Genome-wide association analysis identified molecular markers and candidate genes for flower traits in Chinese orchid (Cymbidium sinense)

The orchid,the champagne of f lowers,brings luxury,elegance,and novelty to nature.Cymbidium sinense is a symbol of gigantic floral variability on account ofwavering shapes and sizes of f loral organs,althoughmarker-trait association(MTA)has not been studied for its f loral traits.We evaluated markers associated with 14 f loral traits of C.sinense through a genome-wide association study(GWAS)of 195 accessions.A total of 65318522 single-nucleotide polymorphisms(SNPs)and 3906176 insertion/deletion(InDel)events were identified through genotyping-by-sequencing.Among these,4694 potential SNPs and 477 InDelswere identified as MTAs at−log10 P>5.The genes related to these SNPs and InDels were largely associated with f loral regulators,hormonal pathways,cell division,and metabolism,playing essential roles in tailoring f loral morphology.Moreover,20 candidate SNPs/InDels linked to 11 genes were verified,8 of which were situated on exons,onewas located in the 5-UTR and twowere positioned in introns.Here,themultitepal trait-related gene RABBIT EARS(RBE)was found to be the most crucial gene.We analyzed the role of CsRBE in the regulation of flower-related genes via efficient transient overexpression in C.sinense protoplasts,and found that the floral homeotic genes CsAP3 and CsPI,as well as organ boundary regulators,including CsCUC and CsTCP genes,were regulated by CsRBE.Thus,we obtained key gene loci for important ornamental traits of orchids using genome-wide association analysis of populations with natural variation.The findings of this study can do a great deal to expedite orchid breeding programs for shape variability.

Transcriptome Analysis Reveals Clues into leaf-like flower mutant in Chinese orchid Cymbidium ensifolium

The floral morphology of Cymbidium ensifolium,a well-known orchid in China,has increasingly attracted horticultural and commercial attention.However,the molecular mechanisms that regulate flower development defects in C.ensifolium mutants are poorly understood.In this work,we examined a domesticated variety of C.ensifolium named‘CuiYuMuDan',or leaf-like flower mutant,which lacks typical characteristics of orchid floral organs but continues to produce sepal-to leaf-like structures along the inflorescence.We used comparative transcriptome analysis to identify 6234 genes that are differentially expressed between mutant and wild-type flowers.The majority of these differentially expre ssed genes are involved in membrane-building,anabolism regulation,and plant hormone signal transduction,implying that in the leaf-like mutant these processes play roles in the development of flower defects.In addition,we identified 152 differentially expre ssed transcription factors,including the bHLH,MYB,MIKC,and WRKY gene families.Moreover,we found 20 differentially expressed genes that are commonly involved in flower development,including MADS-box genes,CLAVATA3(CLV3),WUSCHEL(WUS),and PERIANTHIA(PAN).Among them,floral homeotic genes were further investigated by phylogenetic analysis and expression validation,which displayed distinctive spatial expression patterns and significant changes between the wild type and the mutant.This is the first report on the C.ensifolium leaf-like flower mutant transcriptome.Our results shed light on the molecular regulation of orchid flower development,and may improve our understanding of floral patterning regulation and advance molecular breeding of Chinese orchids.

Advances in Transgenic Breeding of Anthurium andraeanum

At present, transgenic technologies have become important means of plant breeding, and the application and promotion of transgenic technologies have created huge economic and social benefits. Transgenic plant products have significantly affected human life. Anthurium andraeanum is the second major tropical potted flower and its transgenic breeding has a promising prospect of application. In this paper, acceptors, transformation methods and introduced exogenous genes （ including reporter genes, selectable marker genes and target genes） of Anthurium andraeanum were summarized; in addition, several issues related to transforma- tion of Anthurium andraeanum were analyzed, aiming at providing reference for transgenic breeding of Anthurium andraeanum.

Complete Genome Sequence Analysis of Guangdong Isolates of Cymbidium Mosaic Virus

[Objectives]The paper was to explore the genetic information and evolution of CymMV,and to provide an important scientific basis for monitoring and early warning of orchid virus disease and anti-virus genetic engineering of orchid in Guangdong Province.[Methods]RT-PCR and DASELISA were used to detect and identify CymMV from leaves with suspected virus disease of Cymbidium sinense collected from Guangzhou area.The genome sequence assembly,annotation,phylogeny and selection pressure analysis of CymMV isolates were performed with related molecular biology software.[Results]Two CymMV isolates(GZV013 and ZC29)were found in Guangdong Province for the first time in this study.The genome of both GZV013 and ZC29 were 6227 nt in length,encoding 5 functional proteins.The similarity analysis of the full sequence showed that the nucleotide sequence identity of GZV013 and Taiwan isolate M2 was 97.03% and that of ZC29 and Nanjing isolate NJ-1 was 97.11%.The complete genome sequence identity among CymMV isolates ranged from 86.85% to 98.31%,and the differentiation of diverse populations was closely related to host species and geographical isolation.Each region of CymMV genome was affected by negative selection and conformed to the neutral evolution model.The genes encoding RdRp,TGB1 and TGB2 had the highest mutation rates in the genome.[Conclusions]GZV013 was most closely related to Taiwan isolate M2,and ZC29 was most closely related to Nanjing isolate NJ-1,belonging to the same branch of a family.

Cultivation of Hippeastrum vittatum Embryogenic Calli and Their Sensitivity to Antibiotics

In this study, an embryogenic callus induction and proliferation system for Hippeastrum vittatum was established, with the tender bulbs as explants. And then the sensitivity of the explants and calli to kanamycin and hygromycin was evaluated. The results suggested that the embryogenic calli were induced from tender bulbs and cultured in Murashige and Skoog （MS） medium supplemented with 0.5 mg/L N-phenyl-N＇-1,2,3-thiadiazol-5-ylurea （TDZ）, 1.0 mg/L 2,4-diehloro- phenoxyacetic acid （2,4-D）, and 30 g/L sucrose （ pH5. 8） in the dark at 25 ±1℃. Further study of the influence of kanamyein and hygromycin on callus induction and multiplication showed that, the lethal doses of kanamycin and hygromycin to bulb explants were 100 and 30 mg/L, respectively. All explants of H. vittatum died on the medium supplemented with 100 mg/L kanamycin or 30 mg/L hygromycin at the induction stage, and callus proliferation was completely inhibited by 100 mg/L kanamycin or 25 mg,/L hygromyein, and all the calli died at last. These results will provide important reference for further studies of trausgenic H. vittaturn.

A Study on the Sensibility of Embryogenic Suspension Cells of Anthurium andraeanum to Two Antibiotics

In this study,embryogenic cell aggregates obtained from the established embryogenic cell suspension culture system of Anthurium andraeanum‘Alabama’were used as experimental materials to investigate the effects of kanamycin and hygromycin on survival rate of cell aggregates,somatic embryogenesis and plantlet regeneration of A.andraeanum.According to the results,at the embryonic cell propagation stage,lethal doses of kanamycin and hygromycin to embryogenic cell aggregates of A.andraeanum were 200 and 60 mg/L,respectively;at the differentiation stage,either 150 mg/L kanamycin or 40 mg/L hygromycin could inhibit somatic embryogenesis;either 100 mg/L kanamycin or 20 mg/L hygromycin could inhibit plantlet regeneration.These results provided important reference for further studies of transgenic A.andraeanum.

黑鹅绒海芋体细胞胚发生和植株再生

该研究建立了黑鹅绒海芋(Alocasia reginula)体细胞胚胎发生途径的植株再生体系。通过叶柄诱导获得胚性愈伤组织,以此建立胚性细胞悬浮培养系,并实现了高频率的植株再生。叶柄外植体在添加2.0 mg·L^(-1)2,4-D和1.0 mg·L^(-1)TDZ的MS培养基上诱导胚性愈伤组织的效率最高(达81.3%)。将胚性愈伤组织破碎成细胞团,转移至添加2.0 mg·L^(-1)2,4-D和1.0 mg·L^(-1)TDZ的MS液体培养基中进行悬浮培养,2周继代1次,悬浮培养12周后获得大量细胞团。将悬浮培养28周的细胞团转移至不含植物生长调节剂的1/2MS固体培养基上进行分化培养,平均每个细胞团可再生2.3–2.5棵植株。利用光学显微镜和扫描电镜观察体细胞胚的萌发和形成。再生苗移栽至温室4个月后,成活率为95.3%。通过流式细胞术对随机选取的50株成活植株进行检测,未发现染色体倍性变异,核DNA含量为10.94 pg·(2C)^(-1),基因组大小为5 290.12 Mb·C^(-1)。植株移栽到温室直至自然开花,表型无明显变异。研究结果可为黑鹅绒海芋种苗商业化生产和生物技术育种提供良好的技术支持。

An auxin‐responsive endogenous peptide regulates root development in Arabidopsis

Auxin plays critical roles in root formation and development. The components involved in this process, however, are not well understood. Here, we newly identified a peptide encoding gene, auxin-responsive endogenous polypeptide 1 （AREP1）, which is induced by auxin, and mediates root development in Arabidopsis. Expression of AREP1 was specific to the cotyledon and to root and shoot meristem tissues. Amounts of AREP1 transcripts and AREP1-green fluorescent protein fusion proteins were elevated in response to indoleacetic acid treatment. Suppression of AREP1 through RNAi silencing resulted in reduction of primary root length, increase of lateral root number, and expansion of adventitious roots, compared to the observations in wild-type plants in the presence of auxin. By contrast, transgenic plants overexpressing AREP1 showed enhanced growth of the primary root under auxin treatment. Additionally, rootmorphology, including lateral root number and adventitious roots, differed greatly between transgenic and wildtype plants. Further analysis indicated that the expression of auxin-responsive genes, such as IAA3, IAA7, IAA17, GH3.2, GH3.3, and SAUR-AC1, was significantly higher in AREP1 RNAi plants, and was slightly lower in AREP1 overexpressing plants than in wildtype plants. These results suggest that the novel endogenous peptide AREP1 plays an important role in the process of auxinmediated root development.

The genomic and bulked segregant analysis of Curcuma alismatifolia revealed its diverse bract pigmentation

Compared with most flowers where the showy part comprises specialized leaves(petals)directly subtending the reproductive structures,most Zingiberaceae species produce showy“flowers”through modifications of leaves(bracts)subtending the true flowers throughout an inflorescence.Curcuma alismatifolia,belonging to the Zingiberaceae family,a plant species originating from Southeast Asia,has become increasingly popular in the flower market worldwide because of its varied and esthetically pleasing bracts produced in different cultivars.Here,we present the chromosome-scale genome assembly of C.alismatifolia“Chiang Mai Pink”and explore the underlying mechanisms of bract pigmentation.Comparative genomic analysis revealed C.alismatifolia contains a residual signal of whole-genome duplication.Duplicated genes,including pigment-related genes,exhibit functional and structural differentiation resulting in diverse bract colors among C.alismatifolia cultivars.In addition,we identified the key genes that produce different colored bracts in C.alismatifolia,such as F3′5'H,DFR,ANS and several transcription factors for anthocyanin synthesis,as well as chlH and CAO in the chlorophyll synthesis pathway by conducting transcriptomic analysis,bulked segregant analysis using both DNA and RNA data,and population genomic analysis.This work provides data for understanding the mechanism of bract pigmentation and will accelerate breeding in developing novel cultivars with richly colored bracts in C.alismatifolia and related species.It is also important to understand the variation in the evolution of the Zingiberaceae family.