Conservation genetics of an ex situ population of <Emphasis Type="Italic">Primula reinii</Emphasis> var. <Emphasis Type="Italic">rhodotricha</Emphasis>, an endangered primrose endemic to Japan on a limestone mountain

Primula reinii var. rhodotricha is a perennial herb endemic to the limestone slope of Mt. Buko, located approximately 50 km northwest of central Tokyo, Japan. In recent years, its natural population size has decreased markedly due to limestone mining, and this species has been assigned to the ‘Critically Endangered (CR)’ category on the latest Japanese Red List. Although a remnant population of this species has been protected by a mining company outside their historical distribution range on Mt. Buko, the ex situ conservation of this endangered plant has been difficult because of insufficient low seed production. The genetic status of ex situ P. reinii var. rhodotricha and related species were investigated to develop an effective conservation plan for this species. Microsatellite analysis indicated that the ex situ population harbors lower genetic diversity than sister taxa, providing molecular evidence for the recent critical status designation of the ex situ population, whereas the presence of rare alleles may imply further potential for seed reproduction by outcrossing. Therefore, an appropriate propagation strategy that considers genetic diversity is needed for restoration and recovery of this critically endangered ex situ primrose population.     

Non-specific symbiotic germination of <Emphasis Type="Italic">Cynorkis purpurea</Emphasis> (Thouars) Kraezl., a habitat-specific terrestrial orchid from the Central Highlands of Madagascar

Orchids, particularly terrestrial taxa, rely mostly on basidiomycete fungi in the Cantharellales and Sebacinales that trigger the process of seed germination and/or initiate the full development of the seedling. During the course of development, orchids may associate with the same fungus, or they may enlist other types of fungi for their developmental needs leading to resilience in a natural setting. This study examined in vitro seed germination and seedling developmental behavior of Cynorkis purpurea, a terrestrial orchid from the Central Highlands of Madagascar. This species is mostly restricted to gallery forests in the Itremo Massif, in moist substrate between rocks bordering streams. The main objective was to understand the influence of diverse mycorrhizal fungi on seed germination and further development of C. purpurea. The study aims to compare symbiotic versus asymbiotic germination and seedling development with seeds and fungi collected from a 13-km² area in the Itremo region. Seeds collected from the wild were sown with diverse orchid mycorrhizal fungi (OMF) spanning 12 operational taxonomic units (OTUs) in three genera (Tulasnella, Ceratobasidium, and Sebacina) acquired from different habitats. Treatments were assessed in terms of the percentage of germinated seeds and fully developed seedlings against those in asymbiotic control media treatments. Overall, OMF significantly improved seedling development within the 12-week experiment period. Sebacina as a genus was the most effective at promoting seedling development of C. purpurea, as well as having the ability to enter into successful symbiotic relationships with orchids of different life forms; this new knowledge may be especially useful for orchid conservation practiced in tropical areas like Madagascar. A Sebacina isolate from an epiphytic seedling of Polystachya concreta was the most effective at inducing rapid seedling development and was among the five that outperformed fungi isolated from roots of C. purpurea. C. purpurea was found to be a mycorrhizal generalist, despite its specific habitat preference, highlighting the complex interaction between the plant, fungi, and the environment. The potential impact on conservation strategies of understanding the requirements for orchid seed germination and development by identifying and using OMF from diverse sources is discussed in detail.     

The key role of accompanying species in the response of the critically endangered <Emphasis Type="Italic">Naufraga balearica</Emphasis> (Apiaceae) to climatic factors

This study addressed the response of Naufraga balearica, a critically endangered plant endemic to Mallorca (Balearic Islands, western Mediterranean Basin), and its adjacent community to climatic factors by assessing whether climate influences plant cover in N. balearica, including oscillation associated with seasonality, and whether the accompanying species influences its response to climatic factors. The cover of N. balearica and two other accompanying species (Erodium reichardii and Dactylis glomerata) were estimated quarterly from permanent plots over 12 years. The original time series of plant cover and climatic variables were transformed to obtain a smoothed and a detrended time series. The tendency of all variables was assessed using a simple linear regression of the smoothed series. The relationship among plant cover and climatic variables were tested by correlation. Naufraga balearica showed a declining trend, while E. reichardii and D. glomerata experienced a stable and an increasing population trend, respectively. However, all species showed an annual cover oscillation with a similar pattern. A new bioclimatic index combining temperature and precipitation variables was a good predictor although extreme meteorological events substantially altered the trends and patterns of the three species. The response of N. balearica to climatic factors, including extreme weather events, is exacerbated by the presence of other species, mainly those grasses with caespitose growth form and with a faster response to the changing environmental factors. In a climate change scenario, N. balearica would be limited to the shadiest grass-free zones. The current situation emphasises the importance of taking conservation action to guarantee the species conservation.     

High genetic diversity in an endangered medicinal plant, <Emphasis Type="Italic">Saussurea involucrata</Emphasis> (<Emphasis Type="Italic">Saussurea</Emphasis>, Asteraceae), in western Tianshan Mountains,China

Saussurea involucrata (Asteraceae) is a medicinal and second-degree national priority endangered plant that is mainly distributed in the high latitude region of the western Tianshan Mountains. The population is fragmented and isolated, and extensive human impact merits a suitable and specific conservation strategy, which can be compiled based on the genetic diversity, population structure, and demographic history. Phylogeographic studies were conducted on a total of five natural populations and 150 individuals were sampled. Data from three cpDNA intergenic spacer regions (trnL-F, matK, and ndhF-rpl32) and nrDNA ITS sequences showed that twelve haplotypes in cpDNA and five haplotypes in nrDNA indicated high genetic diversity among populations sampled (H _T?=?0.820 and 0.756) and within populations sampled (H _S?=?0.792 and 0.721). Additionally, the high genetic diversity did not mirror genetic structure in either cpDNA (F _ST?=?0.03153, G _ST?>?N _ST, p?<?0.05) or nrDNA (F _ST?=?0.03666, meaningless G _ST and N _ST). Two groups (north and south) were determined for a SAMOVA analysis. Based on this analysis, the demographic history was conducted with a Bayesian Skyline Plot and Isolation with Migration analysis, which showed sustainable and stable extension without a marked bottleneck. Divergence time was indicated at c. 6.25 Mya (90%HPD: 15.30–0.22 Mya) in the Miocene, which is consistent with the formation of the Kelasu section of Tianshan. The southern populations in the Bayanbulak and Gonglu regions require additional attention and transplanting would be an effective way to restore rare cpDNA haplotypes, increase effective population size, and migration rate. Our results suggested that in situ conservation of S. involucrata in western Tianshan should be the main strategy for protection and recovery of the species.     

Host-plant patch qualities and presence of a likely competitor species affect the distribution and abundance of a rare British moth, <Emphasis Type="Italic">Cucullia lychnitis</Emphasis>

Understanding the environmental factors that affect the distribution and abundance of rare and threatened species can help identify priority sites for conservation action such as habitat management. The moth Cucullia lychnitis is identified in UK legislation as a priority species for conservation. Its conspicuous larvae feed on a patchily distributed host plant Verbascum nigrum, but are entirely absent from some host plant patches within the species’ range. Environmental variability among patches was investigated to understand factors affecting (i) patch occupancy by C. lychnitis and (ii) abundance of C. lychnitis, where present. Occupancy of individual V. nigrum plants within occupied patches was also investigated. The likelihood of patch occupancy increased with patch size, and decreased with patch isolation. Abundance of C. lychnitis was negatively correlated with patch isolation and with abundance of the weevil Cionus nigritarsis, which exploits the same host plant and may be a competitor. Within occupied patches, larvae of C. lychnitis were significantly more likely to be found on taller plants with more flower spikes; such plants are typical of established rather than newly-created patches. These results may help to guide efforts to conserve C. lychnitis, highlighting the importance of habitat connectivity and indicating potential management actions to promote features positively associated with larval incidence and abundance.     

Optimization of Nutrients and Culture Conditions for Alkaline Protease Production Using Two Endophytic Micrococci: <Emphasis Type="Italic">Micrococcus aloeverae</Emphasis> and <Emphasis Type="Italic">Micrococcus yunnanensis</Emphasis>

An endophytic species of Micrococcus was isolated from Aloe vera leaf (syn. Aloe barbadensis) and screened for protease production with five other species of Micrococcus. Data indicated that endophytic Micrococcus aloeverae AE-6 MCC 2184^T and Micrococcus yunnanensis DSM 21948^T showed efficient protease production potential and secreted active protease at high salt (10%), temperature (40 °C) and in wide range of pH 8–10. Unlike M. yunnanensis DSM 21948^T, protease production by M. aloeverae AE-6 MCC 2184^T was stringently controlled by pH. Protease induction study using different group of peptides, peptide carbohydrates and peptide macronutrient combinations showed variable response with both the organisms. Result indicated that the amount of protease was not directly related to cell biomass but it depends on nature of inducible peptides. In this study we also developed a modified agar-well assay for semi-quantitative data from large number of replicates.     

Molecular variation and population structure in endangered <Emphasis Type="BoldItalic">Limonium</Emphasis><Emphasis Type="BoldItalic">bicolor</Emphasis>: genetic diversity of microsatellite markers and amplified fragment length polymorphism analysis

Knowledge and analysis of the genetic structure of an endangered species is important for its conservation and evolutionary process. Simple sequence repeats (SSRs) and amplified fragment length polymorphisms (AFLPs) were used in evaluation of the genetic diversity and population differentiation in Limonium bicolor (Plumbaginaceae), an endangered herb with high medicinal and horticulture value. A total of 117 alleles were detected with an average 5.85 alleles per locus using SSR and 222 bands from AFLP were amplified in six populations. It was found that L. bicolor was characterized by high levels of genetic polymorphism (100 and 83.78%), low levels of total genetic diversity (\(H_{\mathrm{t}}=\) 0.2824 and 0.2424), and moderate degrees of genetic differentiation among populations (\(\Phi _{\mathrm{ST}} =\) 0.284 and 0.251). Analysis of molecular variance (AMOVA) revealed that the main variation component existed within populations (71.56%; 74.93%) rather than among populations (28.44%; 25.07%). Four main clusters were displayed in the UPGMA using TFPGA, which was consistent with the result of principal coordinate analysis (PCA) using NTSYS. Mutations or infrequent gene flow among populations can increase the plant slowly, thus in situ conservation policies should be implemented first for effective and sustainable development. At the same time, ex situ measures, such as those individuals with rare alleles, to maintain the relationships between individuals and populations are also proposed.     

Micropropagation of Ajuga species: a mini review

The genus Ajuga L., belonging to Lamiaceae family, is widespread. The demand for Ajuga species has risen sharply because of their medicinal, ornamental, and pharmacological properties. These wide-ranging plants are being rapidly depleted due to over-collection for ornamental and medicinal purposes, as well as by habitat destruction and deforestation. Ajuga boninsimae, A. bracteosa, A. ciliate, A. genevensis, A. incisa, A. makinoi, A. multiflora, A. pyramidalis, A. shikotanensis, A. reptans, and A. vestita are categorized and protected as endangered plants. In vitro plant culture has therefore emerged for the conservation and mass clonal propagation of rare plants. This mini-review covers the current in vitro scenario in the propagation of Ajuga species. Adventitious or axillary shoots are initiated on the leaf, petiole and internodes, as well as roots, nodes, and shoot tip explants. Shoot induction is predominantly dependent on plant growth regulators added to the culture medium. Full- or half-strength Murashige and Skoog medium with or without auxin is used for in vitro rooting. Rooted shoots need to be acclimatized in the greenhouse with an estimated 82–100% survival rate.     

Improvement of seedling and panicle blast resistance in <Emphasis Type="Italic">Xian</Emphasis> rice varieties following <Emphasis Type="Italic">Pish</Emphasis> introgression

Rice blast is a serious disease caused by the filamentous ascomycetous fungus Magnaporthe oryzae. Incorporating disease resistance genes in rice varieties and characterizing the distribution of M. oryzae isolates form the foundation for enhancing rice blast resistance. In this study, the blast resistance gene Pish was observed to be differentially distributed in the genomes of rice sub-species. Specifically, Pish was present in 80.5% of Geng varieties, but in only 2.3% of Xian varieties. Moreover, Pish conferred resistance against only 23.5% of the M. oryzae isolates from the Geng-planting regions, but against up to 63.2% of the isolates from the Xian-planting regions. Thus, Pish may be an elite resistance gene for improving rice blast resistance in Xian varieties. Therefore, near-isogenic lines (NILs) with Pish and the polygene pyramid lines (PPLs) PPL^Pish/Pi1, PPL^Pish/Pi54, and PPL^Pish/Pi33 in the Xian background Yangdao 6 were generated using a molecular marker-assisted selection method. The results suggested that (1) Pish significantly improved rice blast resistance in Xian varieties, which exhibited considerably improved seedling and panicle blast resistance after Pish was introduced; (2) PPLs with Pish were more effective than the NILs with Pish regarding seedling and panicle blast resistance; (3) the PPL seedling and panicle blast resistance was improved by the complementary and overlapping effects of different resistance genes; and (4) the stability of NIL and PPL resistance varied under different environmental conditions, with only PPL^Pish/Pi54 exhibiting highly stable resistance in three natural disease nurseries (Jianyang, Jinggangshan, and Huangshan). This study provides new blast resistance germplasm resources and describes a novel molecular strategy for enhancing rice blast resistance.     

Development and characterization of chloroplast microsatellite markers for <Emphasis Type="BoldItalic">Pinus massoniana</Emphasis> and their application in <Emphasis Type="BoldItalic">Pinus</Emphasis> (Pinaceae) species

Pinus massoniana is one of the important afforestation and pioneer tree species, which is widely distribute in southern China. Chloroplast simple sequence repeat markers (cpSSRs) have been widely used in studies of tree genetics, phylogenetic and breeding. We sequenced the whole chloroplast genome sequences of P. massoniana using PCR and Sanger sequencing. A total of 71 cpSSRs were identified, among which mononucleotide repeats were predominant (70.42%). Seventeen primer pairs were developed and amplification tests were conducted with 15 P. massoniana individuals. Also, cross-species amplification tests were conducted among 15 individuals per Pinus species, including P. elliottii, P. bungeana, P. armandii, P. caribaea, P. tabulaeformis, P. taiwanensis and P. yunnanensis which revealed polymorphic information content ranging from 0.2 to 0.8 and average of haploid diversity (h) ranging from 0.29 to 0.63. In addition, the polymorphic cpSSRs were useful in distinguishing the sampled pine species, and could be powerful tool in phylogenetic studies.     

Cryopreservation of fruit germplasm

Most temperate fruit species are genetically heterozygous and vegetatively propagated. Active collections of fruit genetic resources in Germany are generally maintained in the field, e.g., as potted plants for Fragaria and as trees for pome and stone fruit species. The plant material in active collections should be kept in duplicate to ensure security in case of disease or environmental disaster. The aim of this study was to develop an efficient complementary conservation strategy for fruit genetic resources. Although costly, fruit tree cultivars can be duplicated as field collections at a second site within the framework of the German Fruit Genebank, which is a decentralized fruit-specific network. Wild species accessions, particularly those of the genera Malus spp. (apple) and Fragaria spp. (strawberry) as well as strawberry cultivars, can also be duplicated by means of cryopreservation. In the current study, long-term cryopreservation was initiated for 194 Fragaria genotypes. A protocol combining vitrification with cold acclimation was effective and highly reproducible, with an average regrowth rate of 86%. In Malus, a general cryopreservation protocol based on dormant winter buds was adopted. Based on the results provided in this study, a combination of traditional ex situ conservation and cryopreservation can greatly improve the stability and security of fruit germplasm conservation.     

Disease protection and allelopathic interactions of seed-transmitted endophytic pseudomonads of invasive reed grass (<Emphasis Type="Italic">Phragmites australis</Emphasis>)

Background and aims

Non-native Phragmites australis (haplotype M) is an invasive grass that decreases biodiversity and produces dense stands. We hypothesized that seeds of Phragmites carry microbes that improve seedling growth, defend against pathogens and maximize capacity of seedlings to compete with other plants.

Methods

We isolated bacteria from seeds of Phragmites, then evaluated representatives for their capacities to become intracellular in root cells, and their effects on: 1.) germination rates and seedling growth, 2.) susceptibility to damping-off disease, and 3.) mortality and growth of competitor plant seedlings (dandelion (Taraxacum officionale F. H. Wigg) and curly dock (Rumex crispus L.)).

Results

Ten strains (of 23 total) were identified and characterized; seven were identified as Pseudomonas spp. Strains Sandy LB4 (Pseudomonas fluorescens) and West 9 (Pseudomonas sp.) entered root meristems and became intracellular. These bacteria improved seed germination in Phragmites and increased seedling root branching in Poa annua. They increased plant growth and protected plants from damping off disease. Sandy LB4 increased mortality and reduced growth rates in seedlings of dandelion and curly dock.

Conclusions

Phragmites plants associate with endophytes to increase growth and disease resistance, and release bacteria into the soil to create an environment that is favorable to their seedlings and less favorable to competitor plants.

     

New species and new Chinese records of Bionectriaceae (Hypocreales,Ascomycota)

Collections of bionectriaceous fungi from different areas of China were examined, in which 3 new species were encountered. Bionectria intermedia is characterized by smooth perithecia, 2-layered perithecial wall, cylindrical to clavate asci with an apical ring, ascospore striations composed of separate warts, and dimorphic conidiophores. Hydropisphaera yunnanensis has hairy ascomata which are cupulate when dry, clavate asci with a simple apex, and spinulose and very narrow ascospores fusiform and constricted at septum. Nectriopsis apiosporae possesses laterally pinched perithecia when dry, 1-layered perithecial wall, rough perithecial surface, clavate asci with a simple apex, spinulose ascospores with 3 septa, and on Apiospora sp. Four species, Bionectria epichloë, B. kowhaii, B. subquaternata and Hydropisphaera suffulta, are reported as new to China.     

Identification and pyramiding of QTLs for cold tolerance at the bud bursting and the seedling stages by use of single segment substitution lines in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Cold stress is one of the main constraints in rice production, and damage from cold can occur at different developmental stages in rice. Understanding the genetic basis of cold tolerance is the key for breeding cold-tolerant variety. In this study, we used single segment substitution lines (SSSLs) derived from a cross between cold-tolerant japonica variety “Nan-yang-zhan” and a popular indica variety “Hua-jing-xian 74” to detect and pyramid QTLs for cold tolerance at the bud bursting and the seedling stages. Evaluation of cold tolerance of these SSSLs and their recurrent parent helped identify two cold-tolerant QTLs (qCTBB-5 and qCTBB-6) at the bud bursting stage and two cold-tolerant QTLs (qCTS-6 and qCTS-12) at the seedling stage. The SSSLs carrying these QTLs showed stronger cold tolerance than their recurrent parent HJX74 did in three independent experiments. The qCTBB-6 and qCTS-6 were mapped to the same chromosomal region. QTL pyramiding was performed by intercrossing of SSSLs carrying the respective QTLs for cold tolerance at the bud bursting stage and the seedling stage and marker-assisted selection (MAS). The selected pyramiding line SC1-1 with different cold-tolerant QTLs showed cumulative effects on cold tolerance. Our results suggest that different genes (QTLs) control cold tolerance at bud bursting and seedling stages, and pyramiding of stable expression QTLs for cold tolerance at different developmental stages through MAS is a good strategy to prevent cold damage in rice.     

Sexual reproduction of clonal dwarf shrubs in a forest–tundra ecotone

Successful sexual reproduction may be more important for regeneration of clonal species in high-latitude and -altitude areas than has been previously suggested. We investigated the potential of Vaccinium myrtillus, V. vitis-idaea and Empetrum nigrum ssp. hermaphroditum (E. hermaphroditum) for sexual reproduction at three sites in the forest–tundra ecotone in Finnish Lapland. We studied whether the potential differs between plant communities, whether disturbance enhances germination, and whether seedling emergence is limited by seed availability. We established a field experiment with disturbance and sowing treatments, and monitored seed and seedling numbers and survival rates for two years. The number of mature seeds of V. myrtillus was higher in plants from the tundra heath than in those from the coniferous and mountain birch forests. The number of mature seeds and seedlings emerging from the seed bank of E. hermaphroditum tended also to be higher in the tundra heath. Disturbance marginally increased the seedling emergence of V. myrtillus and E. hermaphroditum, whereas sowing generally increased the seedling numbers. The seedling number of V. myrtillus was lower in the tundra heath and that of E. hermaphroditum was lower in the coniferous forest than at the other sites. Seedling survival was equal for all plant species at all sites. We conclude that the capacity for sexual reproduction varies among plant communities, and seed availability is a stronger constraint than microsite availability for the studied species. Once the crucial early phase of seedling establishment is overcome, seedling survival enables successful recruitment of V. myrtillus, V. vitis-idaea and E. hermaphroditum in the subarctic area.     

Diversity of fungi associated with roots of <Emphasis Type="Italic">Calanthe</Emphasis> orchid species in Korea

While symbiotic fungi play a key role in the growth of endangered Calanthe orchid species, the relationship between fungal diversity and Calanthe species remains unclear. Here, we surveyed root associated fungal diversity of six Calanthe orchid species by sequencing the internal transcribed spacer (ITS) region using 454 pyrosequencing. Our results revealed that Paraboeremia and Coprinopsis are dominant fungal genera among Calanthe species. In terms of overall relative abundance, Paraboeremia was the most common fungal genus associated with Calanthe roots, followed by Coprinopsis. Overall fungal diversity showed a significant degree of variation depending on both location and Calanthe species. In terms of number of different fungal genera detected within Calanthe species, C. discolor had the most diverse fungal community, with 10 fungal genera detected. This study will contribute toward a better understanding of those fungi that are required for successful cultivation and conservation of Korean Calanthe species.     

Adult plant stripe rust resistance gene <Emphasis Type="Italic">Yr71</Emphasis> maps close to <Emphasis Type="Italic">Lr24</Emphasis> in chromosome 3D of common wheat

Australian cultivar Sunco carries three adult plant stripe rust resistance genes. One of these genes corresponded to Yr18 in chromosome 7DS; the second, YrCK, was mapped on chromosome 2D. Here, we describe the characterization of the third adult plant resistance (APR) gene from Sunco. Sunco/2*Avocet S-derived lines SA65 (resistant) and SA67 (susceptible) were crossed and a recombinant inbred line F₆ population was generated. Monogenic segregation among SA65/SA67-derived RIL population was demonstrated and the resistance locus was designated YrSA3. Selective genotyping using an iSelect 90 K Infinium SNP array and SSR markers located YrSA3 on chromosome 3D. Development of KASP markers for SNP loci showing association with YrSA3 allowed construction of a genetic map harboring the resistance gene. Ten KASP markers (KASP_8306, KASP_9142, KASP_10438, KASP_16434, KASP_17207, KASP_20836, KASP_23518, KASP_23615, KASP_57983 and KASP_63653), one SSR marker (gwm114b) and Lr24/Sr24 were mapped 1.8 cM distal to YrSA3. Comparison of marker data indicated that the previously named seedling stripe rust resistance gene Yr45 was located proximal to YrSA3, and therefore the latter was formally designated Yr71. Two recombinants carrying Lr24/Sr24 and Yr71 in combination were identified for use as donor sources in wheat breeding programs. The robustness of gwm114b, KASP_16434, KASP_17207 and KASP_20836 for marker-assisted selection of these genes was demonstrated through tests on 74 Australian wheat cultivars.