Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable.Here,we report a highly useful method using an Agrobacterium-mediated transient ...Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable.Here,we report a highly useful method using an Agrobacterium-mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation.We have also developed a rapid,cost-effective,and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting(HRM)analysis.Using tetraploid tobacco as a model species and the phytoene desaturase(PDS)gene as a target,we successfully created and expediently identified mutant plants,which were verified as tetra-allelic mutants.We produced pds mutant shoots at a rate of 47.5%from tobacco leaf explants,without the use of antibiotic selection.Among these pds plants,17.2%were confirmed to be non-transgenic,for an overall non-transgenic mutation rate of 8.2%.Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction.This method should be applicable to many economically important,heterozygous,perennial crop species that are more difficult to regenerate.展开更多
Bamboo is known for its edible shoots and beautiful texture and has considerable economic and ornamental value.Unique among traditional flowering plants,many bamboo plants undergo extensive synchronized flowering foll...Bamboo is known for its edible shoots and beautiful texture and has considerable economic and ornamental value.Unique among traditional flowering plants,many bamboo plants undergo extensive synchronized flowering followed by large-scale death,seriously affecting the productivity and application of bamboo forests.To date,the molecular mechanism of bamboo flowering characteristics has remained unknown.In this study,a SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1)-like gene,BoMADS50,was identified from Bambusa oldhamii.BoMADS50 was highly expressed in mature leaves and the floral primordium formation period during B.oldhamii flowering and overexpression of BoMADS50 caused early fl owering in transgenic rice.Moreover,BoMADS50 could interact with APETALA1/FRUITFULL(AP1/FUL)-like proteins(BoMADS 14-1/2,BoMADS15-1/2)in vivo,and the expression of BoMADS50 was signi ficantly promoted by BoMADS14-1,further indicating a synergistic effect between BoMADS50 and BoAP1/FUL-like proteins in regulating B.oldhamii flowering.We also identi fi ed four additional transcripts of BoMADS50(BoMADS50-1/2/3/4)with different nucleotide variations.Although the protein-CDS were polymorphic,they had flowering activation functions similar to those of BoMADS50.Yeast one-hybrid and transient expression assays subsequently showed that both BoMADS50 and BoMADS50-1 bind to the promoter fragment of itself and the SHORT VEGETATIVE PHASE(SVP)-like gene BoSVP,but only BoMADS50-1 can positively induce their transcription.Therefore,nucleotide variations likely endow BoMADS50-1 with strong regulatory activity.Thus,BoMADS50 and BoMADS50-1/2/3/4 are probably important positive flowering regulators in B.oldhamii.Moreover,the functional conservatism and speci ficity of BoMADS50 and BoMADS50-1 might be related to the synchronized and sporadic flowering characteristics of B.oldhamii.展开更多
Although most Sinojackia species are endangered, they contribute greatly to the biodiversity of local ecosystems. Sinojackia microcarpa, an endangered species, is distributed only in three provinces of eastern China. ...Although most Sinojackia species are endangered, they contribute greatly to the biodiversity of local ecosystems. Sinojackia microcarpa, an endangered species, is distributed only in three provinces of eastern China. Determining the genetic diversity of S. microcarpa provides key information for germplasm evaluation and species conservation. Here we used simple sequence repeat (SSR) markers to investigate the genetic diversity of eight natural populations of S. microcarpa. Leaf samples were collected from 144 individuals in 8 wild populations. The 156 bands were generated from 14 pairs of informative SSR primers, with an average percentage of polymorphic bands of 45.67%. The average values of Nei’s genetic diversity (He) and Shannon’s diversity index (I) were 0.1007 and 0.1658, respectively. The total genetic variation of S. microcarpa existed mainly within the eight populations, rather than among populations, and reached 86.41%. A cluster analysis showed that the eight wild populations of S. microcarpa could be classified into four groups, at a threshold of 4.0, based on an analysis of the SSR genotypes. Furthermore, there was a significant association between the phylogenetic relationships and the geographic locations of the S. microcarpa populations. In particular, populations from Fuyang, Jiande, and Lin’an in Zhejiang Province had close phylogenetic relationships and geographic distances. In addition, these three populations had the highest genetic diversity and the most individuals, suggesting that these three locations may be the S.microcarpa distribution center. This study serves as a model for studying the genetic diversity of endangered plant species.展开更多
基金We thank the financial support from the USDA National Institute of Food and Agriculture SCRI(grant no.2015-70016-23027)the Florida Citrus Development Foundation(2016-001)+1 种基金the Genetically Modified Organisms Breeding Major Projects of China(2014ZX0801008B-001)The Connecticut-Storrs Agriculture Experimental Station,the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Innovative Research Project of JAAS(ZX-17-2006)also contributed financially to some experiments presented in this manuscript.
文摘Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable.Here,we report a highly useful method using an Agrobacterium-mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation.We have also developed a rapid,cost-effective,and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting(HRM)analysis.Using tetraploid tobacco as a model species and the phytoene desaturase(PDS)gene as a target,we successfully created and expediently identified mutant plants,which were verified as tetra-allelic mutants.We produced pds mutant shoots at a rate of 47.5%from tobacco leaf explants,without the use of antibiotic selection.Among these pds plants,17.2%were confirmed to be non-transgenic,for an overall non-transgenic mutation rate of 8.2%.Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction.This method should be applicable to many economically important,heterozygous,perennial crop species that are more difficult to regenerate.
基金supported by grants from the Natural Science Foundation of Zhejiang Province(LZ20C160002)the National Natural Science Foundation of China(31971735)the State Key Lab oratory of Subtropical Silviculture(ZY20180203).
文摘Bamboo is known for its edible shoots and beautiful texture and has considerable economic and ornamental value.Unique among traditional flowering plants,many bamboo plants undergo extensive synchronized flowering followed by large-scale death,seriously affecting the productivity and application of bamboo forests.To date,the molecular mechanism of bamboo flowering characteristics has remained unknown.In this study,a SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1)-like gene,BoMADS50,was identified from Bambusa oldhamii.BoMADS50 was highly expressed in mature leaves and the floral primordium formation period during B.oldhamii flowering and overexpression of BoMADS50 caused early fl owering in transgenic rice.Moreover,BoMADS50 could interact with APETALA1/FRUITFULL(AP1/FUL)-like proteins(BoMADS 14-1/2,BoMADS15-1/2)in vivo,and the expression of BoMADS50 was signi ficantly promoted by BoMADS14-1,further indicating a synergistic effect between BoMADS50 and BoAP1/FUL-like proteins in regulating B.oldhamii flowering.We also identi fi ed four additional transcripts of BoMADS50(BoMADS50-1/2/3/4)with different nucleotide variations.Although the protein-CDS were polymorphic,they had flowering activation functions similar to those of BoMADS50.Yeast one-hybrid and transient expression assays subsequently showed that both BoMADS50 and BoMADS50-1 bind to the promoter fragment of itself and the SHORT VEGETATIVE PHASE(SVP)-like gene BoSVP,but only BoMADS50-1 can positively induce their transcription.Therefore,nucleotide variations likely endow BoMADS50-1 with strong regulatory activity.Thus,BoMADS50 and BoMADS50-1/2/3/4 are probably important positive flowering regulators in B.oldhamii.Moreover,the functional conservatism and speci ficity of BoMADS50 and BoMADS50-1 might be related to the synchronized and sporadic flowering characteristics of B.oldhamii.
基金supported by the Natural Science Foundation of Zhejiang Province,China(LY13C160007)the Graduate Special Fund Innovative Projects of Jiangxi Province(YC2014-B035)+2 种基金Talent start research projects of Jiyang college of Zhejiang Agriculture and Forestry University(JY2018RC0X)the University Teachers’ Professional Development Project of Zhejiang Province(FX2015050)the Experiment Program of Forest Resources Monitoring Center of Zhejiang Province(2014088)
文摘Although most Sinojackia species are endangered, they contribute greatly to the biodiversity of local ecosystems. Sinojackia microcarpa, an endangered species, is distributed only in three provinces of eastern China. Determining the genetic diversity of S. microcarpa provides key information for germplasm evaluation and species conservation. Here we used simple sequence repeat (SSR) markers to investigate the genetic diversity of eight natural populations of S. microcarpa. Leaf samples were collected from 144 individuals in 8 wild populations. The 156 bands were generated from 14 pairs of informative SSR primers, with an average percentage of polymorphic bands of 45.67%. The average values of Nei’s genetic diversity (He) and Shannon’s diversity index (I) were 0.1007 and 0.1658, respectively. The total genetic variation of S. microcarpa existed mainly within the eight populations, rather than among populations, and reached 86.41%. A cluster analysis showed that the eight wild populations of S. microcarpa could be classified into four groups, at a threshold of 4.0, based on an analysis of the SSR genotypes. Furthermore, there was a significant association between the phylogenetic relationships and the geographic locations of the S. microcarpa populations. In particular, populations from Fuyang, Jiande, and Lin’an in Zhejiang Province had close phylogenetic relationships and geographic distances. In addition, these three populations had the highest genetic diversity and the most individuals, suggesting that these three locations may be the S.microcarpa distribution center. This study serves as a model for studying the genetic diversity of endangered plant species.
