Chinese cherry （Prunus pseudocerasus L.） is an allotetraploid species and exhibits natural self-compatibility.However,the full-length cDNA sequences,functional analysis and the transcripts of S-RNase alleles in Chin...Chinese cherry （Prunus pseudocerasus L.） is an allotetraploid species and exhibits natural self-compatibility.However,the full-length cDNA sequences,functional analysis and the transcripts of S-RNase alleles in Chinese cherry cultivars are not known.In the two cultivars Taixiaohongying and Laiyang Short Cherry with S1S2S3S4 genotypes,two S-RNases were transcribed in Northern blotting,and the two full-length cDNAs of S-RNase were cloned and analyzed.As the result,the transcribed S-RNases were S1-RNase and S2-RNase.The two complete cDNA sequences of S1-RNase and S2-RNase were registered as EU073938 and EU073939,respectively,and had characteristic structure of rosaceous S-RNases based on their sequences indicating that they had normal function for S-RNase in the style.The S3-RNase and S4-RNase were not transcribed in the style and were nonfunctional for S-RNase,so S3m and S4m could be used to represent the nonfunctional S3-RNase and S4-RNase.The phylogenetic analysis implied that the S-RNases of Prunus,including Chinese cherry,had lower intra-specific similarity and diverged earlier than the divergence of species in Prunus.展开更多
Identification of the S genotype of Malus plants will greatly promote the discovery of new genes,the cultivation and production of apple,the breeding of new varieties,and the origin and evolution of self-incompatibili...Identification of the S genotype of Malus plants will greatly promote the discovery of new genes,the cultivation and production of apple,the breeding of new varieties,and the origin and evolution of self-incompatibility in Malus plants.In this experiment,88 Malus germplasm resources,such as Aihuahong,Xishuhaitang,and Reguanzi,were used as materials.Seven gene-specific primer combinations were used in the genotype identification.PCR amplification using leaf DNA produced a single S-RNase gene fragment in all materials.The results revealed that 70 of the identified materials obtained a complete S-RNase genotype,while only one S-RNase gene was found in 18 of them.Through homology comparison and analysis,13 S-RNase genotypes were obtained:S_(1)S_(2)(Aihuahong,etc.),S_(1)S_(28)(Xixian Haitang,etc.),S_(1)S_(51)(Hebei Pingdinghaitang),S_(1)S_(3)(Xiangyangcun Daguo,etc.),S_(2)S_(3)(Zhaiyehaitang,etc.),S_(3)S_(51)(Xishan 1),S_(3)S_(28)(Huangselihaerde,etc.),S_(2)S_(28)(Honghaitang,etc.),S_(4)S_(28)(Bo 11),S_(7)S_(28)(Jiuquan Shaguo),S_(10)S_e(Dongchengguan 13),S_(10)S_(21)(Dongxiangjiao)and S_(3)S_(51)(Xiongyue Haitang).Simultaneously,the frequency of the S gene in the tested materials was analyzed.The findings revealed that different S genes had varying frequencies in Malus resources,as well as varying frequencies between intraspecific and interspecific.S_(3) had the highest frequency of 68.18%,followed by S_(1)(42.04%).In addition,the phylogenetic tree and origin evolution analysis revealed that the S gene differentiation was completed prior to the formation of various apple species,that cultivated species also evolved new S genes,and that the S_(50) gene is the oldest S allele in Malus plants.The S_(1),S_(29),and S_(33) genes in apple-cultivated species,on the other hand,may have originated in M.sieversii,M.hupehensis,and M.kansuensis,respectively.In addition to M.sieversii,M.kansuensis and M.sikkimensis may have also played a role in the origin and evolution of some Chinese apples.展开更多
Self-Incompatibility （SI） Is a genetic mechanism of self/non-self pollen recognition to prevent self-fertilization In many flowering plants and, In most cases, this is controlled by a multl-allellc S-locus. S-RNase ...Self-Incompatibility （SI） Is a genetic mechanism of self/non-self pollen recognition to prevent self-fertilization In many flowering plants and, In most cases, this is controlled by a multl-allellc S-locus. S-RNase and Slocus F box （SLF） proteins have been shown to be the female and male determinants of gametophytlc selfIncompatibility （GSI）, respectively, In the Solanaceae, Scrophulariaceae and Rosaceae. Nevertheless, It is thought that additional factors are required for the SI response. Herein, we constructed a mature anther cDNA library from a self-Incompatible Petunia hybrida Vllm. line of the S3S3 haplotype. Using AhS2-RNase from Antirrhinum hispanicum as a bait for yeast two-hybrid screening, we found that petunia germinating pollen （PGP） S/D3 was capable of Interacting physically with the bait. However, the Interaction lacked haplotype specificity. The PGPS/D3 gene Is a single copy gene that Is expressed In tissues such as the style, ovary, pollen, and leaf. The PGPS/D3：：GFP （green fluorescence protein） construct was detected In both the membrane and cytoplasm. The Implications of these findings In the operation of S-RNase-based SI are discussed.展开更多
Frozen young leaves of apricot(Armeniaca vulgaris) ‘Katy’ and ‘Xinshiji’ were used for isolation of total DNA. Total RNA was isolated from their styles at the balloon stage. DNA and cDNA were amplified through PCR...Frozen young leaves of apricot(Armeniaca vulgaris) ‘Katy’ and ‘Xinshiji’ were used for isolation of total DNA. Total RNA was isolated from their styles at the balloon stage. DNA and cDNA were amplified through PCR using AS1 Ⅱ and ArmyC5R as primers designed according to the conserved (C1,C5) sequences of Rosaceae S-RNases. Three S-RNase genes,P.a S8 from ‘Katy’ and P.a S9,P.a S10 from ‘Xinshiji’,were amplified and cloned. Amplified DNA bands were different sizes: P.a S8 of 927 bp,P.a S9 of 992 bp,P.a S10 of 583 bp,and cDNA bands were 521 bp,521 bp,479 bp,respectively. The results of Blastn in GenBank showed that they were novel S-RNase genes and they have been deposited in GenBank (Accession No.: AY884212,AY864826,AY864825,AY853594 and AY846872). Genomic sequences showed an intron structure between C1 and C5 region. The introns of P.a S8,P.a S9,and P.a S10 were 406 bp,471 bp,104 bp and lay in the hypervariable region (RHV) between C2 and C3. Three genes were compared and displayed similarity at the nucleotide and deduced amino acid level. Most of amino acid sequences of S-RNase gene in Prunoideae (Rosaceae) were used to form their phyligenetic tree. The evolutionary relationships showed S-RNase genes did not form a distinct cluster within species. Intra-species similarity was not higher than inter-species one. Therefore,we speculated that the evolutionary of S-RNase genes in Prunoideae was not consisted with that of species.展开更多
基金supported by the National 863 Program of China (2006AA100108)the Doctoral Fund of Shandong Province,China (2006BS06021)Fine Cultivar Program of Shandong Province,China
文摘Chinese cherry （Prunus pseudocerasus L.） is an allotetraploid species and exhibits natural self-compatibility.However,the full-length cDNA sequences,functional analysis and the transcripts of S-RNase alleles in Chinese cherry cultivars are not known.In the two cultivars Taixiaohongying and Laiyang Short Cherry with S1S2S3S4 genotypes,two S-RNases were transcribed in Northern blotting,and the two full-length cDNAs of S-RNase were cloned and analyzed.As the result,the transcribed S-RNases were S1-RNase and S2-RNase.The two complete cDNA sequences of S1-RNase and S2-RNase were registered as EU073938 and EU073939,respectively,and had characteristic structure of rosaceous S-RNases based on their sequences indicating that they had normal function for S-RNase in the style.The S3-RNase and S4-RNase were not transcribed in the style and were nonfunctional for S-RNase,so S3m and S4m could be used to represent the nonfunctional S3-RNase and S4-RNase.The phylogenetic analysis implied that the S-RNases of Prunus,including Chinese cherry,had lower intra-specific similarity and diverged earlier than the divergence of species in Prunus.
基金financially supported by the Agricultural Science and Technology Innovation Program(CAASASTIP-2021-RIP-02)。
文摘Identification of the S genotype of Malus plants will greatly promote the discovery of new genes,the cultivation and production of apple,the breeding of new varieties,and the origin and evolution of self-incompatibility in Malus plants.In this experiment,88 Malus germplasm resources,such as Aihuahong,Xishuhaitang,and Reguanzi,were used as materials.Seven gene-specific primer combinations were used in the genotype identification.PCR amplification using leaf DNA produced a single S-RNase gene fragment in all materials.The results revealed that 70 of the identified materials obtained a complete S-RNase genotype,while only one S-RNase gene was found in 18 of them.Through homology comparison and analysis,13 S-RNase genotypes were obtained:S_(1)S_(2)(Aihuahong,etc.),S_(1)S_(28)(Xixian Haitang,etc.),S_(1)S_(51)(Hebei Pingdinghaitang),S_(1)S_(3)(Xiangyangcun Daguo,etc.),S_(2)S_(3)(Zhaiyehaitang,etc.),S_(3)S_(51)(Xishan 1),S_(3)S_(28)(Huangselihaerde,etc.),S_(2)S_(28)(Honghaitang,etc.),S_(4)S_(28)(Bo 11),S_(7)S_(28)(Jiuquan Shaguo),S_(10)S_e(Dongchengguan 13),S_(10)S_(21)(Dongxiangjiao)and S_(3)S_(51)(Xiongyue Haitang).Simultaneously,the frequency of the S gene in the tested materials was analyzed.The findings revealed that different S genes had varying frequencies in Malus resources,as well as varying frequencies between intraspecific and interspecific.S_(3) had the highest frequency of 68.18%,followed by S_(1)(42.04%).In addition,the phylogenetic tree and origin evolution analysis revealed that the S gene differentiation was completed prior to the formation of various apple species,that cultivated species also evolved new S genes,and that the S_(50) gene is the oldest S allele in Malus plants.The S_(1),S_(29),and S_(33) genes in apple-cultivated species,on the other hand,may have originated in M.sieversii,M.hupehensis,and M.kansuensis,respectively.In addition to M.sieversii,M.kansuensis and M.sikkimensis may have also played a role in the origin and evolution of some Chinese apples.
基金Supported by the Chinese Academy of Sciences and the National Natural Science Foundation of China （30221002）.Acknowledgements The authors are grateful to Qi Xie （Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences） for help with the construction of the cDNA library and the yeast two-hybrid techniques and Tim Robbins for providing P. hybrida. The authors also thank Jiayang Li （Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences） for the pB1221-35S：GFP vector.
文摘Self-Incompatibility （SI） Is a genetic mechanism of self/non-self pollen recognition to prevent self-fertilization In many flowering plants and, In most cases, this is controlled by a multl-allellc S-locus. S-RNase and Slocus F box （SLF） proteins have been shown to be the female and male determinants of gametophytlc selfIncompatibility （GSI）, respectively, In the Solanaceae, Scrophulariaceae and Rosaceae. Nevertheless, It is thought that additional factors are required for the SI response. Herein, we constructed a mature anther cDNA library from a self-Incompatible Petunia hybrida Vllm. line of the S3S3 haplotype. Using AhS2-RNase from Antirrhinum hispanicum as a bait for yeast two-hybrid screening, we found that petunia germinating pollen （PGP） S/D3 was capable of Interacting physically with the bait. However, the Interaction lacked haplotype specificity. The PGPS/D3 gene Is a single copy gene that Is expressed In tissues such as the style, ovary, pollen, and leaf. The PGPS/D3：：GFP （green fluorescence protein） construct was detected In both the membrane and cytoplasm. The Implications of these findings In the operation of S-RNase-based SI are discussed.
文摘Frozen young leaves of apricot(Armeniaca vulgaris) ‘Katy’ and ‘Xinshiji’ were used for isolation of total DNA. Total RNA was isolated from their styles at the balloon stage. DNA and cDNA were amplified through PCR using AS1 Ⅱ and ArmyC5R as primers designed according to the conserved (C1,C5) sequences of Rosaceae S-RNases. Three S-RNase genes,P.a S8 from ‘Katy’ and P.a S9,P.a S10 from ‘Xinshiji’,were amplified and cloned. Amplified DNA bands were different sizes: P.a S8 of 927 bp,P.a S9 of 992 bp,P.a S10 of 583 bp,and cDNA bands were 521 bp,521 bp,479 bp,respectively. The results of Blastn in GenBank showed that they were novel S-RNase genes and they have been deposited in GenBank (Accession No.: AY884212,AY864826,AY864825,AY853594 and AY846872). Genomic sequences showed an intron structure between C1 and C5 region. The introns of P.a S8,P.a S9,and P.a S10 were 406 bp,471 bp,104 bp and lay in the hypervariable region (RHV) between C2 and C3. Three genes were compared and displayed similarity at the nucleotide and deduced amino acid level. Most of amino acid sequences of S-RNase gene in Prunoideae (Rosaceae) were used to form their phyligenetic tree. The evolutionary relationships showed S-RNase genes did not form a distinct cluster within species. Intra-species similarity was not higher than inter-species one. Therefore,we speculated that the evolutionary of S-RNase genes in Prunoideae was not consisted with that of species.
