Brassica rapa is one of the most important leafy vegetable crops with large cultivated area in China.To increase the availability of DNA markers in B.rapa,we developed insertion-deletion(InDel)markers utilizing high-r...Brassica rapa is one of the most important leafy vegetable crops with large cultivated area in China.To increase the availability of DNA markers in B.rapa,we developed insertion-deletion(InDel)markers utilizing high-resolution melting(HRM)curve analysis.We designed primers for 252 InDels(≥3 bp)evenly distributed in the genome and tested gene polymorphisms with eight accessions.In total,208 markers were specifically amplified,and 148 InDels with polymorphism were genotyped successfully using HRM.We further analyzed the correlation with InDel size,GC number,and predicted the difference in Tm values(Tm)using 208 markers with specific amplification.We found that the success rate of InDel markers was correlated with the GC number of InDel and the predicted-Tm,but not clearly correlated with the length of InDel.When the GC number within InDel was≥8,the successful rate exceeded 90.0%.When the predicted-Tm reached 0.5°C,the success rate was greater than 90.0%,and when it was≥0.6°C,the rate climbed to 100.0%,indicating their role as the optimal parameter for successful development of an applicable InDel marker.The polymorphic InDel markers can be easily genotyped using HRM.They are of great value in genetic analysis,construction of linkage map,and molecular marker-assisted selection in B.rapa.展开更多
Background: Cytoplasmic male sterility in flowering plants is a convenient way to use heterosis via hybrid breeding and may be restored by nuclear restorer-of-fertility(Rf) genes. In most cases, Rf genes encoded penta...Background: Cytoplasmic male sterility in flowering plants is a convenient way to use heterosis via hybrid breeding and may be restored by nuclear restorer-of-fertility(Rf) genes. In most cases, Rf genes encoded pentatricopeptide repeat(PPR) proteins and several Rf genes are present in clusters of similar Rf-PPR-like(RFL) genes. However, the Rf genes in cotton were not fully characterized until now.Results: In total, 35 RFL genes were identified in G. hirsutum, 16 in G. arboreum, and 24 in G. raimondii. Additionally,four RFL-rich regions were identified; the RFL-rich region in Gh05 is the probable location of Rf-PPR genes in cotton and will be studied further in the future. Furthermore, an insertion sequence was identified in the promoter sequence of Gh05 G3392 gene in the restorer line, as compared with the CMS-D2 line and maintainer lines. An InDel-R marker was then developed and could be used to distinguish the restorer line carrying Rfl from other genotypes without the Rf1 allele.Conclusion: In this study, genome-wide identification and analysis of RFL genes have identified the candidate Rf-PPR genes for CMS in Gossypium. The identification and analysis of RFL genes and sequence variation analysis will be useful for cloning Rf genes in the future and also for three-line hybrid breeding in cotton.展开更多
Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility （...Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility （CMS） in japonica rice, a functional marker InDeI-Rf-la based on the difference of nucleotide sequence in Rf-la locus between BT-type CMS lines and restorer lines was developed to detect the genotypes of different rice materials. Conventional indica rice varieties, restorer and maintainer lines without 574 bp deletion could restore the fertility for BT-type CMS in japonica rice. By contrast, most conventional japonica rice varieties except Aichi 106 and Yijing 12, with genotype of rf-larf-la showed the 574 bp deletion maintained sterility for BT-type CMS lines. To further verify the effect of genotyping detection in Rf-la locus, this marker was also used to amplify the genomic DNA in different japonica rice restorer lines, CMS lines, hybrids and F2 segregation population, and three genotypes in Rf-la locus could be distinguished distinctly. Therefore, the marker InDeI-Rf-la could be widely used for genetic id^ntifio.~tinn ~nd m^rkp.r-~.~.~i^fp.d .~.tAr.tinn （MA.~ in hr~=dinn i^nnnir~ r^fnr~=r lin==~展开更多
Soybean [Glycine max(L.) Merrill] is a major plant source of protein and oil. An accurate and well-saturated molecular linkage map is a prerequisite for forward genetic studies of gene function and for modern breeding...Soybean [Glycine max(L.) Merrill] is a major plant source of protein and oil. An accurate and well-saturated molecular linkage map is a prerequisite for forward genetic studies of gene function and for modern breeding for many useful agronomic traits. Next-generation sequence data available in public databases provides valuable information and offers new insights for rapid and efficient development of molecular markers. In this study, we attempted to show the feasibility and facility of using genomic resequencing data as raw material for identifying putative In Del markers. First, we identified 17,613 In Del sites among 56 soybean accessions and obtained 12,619 primer pairs. Second, we constructed a genetic map with a random subset of 2841 primer pairs and aligned 300 polymorphic markers with the 20 consensus linkage groups(LG). The total genetic distance was 2347.3 c M and the number of mapped markers per LG ranged from 10 to 23 with an average of 15 markers. The largest and smallest genetic distances between adjacent markers were 52.3 c M and 0.1 cM, respectively. Finally, we validated the genetic map constructed by newly developed In Del markers by QTL analysis of days to flowering(DTF) under different environments. One major QTL(qDTF4) and four minor QTL(qDTF20, qDTF13, qDTF12,and q DTF11) on 5 LGs were detected. These results demonstrate the utility of the In Del markers developed in this work for map-based cloning and molecular breeding in soybean.展开更多
基金This work is supported by the National Natural Science Foundation of China(Grant No.31630068)the National Program on Key Research Project(Grant No.2016YFD0100307)the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences,the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops,Ministry of Agriculture,China.
文摘Brassica rapa is one of the most important leafy vegetable crops with large cultivated area in China.To increase the availability of DNA markers in B.rapa,we developed insertion-deletion(InDel)markers utilizing high-resolution melting(HRM)curve analysis.We designed primers for 252 InDels(≥3 bp)evenly distributed in the genome and tested gene polymorphisms with eight accessions.In total,208 markers were specifically amplified,and 148 InDels with polymorphism were genotyped successfully using HRM.We further analyzed the correlation with InDel size,GC number,and predicted the difference in Tm values(Tm)using 208 markers with specific amplification.We found that the success rate of InDel markers was correlated with the GC number of InDel and the predicted-Tm,but not clearly correlated with the length of InDel.When the GC number within InDel was≥8,the successful rate exceeded 90.0%.When the predicted-Tm reached 0.5°C,the success rate was greater than 90.0%,and when it was≥0.6°C,the rate climbed to 100.0%,indicating their role as the optimal parameter for successful development of an applicable InDel marker.The polymorphic InDel markers can be easily genotyped using HRM.They are of great value in genetic analysis,construction of linkage map,and molecular marker-assisted selection in B.rapa.
基金financed by National Key Research and Development Program of China(2016YFD0101400)Foundation of State Key Laboratory of Cotton Biology(CB2018C06)
文摘Background: Cytoplasmic male sterility in flowering plants is a convenient way to use heterosis via hybrid breeding and may be restored by nuclear restorer-of-fertility(Rf) genes. In most cases, Rf genes encoded pentatricopeptide repeat(PPR) proteins and several Rf genes are present in clusters of similar Rf-PPR-like(RFL) genes. However, the Rf genes in cotton were not fully characterized until now.Results: In total, 35 RFL genes were identified in G. hirsutum, 16 in G. arboreum, and 24 in G. raimondii. Additionally,four RFL-rich regions were identified; the RFL-rich region in Gh05 is the probable location of Rf-PPR genes in cotton and will be studied further in the future. Furthermore, an insertion sequence was identified in the promoter sequence of Gh05 G3392 gene in the restorer line, as compared with the CMS-D2 line and maintainer lines. An InDel-R marker was then developed and could be used to distinguish the restorer line carrying Rfl from other genotypes without the Rf1 allele.Conclusion: In this study, genome-wide identification and analysis of RFL genes have identified the candidate Rf-PPR genes for CMS in Gossypium. The identification and analysis of RFL genes and sequence variation analysis will be useful for cloning Rf genes in the future and also for three-line hybrid breeding in cotton.
基金supported by the National High-Tech Research and Development Program of China (‘863’ Program) (Grant No. 2011AA10A100)the Key Support Program of Jiangsu Science and Technology, China (Grant No. BE2009303-1)Self-Directed Innovation Fund of Agricultural Science and Technology in Jiangsu Province, China (Grant No. CX[11]4021)
文摘Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility （CMS） in japonica rice, a functional marker InDeI-Rf-la based on the difference of nucleotide sequence in Rf-la locus between BT-type CMS lines and restorer lines was developed to detect the genotypes of different rice materials. Conventional indica rice varieties, restorer and maintainer lines without 574 bp deletion could restore the fertility for BT-type CMS in japonica rice. By contrast, most conventional japonica rice varieties except Aichi 106 and Yijing 12, with genotype of rf-larf-la showed the 574 bp deletion maintained sterility for BT-type CMS lines. To further verify the effect of genotyping detection in Rf-la locus, this marker was also used to amplify the genomic DNA in different japonica rice restorer lines, CMS lines, hybrids and F2 segregation population, and three genotypes in Rf-la locus could be distinguished distinctly. Therefore, the marker InDeI-Rf-la could be widely used for genetic id^ntifio.~tinn ~nd m^rkp.r-~.~.~i^fp.d .~.tAr.tinn （MA.~ in hr~=dinn i^nnnir~ r^fnr~=r lin==~
基金supported by National Natural Science Foundation of China (31430065, 31571686, 31371643, 31071445)National Key Research and Development Program (2016YFD0100401)+4 种基金“Strategic Priority Research Program” of the Chinese Academy of Sciences (XDA08030108)the Open Foundation of the Key Laboratory of Soybean Molecular Design Breeding of Chinese Academy of Sciences“One-hundred Talents” Startup Funds from Chinese Academy of SciencesScientific Research Foundation for Returned Chinese Scholars of Heilongjiang Province, China (LC201417)the Science Foundation for Creative Research Talents of Harbin Science and Technology Bureau, China (2014RFQYJ046)
文摘Soybean [Glycine max(L.) Merrill] is a major plant source of protein and oil. An accurate and well-saturated molecular linkage map is a prerequisite for forward genetic studies of gene function and for modern breeding for many useful agronomic traits. Next-generation sequence data available in public databases provides valuable information and offers new insights for rapid and efficient development of molecular markers. In this study, we attempted to show the feasibility and facility of using genomic resequencing data as raw material for identifying putative In Del markers. First, we identified 17,613 In Del sites among 56 soybean accessions and obtained 12,619 primer pairs. Second, we constructed a genetic map with a random subset of 2841 primer pairs and aligned 300 polymorphic markers with the 20 consensus linkage groups(LG). The total genetic distance was 2347.3 c M and the number of mapped markers per LG ranged from 10 to 23 with an average of 15 markers. The largest and smallest genetic distances between adjacent markers were 52.3 c M and 0.1 cM, respectively. Finally, we validated the genetic map constructed by newly developed In Del markers by QTL analysis of days to flowering(DTF) under different environments. One major QTL(qDTF4) and four minor QTL(qDTF20, qDTF13, qDTF12,and q DTF11) on 5 LGs were detected. These results demonstrate the utility of the In Del markers developed in this work for map-based cloning and molecular breeding in soybean.
