Genome assembly of autotetraploid Actinidia arguta highlights adaptive evolution and enables dissection of important economic traits.

Actinidia arguta, the most widely distributed Actinidia species and the second cultivated species in the genus, can be distinguished from the currently cultivated Actinidia chinensis on the basis of its small and smooth fruit, rapid softening, and excellent cold tolerance. Adaptive evolution of tetraploid Actinidia species and the genetic basis of their important agronomic traits are still unclear. Here, we generated a chromosome-scale genome assembly of an autotetraploid male A. arguta accession. The genome assembly was 2.77 Gb in length with a contig N50 of 9.97 Mb and was anchored onto 116 pseudo-chromosomes. Resequencing and clustering of 101 geographically representative accessions showed that they could be divided into two geographic groups, Southern and Northern, which first diverged 12.9 million years ago. A. arguta underwent two prominent expansions and one demographic bottleneck from the mid-Pleistocene climate transition to the late Pleistocene. Population genomics studies using paleoclimate data enabled us to discern the evolution of the species' adaptation to different historical environments. Three genes (AaCEL1, AaPME1, and AaDOF1) related to flesh softening were identified by multi-omics analysis, and their ability to accelerate flesh softening was verified through transient expression assays. A set of genes that characteristically regulate sexual dimorphism located on the sex chromosome (Chr3) or autosomal chromosomes showed biased expression during stamen or carpel development. This chromosome-level assembly of the autotetraploid A. arguta genome and the genes related to important agronomic traits will facilitate future functional genomics research and improvement of A. arguta.

Characterizing Tetraploid Populations of Actinidia chinensis for Kiwifruit Genetic Improvement.

Understanding genetic diversity and structure in natural populations and their suitable habitat response to environmental changes is critical for the protection and utilization of germplasm resources. We evaluated the genetic diversity and structure of 24 A. chinensis populations using simple sequence repeat (SSR) molecular markers. The potential suitable distribution of tetraploid A. chinensis estimated under the current climate and predicted for the future climate was generated with ecological niche modeling (ENM). The results indicated that the polyploid populations of A.chinensis have high levels of genetic diversity and that there are distinct eastern and western genetic clusters. The population structure of A. chinensis can be explained by an isolation-by-distance model. The results also revealed that potentially suitable areas of tetraploids will likely be gradually lost and the habitat will likely be increasingly fragmented in the future. This study provides an extensive overview of tetraploid A. chinensis across its distribution range, contributing to a better understanding of its germplasm resources. These results can also provide the scientific basis for the protection and sustainable utilization of kiwifruit wild resources.

Cytotype distribution and chloroplast phylogeography of the Actinidia chinensis complex.

BACKGROUND: Plant phylogeographic studies of species in subtropical China have mainly focused on rare and endangered species, whereas few studies have been conducted on taxa with relatively wide distribution, especially polyploid species. We investigated the cytotype and haplotype distribution pattern of the Actinidia chinensis complex, a widespread geographically woody liana with variable ploidy in subtropical China comprising two varieties, with three chloroplast fragments DNA (ndhF-rpl132, rps16-trnQ and trnE-trnT). Macroevolutionary, microevolutionary and niche modeling tools were also combined to disentangle the origin and the demographic history of the species or cytotypes. RESULTS: The ploidy levels of 3338 individuals from 128 populations sampled throughout the species distribution range were estimated with flow cytometry. The widespread cytotypes were diploids followed by tetraploids and hexaploids, whereas triploids and octoploids occurred in a few populations. Thirty-one chloroplast haplotypes were detected. The genetic diversity and genetic structure were found to be high between varieties (or ploidy races) chinensis and deliciosa. Our results revealed that these two varieties inhabit significantly different climatic niche spaces. Ecological niche models (ENMs) indicate that all varieties' ranges contracted during the Last Inter Glacial (LIG), and expanded eastward or northward during the Last Glacial Maximum (LGM). CONCLUSIONS: Pliocene and Plio-Pleistocene climatic fluctuations and vicariance appear to have played key roles in shaping current population structure and historical demography in the A. chinensis complex. The polyploidization process also appears to have played an important role in the historical demography of the complex through improving their adaptability to environmental changes.

Characterization and development of EST-derived SSR markers in Sinowilsonia henryi (Hamamelidaceae).

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed to reveal the genetic diversity of extant populations and the mating system of Sinowilsonia henryi (Hamamelidaceae). METHODS AND RESULTS: In this study, nuclear simple sequence repeat (SSR) markers were developed using the Illumina high-throughput sequencing technique (RNA-Seq). The de novo-assembled transcriptome generated a total of 64,694 unique sequences with an average length of 601 bp. A total of 2941 microsatellite loci were detected. Of the 121 tested loci, 13 loci were polymorphic and eight were monomorphic among 72 individuals representing three natural populations of the species. The number of alleles per locus ranged from one to four, and the observed and expected heterozygosity at population level were 0.00-1.00 and 0.10-0.66, respectively. CONCLUSIONS: The developed expressed sequence tag (EST)-SSRs will be useful for studying genetic diversity of S. henryi as well as assessing the mating system among Sinowilsonia species.

Phylogeography and genetic structure of a Tertiary relict tree species, Tapiscia sinensis (Tapisciaceae): implications for conservation.

BACKGROUND AND AIMS: The phylogeography of plant species in sub-tropical China remains largely unclear. This study used Tapiscia sinensis, an endemic and endangered tree species widely but disjunctly distributed in sub-tropical China, as a model to reveal the patterns of genetic diversity and phylogeographical history of Tertiary relict plant species in this region. The implications of the results are discussed in relation to its conservation management. METHODS: Samples were taken from 24 populations covering the natural geographical distribution of T. sinensis. Genetic structure was investigated by analysis of molecular variance (AMOVA) and spatial analysis of molecular variance (SAMOVA). Phylogenetic relationships among haplotypes were constructed with maximum parsimony and haplotype network methods. Historical population expansion events were tested with pairwise mismatch distribution analysis and neutrality tests. Species potential range was deduced by ecological niche modelling (ENM). KEY RESULTS: A low level of genetic diversity was detected at the population level. A high level of genetic differentiation and a significant phylogeographical structure were revealed. The mean divergence time of the haplotypes was approx. 1·33 million years ago. Recent range expansion in this species is suggested by a star-like haplotype network and by the results from the mismatch distribution analysis and neutrality tests. CONCLUSIONS: Climatic oscillations during the Pleistocene have had pronounced effects on the extant distribution of Tapiscia relative to the Last Glacial Maximum (LGM). Spatial patterns of molecular variation and ENM suggest that T. sinensis may have retreated in south-western and central China and colonized eastern China prior to the LGM. Multiple montane refugia for T. sinense existing during the LGM are inferred in central and western China. The populations adjacent to or within these refugia of T. sinense should be given high priority in the development of conservation policies and management strategies for this endangered species.

The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis.

Actinidia chinensis is an important economic plant belonging to the basal lineage of the asterids. Availability of a complete Actinidia chloroplast genome sequence is crucial to understanding phylogenetic relationships among major lineages of angiosperms and facilitates kiwifruit genetic improvement. We report here the complete nucleotide sequences of the chloroplast genomes for Actinidia chinensis and A. chinensis var deliciosa obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. The total genome size ranges from 155,446 to 157,557 bp, with an inverted repeat (IR) of 24,013 to 24,391 bp, a large single copy region (LSC) of 87,984 to 88,337 bp and a small single copy region (SSC) of 20,332 to 20,336 bp. The genome encodes 113 different genes, including 79 unique protein-coding genes, 30 tRNA genes and 4 ribosomal RNA genes, with 16 duplicated in the inverted repeats, and a tRNA gene (trnfM-CAU) duplicated once in the LSC region. Comparisons of IR boundaries among four asterid species showed that IR/LSC borders were extended into the 5' portion of the psbA gene and IR contraction occurred in Actinidia. The clap gene has been lost from the chloroplast genome in Actinidia, and may have been transferred to the nucleus during chloroplast evolution. Twenty-seven polymorphic simple sequence repeat (SSR) loci were identified in the Actinidia chloroplast genome. Maximum parsimony analyses of a 72-gene, 16 taxa angiosperm dataset strongly support the placement of Actinidiaceae in Ericales within the basal asterids.

High-temperature inhibition of biosynthesis and transportation of anthocyanins results in the poor red coloration in red-fleshed Actinidia chinensis.

In plants, the role of anthocyanins trafficking in response to high temperature has been rarely studied, and therefore poorly understood. Red-fleshed kiwifruit has stimulated the world kiwifruit industry owing to its appealing color. However, fruit in warmer climates have been found to have poor flesh coloration, and the factors responsible for this response remain elusive. Partial correlation and regression analysis confirmed that accumulative temperatures above 25 °C (T25) was one of the dominant factors inhibiting anthocyanin accumulation in red-fleshed Actinidia chinensis, 'Hongyang'. Expression of structural genes, AcMRP and AcMYB1 in inner pericarp sampled from the two high altitudes (low temperature area), was notably higher than the low altitude (high temperature area) during fruit coloration. AcMYB1 and structural genes coordinate expression supported the MYB-bHLH (basic helix-loop-helix)-WD40 regulatory complex mediated downregulation of anthocyanin biosynthesis induced by high temperatures in kiwifruit. Moreover, cytological observations using the light and transmission electronic microscopy showed that there were a series of anthocyanic vacuolar inclusion (AVI)-like structures involved in their vacuolization process and dissolution of the pigmented bodies inside cells of fruit inner pericarp. Anthocyanin transport was inhibited by high temperature via retardation of vacuolization or reduction in AIV-like structure formation. Our findings strongly suggested that complex multimechanisms influenced the effects of high temperature on red-fleshed kiwifruit coloration.

Variation of epimedins A - C and icariin in ten representative populations of Epimedium brevicornu Maxim., and implications for utilization.

The concentration variations of main flavonoids, epimedins A-C and icariin, among ten representative populations of Epimedium brevicornu Maxim. were assessed by HPLC. The populations were collected during the flowering stage and included 419 individual samples. Remarkable variations within and among populations were detected. SXXA Population (see Fig. 1) was an outlier due to its significant low concentrations (<1.00-4.46 mg/g). But even without SXXA, significant concentration differences among populations were still observed in epimedin A (2.31-8.42 mg/g), epimedin B (6.67-55.7 mg/g), epimedin C (5.39-23.0 mg/g), icariin (8.50-39.9 mg/g), and their total (29.1-123 mg/g). All populations except SXXA showed much higher concentrations than the recommended standards (i.e. 5 mg/g for icariin and 13 mg/g for the total). A high-concentration-population structure, estimated both by principal component analysis (PCA) and unweighted pair group method with averaging (UPGMA) cluster analysis, based on Euclidean distances, was observed. Both methods allowed separation of the populations in four groups defined by the concentrations of four main flavonoids. The populations (SXLC and SXQS) located in north of Yellow River were clustered together and characterized by highest concentrations of epimedin B, icariin, and their total. Considering of the high concentrations of main flavonoids and abundant resources, E. brevicornu could be exploited as a good medical resource for Herba Epimedii and would offer a tremendous potential for commercial development, but SXXA population should be paid special attention, and further study is needed.

Microsatellite primers in the endangered quillwort Isoetes hypsophila (Isoetaceae) and cross-amplification in I. sinensis.

PREMISE OF THE STUDY: The first microsatellite primers were developed for Isoetes hypsophila, an endangered quillwort species endemic to the Qinghai-Tibetan Plateau in China, to further describe its genetic variability and population structure. We also examined their cross-amplification in a congeneric species, I. sinensis. METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences COntaining Repeats (FIASCO) protocol, nine microsatellite loci were isolated and characterized in 32 samples from four natural populations of I. hypsophila. The primers amplified di- and hexanucleotide repeats with three to 11 alleles per locus. Seven of nine primers were cross-amplified in I. sinensis with two to seven alleles per locus. CONCLUSION: The microsatellite loci primers will be useful for studies of genetic diversity and gene flow in natural populations of Isoetes species.

Microsatellite primers in the Chinese dove tree, Davidia involucrata (Cornaceae), a relic species of the Tertiary.

PREMISE OF THE STUDY: The first microsatellite primers were developed for Davidia involucrata, an endangered relic species of the Tertiary in China, to further describe its genetic variability and population structure. METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences Containing Repeats (FIASCO) protocol, 15 polymorphic microsatellite loci were isolated and characterized in 20 individuals from the germplasm collections of D. involucrata at the Hunan Forest Botanical Garden. High levels of polymorphism were revealed, with the total number of alleles per locus and the number of alleles per locus per individual ranging from two to 13 and from one to six, respectively. CONCLUSIONS: The multibanded patterns of microsatellite loci obtained in the current study confirmed that D. involucrata might be a polyploid species. The primers will be useful for studies of genetic diversity and for guiding conservation strategies for D. involucrata.

Development of microsatellite markers in Actinidia arguta (Actinidiaceae) based on the NCBI data platform.

PREMISE OF THE STUDY: Expressed sequence tag (EST)-derived microsatellite markers in Actinidia arguta were developed for genotyping and genetic mapping. METHODS AND RESULTS: One hundred and fifty EST-simple sequence repeat (SSR) primer pairs were obtained from the National Center for Biotechnology Information (NCBI) A. chinensis database using SSR Hunter 1.3. With the other 20 reported primers, 170 primer pairs were screened using 16 samples. A total of 72 primers pairs were successively developed for A. arguta. Fifteen of the developed markers were characterized in A. arguta populations from Changbai Mountain and Daba Mountain. The mean number of alleles per locus in the Changbai and Daba populations was 5.133 and 4.133, respectively. CONCLUSIONS: Development of Actinidia ESTs from the NCBI database is an effective method of obtaining SSR markers for A. arguta. These markers will be useful for genome mapping and molecular breeding in A. arguta.

Fourteen microsatellite loci for the Chinese medicinal plant Epimedium sagittatum and cross-species application in other medicinal species.

Epimedium sagittatum is a well-known medicinal plant as well as potential ground cover and ornamental plant. A total of 14 polymorphic microsatellite markers were developed for use in taxonomy, phylogenetics and conservation genetics. Markers were presented between two and eight alleles per locus. The observed heterozygosity (H(O) ) and expected heterozygosity (H(E) ) ranged from 0.03 to 0.81 and from 0.05 to 0.81, respectively. Cross-amplification among other four medicinal species revealed promising results in three to six polymorphic loci. These microsatellite loci contributed to the limited number of useful markers currently available for E. sagittatum.

Simultaneous extraction of epimedin A, B, C and icariin from Herba Epimedii by ultrasonic technique.

An ultrasonic-assisted extraction (UAE) procedure of epimedin A, epimedin B, epimedin C and icariin from Herba Epimedii was developed. The effects of ethanol concentration, ratio of liquid to solid, UAE time, extraction temperature and number of extraction cycles on the extraction yields of the four flavonoids from Herba Epimedii were investigated. The optimal UAE condition was found using orthogonal test: 50% (v/v) ethanol solution, liquid:solid ratio of 30 ml/g, ultrasonication duration 30 min, extraction temperature 50 degrees C and three extraction cycles. The UAE method showed a high reproducibility. Epimedin A, B, C and icariin in the crude extract exhibited photodegradation under ultraviolet irradiation. This UAE method was shown to be highly efficient compared with the conventional Soxhlet extraction and boiling extraction. The effect of ultrasound on cell destruction was examined by scanning electron microscopy. The contents of epimedin A, B, C and icariin in the leaves of 20 Epimedium species were determined using high-performance liquid chromatographic method following UAE method.

Genetic diversity and population differentiation of chestnut blight fungus, Cryphonectria parasitica, in China as revealed by RAPD.

Seventeen Cryphonectria parasitica populations sampled from six regions in China were investigated using RAPD. Across all 169 isolates from the 17 populations evaluated, 52 of the 71 markers (73%) were polymorphic, total genetic diversity (h) was 0.1463, and Shannon's index was 0.2312. Diversity within populations accounted for 74% of total genetic diversity, and genetic differentiation among populations was 0.26 (G (ST) = 0.26). Gene flow was 1.4 among the populations; higher gene flow was found among populations within regions and among regions [N (m) (G (SR)) = 2.8 and N (m) (G (RT)) = 3.5]. The unweighted pair group mean analysis (UPGMA) dendrogram revealed two distinct clusters: the northern China group and the southern China group. The spatial autocorrelation analysis revealed that the variation at most loci was randomly distributed and lacked spatial structure, but several loci and closer distances were spatially structured. Human activity and habitat could also be important factors affecting genetic structure among C. parasitica populations in China. Genetic diversity was highest in Southwest China, descending in an orderly fashion to Northeast China. This pattern indicated that Southwest China might be the center of origin of C. parasitica in China. The present study provides useful information for understanding the origin and spread of chestnut blight fungus in China and valuable data for formulating relevant strategies for controlling the disease in China.

Allozyme diversity and population genetic structure of three medicinal Epimedium species from Hubei.

Three Epimedium species, E. pubescens Maxim., E. sagittatum (Sieb. & Zucc.) Maxim., and E. wushanense T. S. Ying, which are sympatrically distributed in the western Hubei Province, have been used in traditional Chinese medicine (TCM) for about 2,000 years. Genetic variability and population genetic structure of 11 natural populations of these Epimedium species were investigated using isoelectric focusing in thin-layer polyacrylamide slab gels. Of the 22 enzyme systems prescreened, six coding for 13 loci and 45 alleles were resolved, which were used for analyzing genetic diversity and population structure at both intraspecific and interspecific levels. The results showed that: 1) high levels of genetic diversity were observed in all three species (A = 2.6-3.2, P = 69.2%-84.6%, H(O) = 0.274-0.377, H(E) = 0.282-0.369), which were higher than that of other herbaceous and animal-pollinated species with similar life-history characteristics; 2) there was significant deviation from Hardy-Weinberg Equilibrium, with one half of the loci showing heterozygote excess and the other homozygote excess, in all populations, suggesting the complicated breeding system of Epimedium species; 3) the low level of intraspecific and interspecific genetic differentiation (G(ST) = 0.0246-0.0409 and 0.0495-0.1213, respectively) indicated a high level of gene flow among populations and close genetic relationship among the three species; and 4) UPGMA cluster analysis further showed that E. pubescens was more closely related to E. sagittatum than to E. wushanense, which was in good agreement with the morphological characters and the recent phylogenetic analysis of these species. On the basis of these results, it was concluded that the mixed breeding system, long-lived perennial life form, ancient evolutionary history, and seed dispersal by ants in Epimedium are responsible for the genetic variation and population structure of these species.