Genome editing opens a new era of genetic improvement in polyploid crops

Sequence-specific nucleases(SSN) that generate double-stranded DNA breaks(DSBs) in genes of interest are the key to site-specific genome editing in plants. Genome editing has developed into one method of reducing undesirable traits in crops by the induction of knockout mutations. Different SSN-mediated genome-editing systems, including LAGLIDADG homing endonucleases or meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats, are emerging as robust tools for introducing functional mutations in polyploid crops including citrus, wheat, cotton, soybean, rapeseed, potato, grapes, Camelina sativa,dandelion, and tobacco. The approach utilizes knowledge of biological mechanisms for targeted induction of DSBs and their error-prone repair, allowing highly specific changes at designated genome loci. In this review, we briefly describe genome-editing technologies and their application to genetic improvement of polyploid crops.

Induction and characterization of polyploids from seeds of Rhododendron fortunei Lindl.

Most Rhododendron species are ornamental flowering species widely distributed in Asia,North America,and West Europe.Rhododendron fortunei,one of the endemic Rhododendron species in China,has beautiful flowers with bright colors and is being exploited to meet the needs of the flower market.Polyploid plants usually show superiority in growth,disease resistance,and adaption over their diploid relatives.Here,we report the first case of polyploid induction in R.fortunei.In order to induce polyploidy in R.fortunei,germinating seeds were treated with different concentrations of oryzalin for 16 h.By evaluating ploidy level with flow cytometry,a total of 34 polyploid R.fortunei lines,including 27 tetraploid lines and seven octoploid lines,were obtained.A comparison of treatments indicated that 7.5 mg L^-1 oryzalin was the optimal concentration for polyploid induction in seeds of R.fortunei.Compared with diploid plants,tetraploid and octoploid plants exhibited slower growth rates and had thicker and rounder curled leaves with more leaf epidermal hairs.Moreover,larger stomata at lower density were also observed in the leaves of polyploid plants.Chlorophyll contents were also significantly increased in polyploid plants,which leads to a darker green leaf color.Both small and large individuals exhibiting the same characteristics were observed among the obtained tetraploid plants.Overall,our study establishes a feasible method for polyploid induction in R.fortunei,thus providing a basis for breeding new R.fortunei varieties.

Roles of cell fusion, hybridization and polyploid cell formation in cancer metastasis

Cell-cell fusion is a normal biological process playing essential roles in organ formation and tissue differentiation,repair and regeneration.Through cell fusion somatic cells undergo rapid nuclear reprogramming and epigenetic modifications to form hybrid cells with new genetic and phenotypic properties at a rate exceeding that achievable by random mutations.Factors that stimulate cell fusion are inflammation and hypoxia.Fusion of cancer cells with non-neoplastic cells facilitates several malignancy-related cell phenotypes,e.g.,reprogramming of somatic cell into induced pluripotent stem cells and epithelial to mesenchymal transition.There is now considerable in vitro,in vivo and clinical evidence that fusion of cancer cells with motile leucocytes such as macrophages plays a major role in cancer metastasis.Of the many changes in cancer cells after hybridizing with leucocytes,it is notable that hybrids acquire resistance to chemo-and radiation therapy.One phenomenon that has been largely overlooked yet plays a role in these processes is polyploidization.Regardless of the mechanism of polyploid cell formation,it happens in response to genotoxic stresses and enhances a cancer cell’s ability to survive.Here we summarize the recent progress in research of cell fusion and with a focus on an important role for polyploid cells in cancer metastasis.In addition,we discuss the clinical evidence and the importance of cell fusion and polyploidization in solid tumors.

Lycophyte transcriptomes reveal two whole-genome duplications in Lycopodiaceae:Insights into the polyploidization of Phlegmariurus

Lycophytes are an ancient clade of the non-flowering vascular plants with chromosome numbers that vary from tens to hundreds.They are an excellent study system for examining whole-genome duplications(WGDs),or polyploidization,in spore-dispersed vascular plants.However,a lack of genome sequence data limits the reliable detection of very ancient WGDs,small-scale duplications(SSDs),and recent WGDs.Here,we integrated phylogenomic analysis and the distribution of synonymous substitutions per synonymous sites(Ks)of the transcriptomes of 13 species of lycophytes to identify,locate,and date multiple WGDs in the lycophyte family Lycopodiaceae.Additionally,we examined the genus Phlegmariurus for signs of genetic discordance,which can provide valuable insight into the underlying causes of such conflict(e.g.,hybridization,incomplete lineage sorting,or horizontal gene transfer).We found strong evidence that two WGD events occurred along the phylogenetic backbone of Lycopodiaceae,with one occurring in the common ancestor of extant Phlegmariurus(Lycopodiaceae)approximately 22-23 million years ago(Mya)and the other occurring in the common ancestor of Lycopodiaceae around 206-214 Mya.Interestingly,we found significant genetic discordance in the genus Phlegmariurus,indicating that the genus has a complex evolutionary history.This study provides molecular evidence for multiple WGDs in Lycopodiaceae and offers phylogenetic clues to the evolutionary history of Lycopodiaceae.

Comparative genomic analyses reveal cis-regulatory divergence after polyploidization in cotton

Polyploidization has long been recognized as a driver for the evolutionary formation of superior plant traits coupled with gene expression novelty.However,knowledge of the effect of regulatory variation on expression changes following polyploidization remains limited.In this study,we characterized transcriptional regulatory divergence by comparing tetraploid cotton with its putative diploid ancestors.We identified 144,827,99,609,and 219,379 Tn5 transposase-hypersensitive sites(THSs)in Gossypium arboreum,G.raimondii,and G.hirsutum,respectively,and found that the conservation of promoter THSs was associated with coordination of orthologous genes expression.This observation was consistent with analysis of transcription-factor binding sites(TFBS)for 262 known motifs:genes with higher TFBS conservation scores(CS)showed less change than those genes with lower TFBS CS in expression levels.TFBS influenced by genomic variation were involved in the novel regulation networks between transcriptional factors and target genes in tetraploid cotton.We describe an example showing that the turnover of TFBS was linked to expression pattern divergence of genes involved in fiber development(fiber-related genes).Our findings reveal the regulatory divergence of the transcriptional network in cotton after polyploidization and characterizes the regulatory relationships of genes contributing to desirable traits.

Polyploid Gene Expression and Regulation in Polysomic Polyploids

Polyploidization is one of the most crucial pathways in introducing speciation and broadening biodiversity, especially in the Plant Kingdom. Although the majority of studies have focused only on allopolyploid or disomic polyploids, polysomic polyploid species have occurred frequently in higher plants. Due to the occurrence of the capabilities of more copies of alleles in a locus which can have additive dosage effects and/or allelic interactions, polysomic polyploids can lead to unique gene regulations to silence or adjust the expression level to create variations in organ size, metabolic products, and abiotic stress tolerance and biotic stress resistance, etc. This review aims to comprehensively summarize the contemporary understanding and findings concerning the molecular mechanisms of gene expression as well as gene regulation in natural typed and resynthesized polysomic polyploid plants. The review investigates the molecular level of phenomena in polysomic polyploid plants such as 1) typically enlarging organ size and stabilizing meiosis, 2) increasing phytochemical content and metabolic products, 3) enhancing the ability to adapt with biotic and abiotic stress, and 4) changing in gene regulation to silence or adjust the expression levels involve in sequence elimination, methylation, gene suppression, subfunctionalization, neo-functionalization, and transposon activation.

Polyploidization Genetic Mechanism of Sugarcane Genome

The sugarcane genome polyploidization can reduce the pressure of gene evolution selection,promote the fixation of fine traits,and increase the biomass and economic value of sugarcane.This paper mainly introduced the origin of the sugarcane genome,the chromosome composition,the research progress of polyploidization genetic mechanism,in the hope of providing theoretical reference for sugarcane polyploidization breeding.

Induced Polyploidy via Colchicine Treatment Increases Flower Size and Fruit Weight in Cape Gooseberry(Physalis peruviana L.)

An induced polyploid plant through colchicine treatment offers probably the best scope for improvement in flower size and fruit weight.Thus,in the present study,an attempt was made to induce polyploidy in Cape gooseberry using colchicine with the objective of creating more genetic variability.The colchicine concentrations were used as 0.10%(C1),0.20%(C2)and 0.40%(C3)for the duration 12(H1),24(H2)and 36(H3)hours for each concentration with seedling apex dip method(M1),cotton plug method(M2)and lanolin paste method(M3).The plants treated with 0.10%of colchicine by cotton plug method for 12 h showed the better performance during the years 2017-2018 and 2018-2019 in respect of more delay in the flower bud emergence(54 d and 53 d from the date of transplanting),anthesis(19 d and 20 d from the first appearance of bud to full anthesis of flower)and fruit setting(8.00 d and 9.00 d from the date of anthesis),bigger flower size(2.93 cm2 and 3.00 cm2)than the untreated plants.The lower percentage of pollen viability(40.33%and 40.67%)was noticed in the same treatment in comparison to control(70.33%and 72.33%).The fruit maturity was also extended(59 d and 60 d from the date of fruit set)with bigger sized fruits(length:2.53 cm and 2.57 cm,breadth:2.27 cm and 2.33 cm)as well as more fruit weight(8.70 g and 8.33 g)by the application of colchicine at 0.10%with cotton plug method for 12 h.On the basis of results obtained,the application of colchicine at 0.10%for 12 h duration with cotton plug method was found to be the best and effective treatment for induction of polyploidy as well as more flower size and fruit weight in Cape gooseberry.

Studies on the Growth Habits and Characteristics of Two Polyploid Indica-Japonica Hybrid Rice with Powerful Heterosis

Based on a series of polyploid indica-japonica hybrid plant lines obtained from a new breeding strategy of using double predominance of wide cross and polyploidization to breed super rice, two polyploid indica-japonica hybrids, PSR073 and PSR120 were studied in their growth periods to show the powerful heterosis in a larger scale and to study the characteristics of polyploid indica-japonica hybrids more elaborately. The leaf age, tiller growth, flowering habits, and agronomic traits of them were observed to analyze their growth habits and characteristics. The results showed that the agronomic traits of PSR073 and PSR120, such as the plant height, panicle length, grain length, grain width, and 1 000-grain weight, all acquired obvious predominance of polyploidy, and that the seed setting rate was more than 83%. No significant difference was observed between the two tetraploids and common diploids in the leaf age, tiller growth, and flowering habits. It was concluded that the characteristics of the two powerful heterosis polyploid hybrids were different from those of the polyploid rice reported earlier. Wide cross and polyploidization had no negative effects on their growth habits and characteristics; on the contrary these had powerful heterosis. This had provided theoretic and practical evidences for their application to agricultural production.

Induction and Identification of Polyploidy Plants from Superior Individuals of Wild Lonicera edulis Turcz. in Changbai Mountains

[Objectives] This study was conducted to provide high-quality polyploid for returning the grain plots to forestry and promote the rapid development of Lonicera edulis Turcz. [Methods]With superior individuals of wild L. edulis in Changbai Mountains as experimental materials and colchicine as inducer,polyploid induction was performed twice on diploid plants by addition method,and 7 polyploidy plants were identified from 11 regenerated plantlets. [Results]Among the 7 polyploidy plants,four materials were tetraploid,the concentration and treatment time of which were 300 mg/L and 7 and 14 d,respectively; and three materials were octaploid,the concentrations and treatment time of which were 300 and 500 mg/L and 14 d,respectively. [Conclusions]The results of this study showed that colchicine had a higher polyploid induction rate for L. edulis with lower toxic and side effects,and the polyploidy plants could restore to normal growth state after several times of cubculture. Therefore,colchicine is an polyploid inducer suitable for L. edulis.

Natural and artificial polyploids in aquaculture

Genome polyploidy has been revealed to result in evolutionary advantages and novelties,and therefore,polyploid aquatic animals may possess excellent traits of economic interest including rapid growth,extensive adaptability and disease resistance.For this reason,numerous species of natural polyploid fishes,such as common carp,gibel carp,crucian carp,salmon,and sturgeon,were chosen as important target species for aquaculture.Many artificial polyploids have been commercially utilized for aquaculture and most of them were created from natural polyploid fishes of the Cyprinidae and Salmonidae.Thanks to the easy mass production and better economic traits in growth and flesh quality,the synthetized autopolyploids or allopolyploids from natural polyploid species in cyprinid fishes have been extensively applied to aquaculture throughout China.This review outlines polyploidy advantages and innovative opportunities,lists natural polyploid species used in aquaculture,and summarizes artificial polyploids that have been induced or synthetized,and used in aquaculture.Moreover,some main research trends on polyploid utilization and ploidy manipulation of aquaculture animals are also introduced and discussed in the review.

The genome of Orychophragmus violaceus provides genomic insights into the evolution of Brassicaceaepolyploidizationandits distinct traits

Orychophragmus violaceus,referred to as‘‘eryuelan’’(February orchid)in China,is an early-flowering ornamental plant.The high oil content and abundance of unsaturated fatty acids in O.violaceus seeds make it a potential high-quality oilseed crop.Here,we generated a whole-genome assembly for O.violaceus using Nanopore and Hi-C sequencing technologies.The assembled genome of O.violaceus was~1.3 Gb in size,with 12 pairs of chromosomes.Through investigation of ancestral genome evolution,we determined that the genome of O.violaceus experienced a tetraploidization event from a diploid progenitor with the translocated proto-Calepineae karyotype.Comparisons between the reconstructed subgenomes of O.violaceus identified indicators of subgenome dominance,indicating that subgenomes likely originated via allotetraploidy.O.violaceus was phylogenetically close to the Brassica genus,and tetraploidy in O.violaceus occurred approximately 8.57 million years ago,close in time to the whole-genome triplication of Brassica that likely arose via an intermediate tetraploid lineage.However,the tetraploidization in Orychophragmus was independent of the hexaploidization in Brassica,as evidenced by the results from detailed phylogenetic analyses and comparisons of the break and fusion points of ancestral genomic blocks.Moreover,identification of multi-copy genes regulating the production of high-quality oil highlighted the contributions of both tetraploidization and tandem duplication to functional innovation in O.violaceus.These findings provide novel insights into the polyploidization evolution of plant species and will promote both functional genomic studies and domestication/breeding efforts in O.violaceus.

The occurrence,inheritance,and segregation of complex genomic structural variation in synthetic Brassica napus

"Synthetic"allopolyploids recreated by interspecific hybridization play an important role in providing novel genomic variation for crop improvement.Such synthetic allopolyploids often undergo rapid genomic structural variation(SV).However,how such SV arises,is inherited and fixed,and how it affects important traits,has rarely been comprehensively and quantitively studied in advanced generation synthetic lines.A better understanding of these processes will aid breeders in knowing how to best utilize synthetic allopolyploids in breeding programs.Here,we analyzed three genetic mapping populations(735 DH lines)derived from crosses between advanced synthetic and conventional Brassica napus(rapeseed)lines,using whole-genome sequencing to determine genome composition.We observed high tolerance of large structural variants,particularly toward the telomeres,and preferential selection for balanced homoeologous exchanges(duplication/deletion events between the A and C genomes resulting in retention of gene/chromosome dosage between homoeologous chromosome pairs),including stable events involving whole chromosomes("pseudoeuploidy").Given the experimental design(all three populations shared a common parent),we were able to observe that parental SV was regularly inherited,showed genetic hitchhiking effects on segregation,and was one of the major factors inducing adjacent novel and larger SV.Surprisingly,novel SV occurred at low frequencies with no significant impacts on observed fertility and yield-related traits in the advanced generation synthetic lines.However,incorporating genome-wide SV in linkage mapping explained significantly more genetic variance for traits.Our results provide a framework for detecting and understanding the occurrence and inheritance of genomic SV in breeding programs,and support the use of synthetic parents as an important source of novel trait variation.

大果少核椪柑新品种华柑3号的选育

华柑3号是通过鄂柑1号椪柑珠心胚实生苗倍性变异而培育的四倍体大果少核椪柑新品种。果实扁圆形,果实大,平均横径74.08 mm,纵径62.63 mm,平均单果质量179.94 g,平均果皮厚度3.45 mm。果肉橙黄色,肉质脆嫩,酸甜爽口,风味浓,可溶性固形物含量(w,后同)为14.49%,可滴定酸含量为1.16%,可食率为71.6%,化渣性好;种子少,平均2.7粒·果^(-1);树势中等偏强,树姿较直立,结果母枝以春梢和秋梢为主;在湖北武汉地区,12月中下旬果实成熟,可挂树至翌年1月;丰产性中等,按3.0 m×4.0 m密度定植的5年生树,每666.7 m^(2)产量可达1500 kg;适宜在年均温度16℃左右,冬季无霜冻、绝对最低温度在-5℃以上的区域种植。

Allotetraploidization event of Coptis chinensis shared by all Ranunculales

Coptis chinensis Franch.,also named Chinese goldthread is a member of Ranunculaceae in the order Ranunculales and represents an important lineage of early eudicots with traditional medicinal value.In our study,by using syntenic analysis combined with phylogenomic analysis of C.chinensis and four other representative genomes from basal and core eudicots,we confirmed that the WGD event in C.chinensis was shared by Aquilegia coerulea and Papaver somniferum L.and quickly occurred after Ranunculales diverged from other eudicots,likely a Ranunculales common tetraploidization(RCT).The synonymous nucleotide substitutions at synonymous sites distribution of syntenic blocks across these genomes showed that the evolutionary rate of the P.somniferum genome is faster than that of the C.chinensis genome by approximately 13.7%,possibly due to Papaveraceaes having an additional special tetraploidization event(PST).After Ks correction,the RCT dated to 115—130 million years ago(MYA),which was close to the divergence of Ranunculaceaes and Papaveraceaes approximately115.45—130.51 MYA.Moreover,we identified homologous genes related to polyploidization and speciation and constructed multiple sequence alignments with different reference genomes.Notably,the event-related subgenomes in the basal genomes all showed genomic fractionation bias,suggesting a likely allopolyploid nature of the RCT,PST and T-Alpha and T-Beta events in Tetracentron sinense.In addition,we detected that the sixteen P450 subfamilies were markedly expanded in the genomes of Ranunculales,and most of them were related to the RCT and PST events.We constructed a new platform for Early Eudicot Comparative Genomic Research(http://www.cgrpoee.top/index.html)to store more information.In summary,our findings support the WGD of C.chinensis shared by Ranunculales,which is likely an allotetraploidization event.This present effort offered new insights into the evolution of key polyploidization events and the genes related to secondary metabolites during the diversification of early eudicots.

匍茎百合再生体系的建立及多倍体诱导

为了保护匍茎百合(Lilium lankongense)的野生种质资源并提供简单有效的快繁方法,以匍茎百合为植物材料,建立以鳞茎为外植体的高效再生体系,并利用秋水仙素处理种子,诱导出多倍体植株。通过调节培养基中的植物生长激素和蔗糖的质量浓度,确定了匍茎百合不定芽诱导、小鳞茎膨大和生根的最佳培养基。结果表明:最佳不定芽诱导培养基为MS培养基+6-苄氨基嘌呤(6-BA)1.0 mg·L^(-1)+萘乙酸(NAA)0.7 mg·L^(-1)+蔗糖30 g·L^(-1),不定芽诱导系数为2.33,显著高于其他处理组合;最佳小鳞茎膨大培养基为MS培养基+蔗糖60 g·L^(-1),可显著提高小鳞茎的直径和鲜质量;最佳生根培养基为0.5倍浓度的MS培养基+萘乙酸(NAA)0.4 mg·L^(-1)+吲哚丁酸(IBA)0.1 mg·L^(-1),可显著增加根长和根数。用质量分数为0.03%的秋水仙素浸泡18 h,种子的成活率最高,为93.15%,再通过形态学、生理学和流式细胞仪鉴定,最终鉴定出4个四倍体植株和16个嵌合体植株。

Plant Polyploidy: Origin, Evolution, and Its Influence on Crop Domestication

The prevalence and recurrence of polyploidization in plant species make it one of the most important evolutionary events in plants, and as a result, polyploidization is an extensively investigated research field. Due to the rapid development of sequencing technologies, there is increased evidence to support that polyploidization plays an important role in the diversification of plant species, evolution of genes, and the domestication of crops. Here, we reviewed the influence of polyploidization on various aspects of plant evolution, mainly focused on polyploid origin, characteristics, subsequent genome divergence, and its impact on gene function innovation and crop domestication. The occurrence of many independent polyploidization events in plants was found to be tightly associated with the timing of extreme climate events or natural disasters on earth, leading to mass extinction while possibly facilitating increased polyploidization. Following allo-polyploidization, a distinct phenomenon known as sub-genome dominance occurred during sub-genome evolution, which was found to be associated with the methylation of transposons. Extensive gene fractionations(lost) following polyploidization were reported in almost all paleo-polyploids, and the evolutionary fates of multi-copy genes, such as sub-/neo-functionalization, were further proposed to illustrate their underlying mechanisms. Moreover,polyploidization was found to significantly impact species diversification, with subsequent effects on crop domestication and the development of traits with agronomic importance. Based on the progress of plant polyploidization studies, we discussed several main topics that might further improve our understanding of polyploid evolution and that are likely contribute to the application of polyploidization in crop breeding in the near future.

Cyprininae phylogeny revealed independent origins of the Tibetan Plateau endemic polyploid cyprinids and their diversifications related to the Neogene uplift of the plateau

Origin and diversification of the Tibetan polyploid cyprinids(schizothoracins) may help us to explore relationships between diversification of the cyprinids and the Tibetan Plateau uplift. Cyprininae phylogeny was analyzed using mitochondrial and nuclear DNA sequences to trace origins of polyploidy and diversifications of schizothoracins. Ancestral states reconstruction for ploidy levels indicated that the Cyprininae was diploid origin and the schizothoracin clades tetraploid origins. There were two diversification rate shifts along with diversification of the cyprinine fishes in response to the Tibetan uplift. The unusual diversification shifts were located to branches subtending the clades of Tibetan polyploid cyprinids. Our analyses suggested that(i) phylogeny of Cyprininae recovered two independent origins of the Tibetan polyploidy schizothoracins;(ii) diversifications of the schizothoracins were closely related to the Neogene uplift of the Tibetan plateau in the following ways: the relatively ancient Late Oligocene-Middle Miocene adaptive radiation may be associated with the uplift of the southern Tibet and Himalaya; the Middle Miocene-Early Pleistocene lineage-specific diversification broadly coincident with major phase of the Neogene Tibetan uplift; and the most recent Pleistocene diversification shift in Schizothorax closely coincident with the successive Kunlun-Huanghe and Gonghe movements of the Tibetan uplift and the glaciation-induced climate oscillations on the plateau.

Genome sequences of five Sitopsis species of Aegilops and the origin of polyploid wheat B subgenome

Common wheat(Triticum aestivum,BBAADD)is a major staple food crop worldwide.The diploid progenitors of the A and D subgenomes have been unequivocally identified;that of B,however,remains ambiguous and controversial but is suspected to be related to species of Aegilops,section Sitopsis.Here,we report the assembly of chromosome-level genome sequences of all five Sitopsis species,namely Aegilops bicornis,Ae.longissima,Ae.searsii,Ae.sharonensis,and Ae.speltoides,as well as the partial assembly of the Amblyopyrum muticum(synonym Aegilops mutica)genome for phylogenetic analysis.Our results reveal that the donor of the common wheat B subgenome is a distinct,and most probably extinct,diploid species that diverged from an ancestral progenitor of the B lineage to which the still extant Ae.speltoides and Am.muticum belong.In addition,we identified interspecific genetic introgressions throughout the evolution of the Triticum/Aegilops species complex.The five Sitopsis species have various assembled genome sizes(4.11-5.89 Gb)with high proportions of repetitive sequences(85.99%-89.81%);nonetheless,they retain high collinearity with other genomes or subgenomes of species in the Triticum/Aegilops complex.Differences in genome size were primarily due to independent post-speciation amplification of transposons.We also identified a set of Sitopsis genes pertinent to important agronomic traits that can be harnessed for wheat breeding.These newly assembled genome resources provide a new roadmap for evolutionary and genetic studies of the Triticum/Aegilops complex,as well as for wheat improvement.