Pusajaikisan and Varuna, hold the most positive allele in all the six C-QTL. In silico mapping of candidate genes with the consensus QTL localized 11 genes known to influence seed weight in Arabidopsis thaliana and also showed conserved crucifer blocks harboring seed weight QTL between the A subgenomes of B. juncea and B. rapa. These findings pave the way for a better understanding of the genetics of seed weight in the oilseed crop B. juncea and reveal the scope available for improvement of seed weight through marker-assisted breeding.

     

Biosynthesis of <Emphasis Type="Italic">Veratrum californicum</Emphasis> specialty chemicals in <Emphasis Type="Italic">Camelina sativa</Emphasis> seed

Economically feasible systems for heterologous production of complex secondary metabolites originating from difficult to cultivate species are in demand since Escherichia coli and Saccharomyces cerevisiae are not always suitable for expression of plant and animal genes. An emerging oilseed crop, Camelina sativa, has recently been engineered to produce novel oil profiles, jet fuel precursors, and small molecules of industrial interest. To establish C. sativa as a system for the production of medicinally relevant compounds, we introduced four genes from Veratrum californicum involved in steroid alkaloid biosynthesis. Together, these four genes produce verazine, the hypothesized precursor to cyclopamine, a medicinally relevant steroid alkaloid whose analogs are currently being tested for cancer therapy in clinical trials. The future supply of this potential cancer treatment is uncertain as V. californicum is slow-growing and not amendable to cultivation. Moreover, the complex stereochemistry of cyclopamine results in low-yield syntheses. Herein, we successfully engineered C. sativa to synthesize verazine, as well as other V. californicum secondary metabolites, in seed. In addition, we have clarified the stereochemistry of verazine and related V. californicum metabolites.