Tetraploid Species Research Articles

Nomenclatural and taxonomic comments on some taxa of Chenopodiaceae of the Himalayas and Tibet/Xizang

Nomenclatural corrections and comments are provided on several taxa of Chenopodiaceae occurring in the Himalayas and Xizang/Tibet and adjacent areas, following the recent monographic revision of the family in that region and earlier publications. In particular, the original identity of the name Atriplex bengalensis (Chenopodium bengalense) is discussed and it is confirmed, based on additional evidence, that the name was originally (before its epitypification in 2014) applicable to a robust diploid of the Chenopodium ficifolium aggregate, not to the robust hexaploid currently known as C. giganteum. It is thus also concluded that the recent proposal by Mosyakin and Mandák (2018) to conserve the name C. giganteum with a conserved type corresponding to the current understanding and application of that name will best serve nomenclatural stability. A nomenclatural solution alternative to the proposal to reject the name A. bengalensis might be the following: (1) to conserve the name Atriplex bengalensis with a conserved C. ficifolium against C. bengalense. The nomenclaturally paradoxical situation with the names Chenopodium pallidum, C. harae, and Atriplex pallida (all now considered homotypic, as justified by Mosyakin and McNeill in 2018), which emerged from the conflicting lectotypification and epitypification of the name C. pallidum, is revisited and reconsidered. Possible options for dealing with that nomenclatural problem are outlined: (1) keeping the status quo, (2) proposing to conserve the name C. pallidum with a conserved type other than the standing lectotype, and (3) proposing to reject the name C. pallidum. The last option is considered preferable. Additional considerations are presented on a possible taxonomic identity of Chenopodium strictum as originally described by Roth; it is confirmed that that name was misapplied to a widespread Eurasian tetraploid species now properly known as C. betaceum. The identity of the name Bassia fiedleri is discussed; being a replacement name for Echinopsilon divaricatum, it is homotypic with Bassia divaricata (Kar. & Kir.) Kuntze (nom. illeg., non F. Muell.) and is a taxonomic synonym of Grubovia dasiphylla (as correctly stated by Kadereit and Freitag in 2011), but not a synonym of Bassia scoparia. Several comments on type designations of selected taxa of Chenopodiaceae from the Sino-Himalayan region are provided as well; e.g., for Acroglochin persicarioides and associated names, Chenopodium karoi, and Salsola monoptera.

Some Mechanisms Modulating the Root Growth of Various Wheat Species under Osmotic-Stress Conditions.

The role of the root in water supply and plant viability is especially important if plants are subjected to stress at the juvenile stage. This article describes the study of morphophysiological and cytological responses, as well as elements of the anatomical structure of primary roots of three wheat species, Triticum monococcum L., Triticum dicoccum Shuebl., and Triticum aestivum L., to osmotic stress. It was shown that the degree of plasticity of root morphology in water deficit affected the growth and development of aboveground organs. It was found that in conditions of osmotic stress, the anatomical root modulations were species-specific. In control conditions the increase in absolute values of root diameter was reduced with the increase in the ploidy of wheat species. Species-specific cytological responses to water deficit of apical meristem cells were also shown. The development of plasmolysis, interpreted as a symptom of reduced viability apical meristem cells, was revealed. A significant increase in enzymatic activity of superoxide dismutase under osmotic stress was found to be one of the mechanisms that could facilitate root elongation in adverse conditions. The tetraploid species T. dicoccum Shuebl. were confirmed as a source of traits of drought tolerant primary root system for crosses with wheat cultivars.

Collections‐based systematics and biogeography in the 21st century: A tribute to Dr. Vicki Funk

Collections‐based systematics and biogeography in the 21st century: A tribute to Dr. Vicki Funk

Differentiate responses of tetraploid and hexaploid wheat (Triticum aestivum L.) to moderate and severe drought stress: a cue of wheat domestication

ABSTRACT Differentiate mechanism of wheat species in response to contrasting drought stress gradients implies a cue of its long-term domestication. In the present study, three water regimes including well-watered control (WW, 80% field water capacity (FC)), moderate drought stress (MS, 50% FC,) and severe drought stress (SS, 30% FC) were designed to reveal different responses of eight wheat species (four tetraploid and four hexaploid) representing different breeding decades and genetic origins to drought stresses. The data indicated that 50% FC and 30% FC fell into the soil moisture threshold range of non-hydraulic and hydraulic root signal occurrence, respectively. In general, grain yield, grain number/spike weight per plant, aboveground biomass, harvest index (HI) and water use efficiency (WUE) were significantly higher in hexaploid species than those of tetraploid species under drought stress (P < .05). Particularly, non-hydraulic root signal was triggered and continuously operated under 50% FC, while hydraulic root signal was observed under 30% FC, respectively. Under 80% FC, the allometric exponent (ɑ) of Maboveground vs Mroot decreased from tetraploid to hexaploid (both were of <1), indicating that during the domestication, the hexaploid species allocated less biomass to root system. For the relationship of Mear vs Mvegetative, the ɑ value was significantly greater in the hexaploid species, showing that hexaploid wheat distributed more biomass to ear than tetraploid to improve yield. Under 50% FC, this trend was enhanced. However, under 30% FC, there was no significant difference in the ɑ value between two species. Additionally, correlation analyses on yield formation affirmed the above results. Therefore, drought tolerance tended to be enhanced in hexaploid species under the pressure of artificial selection than that of tetraploid species. When drought stress exceeded a certain threshold, both species would be negatively seriously affected and followed a similar mechanism for better survival.

Naturally occurring triploid birch hybrids from woodlands in Iceland are partially fertile

Woodlands in Iceland are characterized by shrub-like birch, a result of introgressive hybridization between diploid dwarf birch, Betula nana, and tetraploid downy birch, B. pubescens. Introgression occurs via triploid hybrids. We evaluated the fertility of triploid birch by examining pollen viability and seed germination. Pollen samples were collected from woodlands throughout Iceland from 99 plants, including 22 triploid hybrids. The germination of pollen from the triploid hybrids was about one third that of the diploid or tetraploid species (mean values: 9% vs. 26% and 29%). Most triploid plants had a pollen germination of less than 2%. However, some showed exceptionally high pollen fertility (six plants: 11–79%). Microscopic examination revealed normal pollen tube growth and elongation in hybrids with low germination, while other hybrids showed abnormal tube growth. Fluorescence microscopy indicated pollen viability in all ploidy groups, with green autofluorescence from the cytoplasmic contents and red fluorescence from the exine wall. The Aniline test of pollen viability showed that grains with three pores (normal, triporate grains) stained positively, but non-triporate grains were unstained and empty. Germination tests were performed on seeds collected from 246 plants, including 21 triploid hybrids. The germination percentage of seeds from triploid hybrid plants was about 20 times lower than that of the diploid or the tetraploid species (population means: 0–4% vs. 3–41% and 6–54%). The present study shows that triploid birch hybrids are not sterile. Both the paternal and maternal fertility of the hybrids should be sufficient to facilitate gene flow via backcrossing with the parental species.

Recent genome duplications facilitate the phenotypic diversity of Hb repertoire in the Cyprinidae.

Whole-genome duplications (WGDs) are an important contributor to phenotypic innovations in evolutionary history. The diversity of blood oxygen transport traits is the perfect reflection of physiological versatility for evolutionary success among vertebrates. In this study, the evolutionary changes of hemoglobin (Hb) repertoire driven by the recent genome duplications were detected in representative Cyprinidae fish, including eight diploid and four tetraploid species. Comparative genomic analysis revealed a substantial variation in both membership composition and intragenomic organization of Hb genes in these species. Phylogenetic reconstruction analyses were conducted to characterize the evolutionary history of these genes. Data were integrated with the expression profiles of the genes during ontogeny. Our results indicated that genome duplications facilitated the phenotypic diversity of the Hb gene family; each was associated with species-specific changes in gene content via gene loss and fusion after genome duplications. This led to repeated evolutionary transitions in the ontogenic regulation of Hb gene expression. Our results revealed that genome duplications helped to generate phenotypic changes in Cyprinidae Hb systems.

Evolutionary dynamics of transposable elements and satellite DNAs in polyploid Spartina species

Repeated sequences and polyploidy play a central role in plant genome dynamics. Here, we analyze the evolutionary dynamics of repeats in tetraploid and hexaploid Spartina species that diverged during the last 10 million years within the Chloridoideae, one of the poorest investigated grass lineages. From high-throughput genome sequencing, we annotated Spartina repeats and determined what sequence types account for the genome size variation among species. We examined whether differential genome size evolution correlated with ploidy levels and phylogenetic relationships. We also examined the tempo of repeat sequence dynamics associated with allopatric speciation over the last 3–6 million years between hexaploid species that diverged on the American and European Atlantic coasts and tetraploid species from North and South America. The tetraploid S. spartinae, whose phylogenetic placement has been debated, exhibits a similar repeat content as hexaploid species, suggesting common ancestry. Genome expansion or contraction resulting from repeat dynamics seems to be explained mostly by the contrasting divergence times between species, rather than by genome changes triggered by ploidy level change per se. One 370 bp satellite may be exhibiting ‘meiotic drive’ and driving chromosome evolution in S. alterniflora. Our results provide crucial insights for investigating the genetic and epigenetic consequences of such differential repeat dynamics on the ecology and distribution of the meso- and neopolyploid Spartina species.

Differentiate effects of non-hydraulic and hydraulic root signaling on yield and water use efficiency in diploid and tetraploid wheat under drought stress

Non-hydraulic root signals (nHRS) and hydraulic root signals (HRS) are important mechanisms that play a major role in determining plant productivity under drought stress. In the present study, six wheat varieties (three diploid and three tetraploid) were used to investigate the variation of yield, its components, water use efficiency (WUE) and biomass allocation under two signals. At the jointing stage, three water treatments were set up: i) well-watered control (WW, 80 % field water capacity (FC)), ii) moderate water stress (MS, 50 % FC) and iii) severe water stress (SS, 30 % FC). The results showed that around 50 % FC, wheat activated the nHRS to respond to drought. While around 30 % FC, wheat activated the HRS as a response to deeper drought. Under the operation of nHRS, the reduction of yield, aboveground biomass and WUE of diploid species was greater than that of tetraploid ones, but there were no differences under the operation of HRS. It could be argued that two different wheat species followed differentiate positive defense strategy in response to nHRS, but fell into a similar negative adaptive strategy under the HRS. The ploidy level of wheat also had a significant effect on wheat yield composition, WUE and biomass allocation model. As such, grain yield, grain number and spike weight per plant, aboveground biomass, harvest index (HI) and WUE were significantly greater in the tetraploid species than the diploids ones. In addition, the retention rate of aboveground biomass and grain decreased under the operation of nHRS and HRS. Path analysis showed that spikelet number, thousand grain weight and grain number were the main factors affecting yield formation. Grain number had the greatest direct effect on yield, while spikelet number and its grain number exerted the main effect on yield. These results were consistent under both signals.

A competence of embryo-derived tissues of tetraploid cultivated wheat species Triticum dicoccum and Triticum timopheevii for efficient and stable transgenesis mediated by particle inflow gun

BackgroundThe ability to engineer cereal crops by gene transfer technology is a powerful and informative tool for discovering and studying functions of genes controlling environmental adaptability and nutritional value. Tetraploid wheat species such as emmer wheat and Timopheevi wheat are the oldest cereal crops cultivated in various world areas long before the Christian era. Nowadays, these hulled wheat species are gaining new interest as donors for gene pools responsible for the improved grain yield and quality, tolerance for abiotic and biotic stress, resistance to pests and disease. The establishing of efficient gene transfer techniques for emmer and Timopheevi wheat may help in creation of modern polyploid wheat varieties.ResultsIn the present study, we describe a robust protocol for the production of fertile transgenic plants of cultivated emmer wheat (Russian cv. ‘Runo’) using a biolistic delivery of a plasmid encoding the gene of green fluorescent protein (GFP) and an herbicide resistance gene (BAR). Both the origin of target tissues (mature or immature embryos) and the type of morphogenic calli (white or translucent) influenced the efficiency of stable transgenic plant production in emmer wheat. The bombardment of nodular white compact calluses is a major factor allowed to achieve the highest transformation efficiency of emmer wheat (on average, 12.9%) confirmed by fluorescence, PCR, and Southern blot. In the absence of donor plants for isolation of immature embryos, mature embryo-derived calluses could be used as alternative tissues for recovering transgenic emmer plants with a frequency of 2.1%. The biolistic procedure based on the bombardment of immature embryo-derived calluses was also successful for the generation of transgenic Triticum timopheevii wheat plants (transformation efficiency of 0.5%). Most of the primary events transmitted the transgene expression to the sexual progeny.ConclusionThe procedures described here can be further used to study the functional biology and contribute to the agronomic improvement of wheat. We also recommend involving in such research the Russian emmer wheat cv. ‘Runo’, which demonstrates a high capacity for biolistic-mediated transformation, exceeding the previously reported values for different genotypes of polyploid wheat.

Variable yet vague: Questioning the utility of PHYB for barcoding in Potamogeton

Abstract Potamogeton is a cosmopolitan genus of aquatic plants comprising ca. 72 species and 99 hybrids. Hybridization and polyploidization events are characteristic of its evolutionary history, thus molecular approach is desirable for confirmation of species and hybrids determinations. In this study, we tested the suitability of low-copy nuclear gene PHYB as a barcoding marker, as this gene was shown to have sufficient level of variability in Potamogeton in preceding studies. We cloned and sequenced part of gene PHYB exon 1 for 19 samples of 8 Potamogeton species. Our sampling took place in East Europe, Siberia, Russian Far East and NE North America. The obtained sequences were used to construct a genetic network. We report some features of PHYB which hamper its application as a barcode in Potamogeton. Most of the tetraploid species provided two sets of dissimilar PHYB sequences, which probably stand for two paralogs. PCR-derived errors, such as recombination and random substitutions, shifted the position of some sequences in the network. In addition, one haplotype was shared between two closely related species, breaking the species-specificity of PHYB barcode. Even though it may provide higher resolution for hybrid identification than ITS or 5S-NTS, using it as a marker for standard barcoding in this genus is not the best option.

Maintenance of Species Differences in Closely Related Tetraploid Parasitic Euphrasia (Orobanchaceae) on an Isolated Island

Polyploidy is pervasive in angiosperm evolution and plays important roles in adaptation and speciation. However, polyploid groups are understudied due to complex sequence homology, challenging genome assembly, and taxonomic complexity. Here, we study adaptive divergence in taxonomically complex eyebrights (Euphrasia), where recent divergence, phenotypic plasticity, and hybridization blur species boundaries. We focus on three closely related tetraploid species with contrasting ecological preferences that are sympatric on Fair Isle, a small isolated island in the British Isles. Using a common garden experiment, we show a genetic component to the morphological differences present between these species. Using whole-genome sequencing and a novel k-mer approach we call “Tetmer”, we demonstrate that the species are of allopolyploid origin, with a sub-genome divergence of approximately 5%. Using ∼2 million SNPs, we show sub-genome homology across species, with a very low sequence divergence characteristic of recent speciation. This genetic variation is broadly structured by species, with clear divergence of Fair Isle heathland Euphrasia micrantha, while grassland Euphrasia arctica and coastal Euphrasia foulaensis are more closely related. Overall, we show that tetraploid Euphrasia is a system of allopolyploids of postglacial species divergence, where adaptation to novel environments may be conferred by old variants rearranged into new genetic lineages.

Hepatica transsilvanica Fuss (Ranunculaceae) is an Allotetraploid Relict of the Tertiary Flora in Europe – Molecular Phylogenetic Evidence

The &lt;em&gt;Hepatica &lt;/em&gt;section &lt;em&gt;Angulosa &lt;/em&gt;consists of mainly tetraploid (2&lt;em&gt;n &lt;/em&gt;= 28) species that are distributed disjunctly throughout Eurasia. Karyological evidence proves the hybrid origin of the polyploid species of this section. &lt;em&gt;Hepatica transsilvanica &lt;/em&gt;is a member of this species group with a conspicuous distribution restricted to the Eastern Carpathians. Based on genome size and cytotypes, the paternal parent of &lt;em&gt;H. transsilvanica &lt;/em&gt;is described to be the only diploid species in section &lt;em&gt;Angulosa&lt;/em&gt;, &lt;em&gt;H. falconeri&lt;/em&gt;. The maternal species is hypothesized to be &lt;em&gt;H. nobilis&lt;/em&gt;, a European species with entirely lobed leaves and a wider distribution area. Although the hybrid origin of &lt;em&gt;H. transsilvanica &lt;/em&gt;is well documented by karyological evidence, the time of hybridization has never been studied. By using sequences of both the nuclear and plastid genome, we reconstructed the phylogenetic relationships and divergence times of &lt;em&gt;H. transsilvanica &lt;/em&gt;and its parental species. The identity of the parental species is corroborated by discordant gene tree topologies of the nrITS and plastid sequences. Moreover, both gene copies of the parental species could be identified with the low-copy nuclear gene, &lt;em&gt;MLH1&lt;/em&gt;. Divergence dating analysis using Bayesian phylogenetic methods strongly supported the long-term survival of &lt;em&gt;H. transsilvanica &lt;/em&gt;in the Southeastern Carpathians, as the most recent common ancestor of the hybrid and parent species existed not later than the beginning of the Pleistocene, ca. 3 million years ago. These results not only highlight the biogeographic importance of the Southeastern Carpathians in the Quaternary glaciation periods, but also emphasize that Tertiary lineages could have survived in a Central European cryptic refugium.

Structural Variations Affecting Genes and Transposable Elements of Chromosome 3B in Wheats.

Structural variations (SVs) such as copy number and presence–absence variations are polymorphisms that are known to impact genome composition at the species level and are associated with phenotypic variations. In the absence of a reference genome sequence, their study has long been hampered in wheat. The recent production of new wheat genomic resources has led to a paradigm shift, making possible to investigate the extent of SVs among cultivated and wild accessions. We assessed SVs affecting genes and transposable elements (TEs) in a Triticeae diversity panel of 45 accessions from seven tetraploid and hexaploid species using high-coverage shotgun sequencing of sorted chromosome 3B DNA and dedicated bioinformatics approaches. We showed that 23% of the genes are variable within this panel, and we also identified 330 genes absent from the reference accession Chinese Spring. In addition, 60% of the TE-derived reference markers were absent in at least one accession, revealing a high level of intraspecific and interspecific variability affecting the TE space. Chromosome extremities are the regions where we observed most of the variability, confirming previous hypotheses made when comparing wheat with the other grasses. This study provides deeper insights into the genomic variability affecting the complex Triticeae genomes at the intraspecific and interspecific levels and suggests a phylogeny with independent hybridization events leading to different hexaploid species.

Revisiting the evolution of the North American tetraploid treefrog (Hyla versicolor)

Hyla chrysoscelis and H. versicolor are common treefrogs in eastern North America and are a cryptic diploid-tetraploid species pair. They are morphologically identical but H. versicolor is a tetraploid. They can be identified acoustically by the male's advertisement mating call, which has a pulse repetition rate that has twice as many pulses per second in the diploid species, H. chrysoscelis. We used isozymes, microsatellite DNA alleles, and mitochondrial cytochrome b sequences to test the hypothesis that gene exchange occurs between the diploid and tetraploid species in sympatric populations. Each method provided results that are best explained by occasional hybridization of female H. versicolor and male H. chrysoscelis. We propose that H. versicolor first arose from an autotriploid H. chrysoscelis female that produced unreduced triploid eggs. After H. versicolor became established, genes could be passed from H. chrysoscelis to H. versicolor in sympatric populations when these species hybridize. Their F1 female progeny produce unreduced triploid eggs that are fertilized by haploid H. chrysoscelis sperm to reconstitute H. versicolor. Genes can be passed from diploid H. chrysoscelis to tetraploid H. versicolor in sympatric populations.

Heterogeneity of synonymous substitution rates in the Xenopus frog genome.

With the increasing availability of high quality genomic data, there is opportunity to deeply explore the genealogical relationships of different gene loci between closely related species. In this study, we utilized genomes of Xenopus laevis (XLA, a tetraploid species with (L) and (S) sub-genomes) and X. tropicalis (XTR, a diploid species) to investigate whether synonymous substitution rates among orthologous or homoeologous genes displayed any heterogeneity. From over 1500 orthologous/homoeologous genes collected, we calculated proportion of synonymous substitutions between genomes/sub-genomes (k) and found variation within and between chromosomes. Within most chromosomes, we identified higher k with distance from the centromere, likely attributed to higher substitution rates and recombination in these regions. Using maximum likelihood methods, we identified further evidence supporting rate heterogeneity, and estimated species divergence times and ancestral population sizes. Estimated species divergence times (XLA.L-XLA.S: ~25.5 mya; XLA-XTR: ~33.0 mya) were slightly younger compared to a past study, attributed to consideration of population size in our study. Meanwhile, we found very large estimated population size in the ancestral populations of the two species (NA = 2.55 x 106). Local hybridization and population structure, which have not yet been well elucidated in frogs, may be a contributing factor to these possible large population sizes.

Unravelling Cotton Nonexpressor of Pathogenesis-Related 1(NPR1)-Like Genes Family: Evolutionary Analysis and Putative Role in Fiber Development and Defense Pathway.

The nonexpressor of pathogenesis-related 1 (NPR1) family plays diverse roles in gene regulation in the defense and development signaling pathways in plants. Less evidence is available regarding the significance of the NPR1-like gene family in cotton (Gossypium species). Therefore, to address the importance of the cotton NPR1-like gene family in the defense pathway, four Gossypium species were studied: two tetraploid species, G. hirsutum and G. barbadense, and their two potential ancestral diploids, G. raimondii and G. arboreum. In this study, 12 NPR1-like family genes in G. hirsutum were recognized, including six genes in the A-subgenome and six genes in the D-subgenome. Based on the phylogenetic analysis, gene and protein structural features, cotton NPR-like proteins were grouped into three different clades. Our analysis suggests the significance of cis-regulatory elements in the upstream region of cotton NPR1-like genes in hormonal signaling, biotic stress conditions, and developmental processes. The quantitative expression analysis for different developmental tissues and fiber stages (0 to 25 days post-anthesis), as well as salicylic acid induction, confirmed the distinct function of different cotton NPR genes in defense and fiber development. Altogether, this study presents specifications of conservation in the cotton NPR1-like gene family and their functional divergence for development of fiber and defense properties.

Examination of unidentifiable spined loach individuals found in the overlap zone of two tetraploid species within a single river in Japan

An endangered tetraploid spined loach species, Cobitis takenoi (Cypriniformes: Cobitidae; hereafter called Tango loach) is known to inhabit only a single river in Kyoto Prefecture, Japan. Since Tango loach was discovered recently, in 2010, and only described in 2016, its morphology, ecology, and genetics are not well studied. Another tetraploid spined loach species Cobitis sp. BIWAE type A (hereafter, called Ohshima loach) inhabits the same river. The two loaches are reported as morphologically distinguishable from each other. Although the habitats of the two species in the river are segregated (Ohshima loach and Tango loach inhabit the upper and lower reaches, respectively), they overlap to a small degree in the boundary area. Recently, some individuals with morphological characteristics that are intermediate between the two species were found in the overlap zone. It was suspected that hybrids between the two species were produced since breeding seasons of the two species overlapped. To investigate whether the two species produce hybrids, we performed mitochondrial and nuclear DNA analyses on the unidentifiable individuals. Eight individuals unidentifiable to the species level collected in the river between 2017 and 2018 were examined and compared with the Tango and Ohshima loach species. Using mitochondrial DNA (mtDNA) cytochrome b analysis, we found that six individuals had mtDNA types identical to Tango loach and two individuals had mtDNA types identical to Ohshima loach. Furthermore, sequencing analysis of nuclear recombination activating gene 1 (RAG-1) revealed that each species had species-specific alleles. The phylogenetic analysis indicated that alleles in Tango loach were divided into two clusters and those from Ohshima loach formed a single cluster. There were no discrepancies in the combination between mtDNA and nuclear DNA species types within each specimen. DNA fingerprinting analysis (AFLP) showed that the species-unidentifiable individuals exhibited distinctly segregated genetic groups corresponding with Tango and Ohshima loaches. In summary, no hybrids were detected from among any unidentifiable individual examined in this study. New conventional genetic method for discriminating the two sympatric loach species developed here can be effective tool for the conservation of the Tango loach since there was no strict diagnostic morphological character between them.

Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa).

Alfalfa (Medicago sativa) is an outcrossing tetraploid legume species widely cultivated in the world. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has been successfully used for genome editing in many plant species. However, the use of CRISPR/Cas9 for gene knockout in alfalfa is still very challenging. Our initial single gRNA-CRISPR/Cas9 system had very low mutagenesis efficiency in alfalfa with no mutant phenotype. In order to develop an optimized genome editing system in alfalfa, we constructed multiplex gRNA-CRISPR/Cas9 vectors by a polycistronic tRNA-gRNA approach targeting the Medicago sativa stay-green (MsSGR) gene. The replacement of CaMV35S promoter by the Arabidopsis ubiquitin promoter (AtUBQ10) to drive Cas9 expression in the multiplex gRNA system led to a significant improvement in genome editing efficiency, whereas modification of the gRNA scaffold resulted in lower editing efficiency. The most effective multiplex system exhibited 75% genotypic mutagenesis efficiency, which is 30-fold more efficient than the single gRNA vector. Importantly, phenotypic change was easily observed in the mutants, and the phenotypic mutation efficiency reached 68%. This highly efficient multiplex gRNA-CRISPR/Cas9 genome editing system allowed the generation of homozygous mutants with a complete knockout of the four allelic copies in the T0 generation. This optimized system offers an effective way of testing gene functions and overcomes a major barrier in the utilization of genome editing for alfalfa improvement.

Current and Future Potential Distribution of Wild Strawberry Species in the Biodiversity Hotspot of Yunnan Province, China

Based on 243 current valid distribution records for six wild strawberry species in China and data on 20 environmental variables, the geographical distributions of and potentially suitable areas for the wild strawberry species in Yunnan Province (China) under the current climate scenario were explored using the MaxEnt model and ArcGIS software, and major environmental variables affecting their geographical distributions were evaluated. In addition, the spatio-temporal dynamic patterns of the suitable areas for the six wild strawberry species in Yunnan Province in the 2050s and 2070s under the two climate models of RCP2.6 and RCP8.5 were predicted. Under the current climate scenario, the six wild strawberry species have suitable areas in Yunnan Province, which were mainly distributed in the high-altitude and low-temperature regions in the northwest and northeast, such as Diqing and Zhaotong. In addition, the average size of the highly suitable area for diploid wild strawberry species was greater than that for tetraploid species. Under the future climate scenarios, the average size of the highly suitable area for diploid species showed a tendency to expand, while that of tetraploid species showed a tendency to shrink. Altitude was a critical variable affecting the distribution of tetraploid species. Under the two future climate models of RCP2.6 and RCP8.5, the suitable areas for wild strawberry species shifted to the regions of high latitude, high altitude, and low temperature. In addition, the average distance in the shift of the suitable area for tetraploid strawberry species was greater than that for the suitable area for diploid strawberry species. The above results provide valuable information for the management and protection of the germplasm resources of Fragaria.

The Genomes of the Allohexaploid Echinochloa crus-galli and Its Progenitors Provide Insights into Polyploidization-Driven Adaptation

The hexaploid species Echinochloa crus-galli is one of the most detrimental weeds in crop fields, especially in rice paddies. Its evolutionary history is similar to that of bread wheat, arising through polyploidization after hybridization between a tetraploid and a diploid species. In this study, we generated and analyzed high-quality genome sequences of diploid (E. haploclada), tetraploid (E. oryzicola), and hexaploid (E. crus-galli) Echinochloa species. Gene family analysis showed a significant loss of disease-resistance genes such as those encoding NB-ARC domain-containing proteins during Echinochloa polyploidization, contrary to their significant expansionduring wheat polyploidization, suggesting that natural selection might favor reduced investment in resistance in this weed to maximize its growth and reproduction. In contrast to the asymmetric patterns of genome evolution observed in wheat and other crops, no significant differences in selection pressure were detected between the subgenomes in E. oryzicola and E. crus-galli. In addition, distinctive differences in subgenome transcriptome dynamics during hexaploidization were observed between E. crus-galli and bread wheat. Collectively, our study documents genomic mechanisms underlying the adaptation of a major agricultural weed during polyploidization. The genomic and transcriptomic resources of three Echinochloa species and new insights into the polyploidization-driven adaptive evolution would be useful for future breeding cereal crops.