In 1988, we found strain 9003 from compositehybridization of indica and japonica. 9003 pos-sesses characteristics of twin seedlings withfrequency of 15-20% of the investigated popu-lation. The highest frequency was 45...In 1988, we found strain 9003 from compositehybridization of indica and japonica. 9003 pos-sesses characteristics of twin seedlings withfrequency of 15-20% of the investigated popu-lation. The highest frequency was 45.68%.Afterwards, we discovered that some twinseedlings had variabilities in chromosome set.As to a pair of twin plants, it may be 2n-1n,2n-3n, 3n-3n or 1n-1n. The changes in chro-mosome set also occurred in the plants devel-oped from mono-embryo seeds. All of thesevariants nearly account for 1% in our con-trolled experiment. We named the triploidplants SAR-3 and found that SAR-3 could be aresource of diploid true breeding hybrid rice.展开更多
A total of five pairs of diploidotriploid twin-seedlings （a diploid seedling and a triploid seedling emerged from a grain） were selected out from 4500 pairs of seedlings from SARII-628, a twin-seedling rice line. SS...A total of five pairs of diploidotriploid twin-seedlings （a diploid seedling and a triploid seedling emerged from a grain） were selected out from 4500 pairs of seedlings from SARII-628, a twin-seedling rice line. SSR analysis indicated that no difference between the diploid seedling and corresponding triploid seedling in a twin-seedling was found at the 310 loci, indicating that there was no obvious change in DNA primary structure. A modified AFLP technique ＇MSAP （methylation-sensitive AFLP）＇ was used to analyze methylation mutation. Although no methylation mutation was noted among the five diploids, 29 methylation mutation loci were found from the corresponding triploids. This suggested that methylation mutation happened rapidly on Mogeneration after natural homologous triploidization. The mutations were classified into 10 types, including 3 increased types, 3 decreased types and 4 undecided types of methylation-degrees. The bands of 22 loci were sequenced and then those sequences were searched through website. The result showed that the methylation mutation involved into the whole rice genome and the 12 pairs of chromosomes. The mutation trend was site-related and there were different mutation loci for different triploids, which foretold that SARII-628 would have different evolution fates after natural homologous triploidization.展开更多
As maternal parents, diploid （L202-2x） and autotetraploid （L202-4x） of Oryza sativa cv. L2O2 were crossed with O. officinalis. Embryo development and fertilization in these two crosses were comparatively studied. ...As maternal parents, diploid （L202-2x） and autotetraploid （L202-4x） of Oryza sativa cv. L2O2 were crossed with O. officinalis. Embryo development and fertilization in these two crosses were comparatively studied. There were no mature hybrid seeds obtained because all the hybridized spikelets died 30 days after pollination. The main reasons for no seed set were abnormal fertilization and development of the embryos and endosperms in the interspecific hybrids. There were doublefertilization, egg cell single-fertilization and non-fertilization in these crosses. Although 59.45% and 54.87% of hybrid embryos produced in the crosses of L202-2x/O. officinalis and L202-4x/O. officinalis, respectively, hybrid embryos ceased to develop or degenerated and plenty of free endosperm nuclei were in disaggregating state without developing cellular endosperms three days after pollination. Besides, some embryological differences in these two crosses were found, that is, the rate of double-fertilization and total rate of doubleand single-fertilization in L202-2x/O. officinalis were higher than those in L202-4x/O. officinalis. The embryo and endosperm of hybrids developed more slowly, and embryos and free endosperm nuclei were more severely degenerated in L202-4x/O. officinalis than in L202-2x/O. officinalis. Five days after pollination, a few of embryos in L202-2x/O. officinalis developed into pear-shaped ones, however, embryos in L202-4x/O. officinalis were all degenerated. Therefore, it is more difficult to obtain interspecific hybrids by wide crosses between autotetraploid of O. sativa and O. officinalis.展开更多
Eighteen pairs of diploid-haploid twin-seedlings were identified and screened out fromspecial rice SARII-628 population. Five pairs of themwere selected and randomly designated as A, B, C, Dand E. Simple sequence repe...Eighteen pairs of diploid-haploid twin-seedlings were identified and screened out fromspecial rice SARII-628 population. Five pairs of themwere selected and randomly designated as A, B, C, Dand E. Simple sequence repeats (SSR) analysisshowed that there was no difference among 310 siteswhich indicated that there was no base mutation onDNA primary structure. DNA methylation plays animportant role in gene expression regulation duringgrowth and development stages in eukaryotes. Amodified AFLP technique (methylation-sensitiveAFLP, MSAP) was employed to detect the DNA me-thylation patterns in the 5′-CCGG sites of the fivepairs of twin-seedlings. Although no methylationmutation was detected among the five diploids,forty-three methylation mutation sites were foundfrom the corresponding haploids. The MSAP ratios,which were the ratios of MSAP sites to the total am-plified sites, in five haploids were 18.79%, 19.35%,18.49%, 18.45% and 18.75%, respectively. And cor-responding full methylation levels (5′-CmCGG indouble strands) of those haploids were 10.58%,11.3%, 10.11%, 10.09% and 10.34%, respectively.Both MSAP and full methylation levels in the fivehaploids were higher than that of their correspondingdiploids, which suggested that hypermethylation oc-curred in some 5′-CCGG sites. Five types of MASPpatterns among the five pairs of twin-seedlings weredetected as follows: (1) no changes, methylation lev-els were the same in both haploids and diploids; (2) demethylation, diploid was methylated but no me-thylation in the same site in haploid; (3) hypermethy-lation, the methylation level in haploid was higher than those in diploid; (4) hypomethylation, methyla-tion in haploid was lower than those in diploid; (5) undecided pattern, change trend of methylation lev-els in haploids was not decided. The bands of 18 sites were reclaimed, then sequenced and searched on website to determine the sites of those sequences on rice chromosomes. The result showed that the methylation mutation involved the whole rice genome and 12 pairs of chromosomes. The mutation was site-related and there were different mutation sites for different haploids. Compared to diploids, the higher methylation level in haploids might be a readjusting reaction to the decrease in ploidy for the sake of sur-vival.展开更多
In this study, microarray technique was employed to analyze the gene expression at the RNA level between haploids and corresponding diploids derived from a rice twin-seedling line SARII-628. Differ- ent degrees of exp...In this study, microarray technique was employed to analyze the gene expression at the RNA level between haploids and corresponding diploids derived from a rice twin-seedling line SARII-628. Differ- ent degrees of expression variations were observed in the plant after haploidization. The main results are as follows: (1) after haploidization, the ratio of the sensitive loci was 2.47% of the total loci designed on chip. Those loci were randomly distributed on the 12 pairs of rice chromosomes and the activated loci were more than the silenced ones. (2) Gene clusters on chromosome were observed for 33 se- quences. (3) GoPipe function classification for 575 sensitive loci revealed an involvement in the bio- logical process, cell component and molecular function. (4) RT-PCR generally validated the result from microarray with a coincidence rate of 83.78%. And for the randomly-selected activated or silenced loci in chip analysis, the coincidence rate was up to 91.86%.展开更多
Both cDNA and a genomic DNA fragment encoding a new potato proteinase inhibitor Ⅱ were isolated from a diploid potato IVP101 （Solanum phurejia L.） and named PINII-2x. Nucleotlde sequencing confirmed that the DNA fr...Both cDNA and a genomic DNA fragment encoding a new potato proteinase inhibitor Ⅱ were isolated from a diploid potato IVP101 （Solanum phurejia L.） and named PINII-2x. Nucleotlde sequencing confirmed that the DNA fragment of PINll-2xwas 580 bp, including a 115-bp intron and two exons. The deduced PINII-2x proteln contained an Intact signal peptide and two active sites. The PINII-2x gene and its deduced PINII-2x protein had 88% and 93% homology with another tetraploid potato proteinase inhibitor Ⅱ, respectively. Northern blotting analysis Indicated that the mRNA of PINII-2x gene was wound induced in potato leaves. Binary vector pNAR301 and pNAR302 were constructed for rice transformation, in which the PINII-2x cDNA was driven, respectively, by rice actin I promoter （Actl） and maize ubiquitin promoter （Ubll）. Via an Agrobacteriummediated method, these two constructs were transferred into japonica rice cv. Xiushui 63. PCR and Southern blotUng analysis for transgenic rice revealed the integration of the PINII-2x gene. Northern blotting analysis also confirmed transcripts of the PINII.2x gene in transgenic rice plants. Insect bloassays using stripe stem borer （Chilo auppressalis Walker） demonstrated that the average weight and body length of larvae In transgenic plants were only nearly 50% and 61% of those of larvae in control plants, respectively. These results Indicate that the PINII-2x gene should be an effective insect-resistance gene and could be valuable for application in crop breeding for insect resistance.展开更多
文摘In 1988, we found strain 9003 from compositehybridization of indica and japonica. 9003 pos-sesses characteristics of twin seedlings withfrequency of 15-20% of the investigated popu-lation. The highest frequency was 45.68%.Afterwards, we discovered that some twinseedlings had variabilities in chromosome set.As to a pair of twin plants, it may be 2n-1n,2n-3n, 3n-3n or 1n-1n. The changes in chro-mosome set also occurred in the plants devel-oped from mono-embryo seeds. All of thesevariants nearly account for 1% in our con-trolled experiment. We named the triploidplants SAR-3 and found that SAR-3 could be aresource of diploid true breeding hybrid rice.
文摘A total of five pairs of diploidotriploid twin-seedlings （a diploid seedling and a triploid seedling emerged from a grain） were selected out from 4500 pairs of seedlings from SARII-628, a twin-seedling rice line. SSR analysis indicated that no difference between the diploid seedling and corresponding triploid seedling in a twin-seedling was found at the 310 loci, indicating that there was no obvious change in DNA primary structure. A modified AFLP technique ＇MSAP （methylation-sensitive AFLP）＇ was used to analyze methylation mutation. Although no methylation mutation was noted among the five diploids, 29 methylation mutation loci were found from the corresponding triploids. This suggested that methylation mutation happened rapidly on Mogeneration after natural homologous triploidization. The mutations were classified into 10 types, including 3 increased types, 3 decreased types and 4 undecided types of methylation-degrees. The bands of 22 loci were sequenced and then those sequences were searched through website. The result showed that the methylation mutation involved into the whole rice genome and the 12 pairs of chromosomes. The mutation trend was site-related and there were different mutation loci for different triploids, which foretold that SARII-628 would have different evolution fates after natural homologous triploidization.
基金supported by the Guangdong Provincial Key Project of Natural Science Foundation, China (Grant No. 021037)Guangdong Province Natural Science Foundation, China (Grant No. 7301008)+1 种基金South China Agricultural University President Foundation, China (Grant No. 2007K036)The Confocal Laser Scanning Microscope was provided by the Testing Center of South China Agricultural University, Guangzhou, China
文摘As maternal parents, diploid （L202-2x） and autotetraploid （L202-4x） of Oryza sativa cv. L2O2 were crossed with O. officinalis. Embryo development and fertilization in these two crosses were comparatively studied. There were no mature hybrid seeds obtained because all the hybridized spikelets died 30 days after pollination. The main reasons for no seed set were abnormal fertilization and development of the embryos and endosperms in the interspecific hybrids. There were doublefertilization, egg cell single-fertilization and non-fertilization in these crosses. Although 59.45% and 54.87% of hybrid embryos produced in the crosses of L202-2x/O. officinalis and L202-4x/O. officinalis, respectively, hybrid embryos ceased to develop or degenerated and plenty of free endosperm nuclei were in disaggregating state without developing cellular endosperms three days after pollination. Besides, some embryological differences in these two crosses were found, that is, the rate of double-fertilization and total rate of doubleand single-fertilization in L202-2x/O. officinalis were higher than those in L202-4x/O. officinalis. The embryo and endosperm of hybrids developed more slowly, and embryos and free endosperm nuclei were more severely degenerated in L202-4x/O. officinalis than in L202-2x/O. officinalis. Five days after pollination, a few of embryos in L202-2x/O. officinalis developed into pear-shaped ones, however, embryos in L202-4x/O. officinalis were all degenerated. Therefore, it is more difficult to obtain interspecific hybrids by wide crosses between autotetraploid of O. sativa and O. officinalis.
文摘Eighteen pairs of diploid-haploid twin-seedlings were identified and screened out fromspecial rice SARII-628 population. Five pairs of themwere selected and randomly designated as A, B, C, Dand E. Simple sequence repeats (SSR) analysisshowed that there was no difference among 310 siteswhich indicated that there was no base mutation onDNA primary structure. DNA methylation plays animportant role in gene expression regulation duringgrowth and development stages in eukaryotes. Amodified AFLP technique (methylation-sensitiveAFLP, MSAP) was employed to detect the DNA me-thylation patterns in the 5′-CCGG sites of the fivepairs of twin-seedlings. Although no methylationmutation was detected among the five diploids,forty-three methylation mutation sites were foundfrom the corresponding haploids. The MSAP ratios,which were the ratios of MSAP sites to the total am-plified sites, in five haploids were 18.79%, 19.35%,18.49%, 18.45% and 18.75%, respectively. And cor-responding full methylation levels (5′-CmCGG indouble strands) of those haploids were 10.58%,11.3%, 10.11%, 10.09% and 10.34%, respectively.Both MSAP and full methylation levels in the fivehaploids were higher than that of their correspondingdiploids, which suggested that hypermethylation oc-curred in some 5′-CCGG sites. Five types of MASPpatterns among the five pairs of twin-seedlings weredetected as follows: (1) no changes, methylation lev-els were the same in both haploids and diploids; (2) demethylation, diploid was methylated but no me-thylation in the same site in haploid; (3) hypermethy-lation, the methylation level in haploid was higher than those in diploid; (4) hypomethylation, methyla-tion in haploid was lower than those in diploid; (5) undecided pattern, change trend of methylation lev-els in haploids was not decided. The bands of 18 sites were reclaimed, then sequenced and searched on website to determine the sites of those sequences on rice chromosomes. The result showed that the methylation mutation involved the whole rice genome and 12 pairs of chromosomes. The mutation was site-related and there were different mutation sites for different haploids. Compared to diploids, the higher methylation level in haploids might be a readjusting reaction to the decrease in ploidy for the sake of sur-vival.
基金the National Natural Science Foundation of China (Grant No. 30771157)the Changjiang Scholars and Innovative Research Team in University (Grant No. IRT0453)
文摘In this study, microarray technique was employed to analyze the gene expression at the RNA level between haploids and corresponding diploids derived from a rice twin-seedling line SARII-628. Differ- ent degrees of expression variations were observed in the plant after haploidization. The main results are as follows: (1) after haploidization, the ratio of the sensitive loci was 2.47% of the total loci designed on chip. Those loci were randomly distributed on the 12 pairs of rice chromosomes and the activated loci were more than the silenced ones. (2) Gene clusters on chromosome were observed for 33 se- quences. (3) GoPipe function classification for 575 sensitive loci revealed an involvement in the bio- logical process, cell component and molecular function. (4) RT-PCR generally validated the result from microarray with a coincidence rate of 83.78%. And for the randomly-selected activated or silenced loci in chip analysis, the coincidence rate was up to 91.86%.
基金Supported by the Project of Jiangsu Province(BG2001305)the Program for Changjiang Scholars and Innovative Research Team in University
文摘Both cDNA and a genomic DNA fragment encoding a new potato proteinase inhibitor Ⅱ were isolated from a diploid potato IVP101 （Solanum phurejia L.） and named PINII-2x. Nucleotlde sequencing confirmed that the DNA fragment of PINll-2xwas 580 bp, including a 115-bp intron and two exons. The deduced PINII-2x proteln contained an Intact signal peptide and two active sites. The PINII-2x gene and its deduced PINII-2x protein had 88% and 93% homology with another tetraploid potato proteinase inhibitor Ⅱ, respectively. Northern blotting analysis Indicated that the mRNA of PINII-2x gene was wound induced in potato leaves. Binary vector pNAR301 and pNAR302 were constructed for rice transformation, in which the PINII-2x cDNA was driven, respectively, by rice actin I promoter （Actl） and maize ubiquitin promoter （Ubll）. Via an Agrobacteriummediated method, these two constructs were transferred into japonica rice cv. Xiushui 63. PCR and Southern blotUng analysis for transgenic rice revealed the integration of the PINII-2x gene. Northern blotting analysis also confirmed transcripts of the PINII.2x gene in transgenic rice plants. Insect bloassays using stripe stem borer （Chilo auppressalis Walker） demonstrated that the average weight and body length of larvae In transgenic plants were only nearly 50% and 61% of those of larvae in control plants, respectively. These results Indicate that the PINII-2x gene should be an effective insect-resistance gene and could be valuable for application in crop breeding for insect resistance.
