Hybrids between the indica and japonica subspecies of rice （Oryza sativa） are usually sterile, which hinders utilization of heterosis in the inter-subspecific hybrid breeding. The complex locus Sa comprises two adja...Hybrids between the indica and japonica subspecies of rice （Oryza sativa） are usually sterile, which hinders utilization of heterosis in the inter-subspecific hybrid breeding. The complex locus Sa comprises two adjacently located genes, SaF and SaM, which interact to cause abortion of pollen grains carrying the japonica allele in japonica-indica hybrids. Here we showed that silencing of SaF or SaM by RNA interference restored male fertility in indica-japonica hybrids with heterozygous Sa. We further used clustered regularly interspaced short palindromic repeats （CRISPR）/Cas9-based genome editing to knockout the SaF and SaM alleles, respectively, of an indica rice line to create hybrid-compatible lines. The resultant artificial neutral alleles did not affect pollen viability and other agricultural traits, but did break down the reproductive barrier in the hybrids. We found that some rice lines have natural neutral allele Sa-n, which was compatible with the typical japonica or indica Sa alleles in hybrids. Our results demonstrate that SaF and SaM are required for hybrid male sterility, but are not essential for pollen development. This study provides effective approaches for the generation of hybrid-compatible lines by knocking out the Sa locus or using the natural Sa-n allele to overcome hybrid male sterility in rice breeding.展开更多
Despite continuous improvements,it is difficult to efficiently amplify large sequences from complex templates using current PCR methods.Here,we developed a suppression thermo-interlaced(STI)PCR method for the efficien...Despite continuous improvements,it is difficult to efficiently amplify large sequences from complex templates using current PCR methods.Here,we developed a suppression thermo-interlaced(STI)PCR method for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools.This method uses site-specific primers containing a common 50 tag to generate a stem-loop structure,thereby repressing the amplification of smaller non-specific products through PCR suppression(PS).However,large target products are less affected by PS and show enhanced amplification when the competitive amplification of non-specific products is suppressed.Furthermore,this method uses nested thermointerlaced cycling with varied temperatures to optimize strand extension of long sequences with an uneven GC distribution.The combination of these two factors in STI PCR produces a multiplier effect,markedly increasing specificity and amplification capacity.We also developed a webtool,calGC,for analyzing the GC distribution of target DNA sequences and selecting suitable thermo-cycling programs for STI PCR.Using this method,we stably amplified very long genomic fragments(up to 38 kb)from plants and human and greatly increased the length of de novo DNA synthesis,which has many applications such as cloning,expression,and targeted genomic sequencing.Our method greatly extends PCR capacity and has great potential for use in biological fields.展开更多
基金supported by grants from the National Key Research and Development Program of China(2016YFD0100804)the National Natural Science Foundation of China(31471564)
文摘Hybrids between the indica and japonica subspecies of rice （Oryza sativa） are usually sterile, which hinders utilization of heterosis in the inter-subspecific hybrid breeding. The complex locus Sa comprises two adjacently located genes, SaF and SaM, which interact to cause abortion of pollen grains carrying the japonica allele in japonica-indica hybrids. Here we showed that silencing of SaF or SaM by RNA interference restored male fertility in indica-japonica hybrids with heterozygous Sa. We further used clustered regularly interspaced short palindromic repeats （CRISPR）/Cas9-based genome editing to knockout the SaF and SaM alleles, respectively, of an indica rice line to create hybrid-compatible lines. The resultant artificial neutral alleles did not affect pollen viability and other agricultural traits, but did break down the reproductive barrier in the hybrids. We found that some rice lines have natural neutral allele Sa-n, which was compatible with the typical japonica or indica Sa alleles in hybrids. Our results demonstrate that SaF and SaM are required for hybrid male sterility, but are not essential for pollen development. This study provides effective approaches for the generation of hybrid-compatible lines by knocking out the Sa locus or using the natural Sa-n allele to overcome hybrid male sterility in rice breeding.
基金supported by the National Natural Science Foundation of China(31991222,32030080 and 31760300)the Key Research Program of the Guangzhou Science,Technology and Innovation Commission(201904020030)+1 种基金the Major Program of Guangdong Basic and Applied Basic Research(2019B030302006)the China Postdoctoral Science Foundation(2020M682730).
文摘Despite continuous improvements,it is difficult to efficiently amplify large sequences from complex templates using current PCR methods.Here,we developed a suppression thermo-interlaced(STI)PCR method for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools.This method uses site-specific primers containing a common 50 tag to generate a stem-loop structure,thereby repressing the amplification of smaller non-specific products through PCR suppression(PS).However,large target products are less affected by PS and show enhanced amplification when the competitive amplification of non-specific products is suppressed.Furthermore,this method uses nested thermointerlaced cycling with varied temperatures to optimize strand extension of long sequences with an uneven GC distribution.The combination of these two factors in STI PCR produces a multiplier effect,markedly increasing specificity and amplification capacity.We also developed a webtool,calGC,for analyzing the GC distribution of target DNA sequences and selecting suitable thermo-cycling programs for STI PCR.Using this method,we stably amplified very long genomic fragments(up to 38 kb)from plants and human and greatly increased the length of de novo DNA synthesis,which has many applications such as cloning,expression,and targeted genomic sequencing.Our method greatly extends PCR capacity and has great potential for use in biological fields.
