Flavobacterium columnare, the etiological agent of colunmaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable marke...Flavobacterium columnare, the etiological agent of colunmaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers （chloramphenicol and spectinomycin resistance） for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G4 via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.展开更多
[Objective] New SSR primers were designed and screened to apply in the backcross breeding for modified resistance against rice stripe virus.[Method] The conventional late japonica rice varieties including 502 with hig...[Objective] New SSR primers were designed and screened to apply in the backcross breeding for modified resistance against rice stripe virus.[Method] The conventional late japonica rice varieties including 502 with high resistance to stripe virus,Xiushui 09 with high susceptibility to stripe virus and their derived strains were adopted as the test materials,SSR and SAPR markers were used to locate RSV1 gene with high resistance against stripe virus,and three pairs of SSR markers （M-11-1,M-11-2,M-11-3） were further designed.Through screening and analysis,M-11-3 was selected as the RSV1 detection marker gene for tracking RSV1 gene,thus RSV1 gene was successfully introduced to the backcross breeding of late japonica rice varieties such as Xiushui 09,and the resistance expression of different strains was identified.[Result]The resistance of improved strains against stripe virus was significantly higher than Xiushui 09,the resistance of most strains was close to the level of donor,and the expression of resistance among years was stable.Therefore,the resistance effect of RSV1 gene used in the test was very obvious,which was accurate with the assisted selection of RSV1 gene linked markers M-11-3.[Conclusion]The study certified the feasibility of molecular markers application in resistance improvement against rice stripe virus,which also showed that optimization and development of new marker genes could effectively improve the efficiency of marker-assisted selection.展开更多
Pichia membranefaciens, which was isolated from the surface of peach fruits, showed effective biocontrol capabilityagainst rhizopus rot of peach fruits. Aminoglycoside antibiotic G418 can inhibit the growth of P. memb...Pichia membranefaciens, which was isolated from the surface of peach fruits, showed effective biocontrol capabilityagainst rhizopus rot of peach fruits. Aminoglycoside antibiotic G418 can inhibit the growth of P. membranefaciens. Theminimal inhibitory concentration of G418 to P. membranefaciens in YPD medium was 100g mL-1. We constructed aphosphoglycerate kinase (PGK) promoter-driven neoR expression cassette, which was called pFL61-neo. The biocontrolyeast P. membranefaciens was transformed with pFL61-neo by lithium acetate method. Expression vector pFL61-neoconferred P. membranefaciens drug resistance to 100g mL-1 G418. The transformant could keep a high percentage ofplasmid-containing of transformant with 67.87% after 50 generations in non-selective medium. The result showed that P.membranefaciens could recognize the promoter and terminator of PGK and direct the expression of heterologous neoRgene. Expression vector pFL61-neo could exist stably in P. membranefaciens. Therefore, it is feasible to utilize G418-resistance as a dominant selectable marker for heterogenous gene expression in antagonist P. membranefaciens.展开更多
Carotenoid biosynthesis and accumulation are important in determining nutritional and commercial value of crop products.Yellow pigmentation of mature kernels caused by carotenoids is considered a vital quality trait i...Carotenoid biosynthesis and accumulation are important in determining nutritional and commercial value of crop products.Yellow pigmentation of mature kernels caused by carotenoids is considered a vital quality trait in foxtail millet,an ancient and widely cultivated cereal crop across the world.Genomic regions associated with yellow pigment content(YPC),lutein and zeaxanthin in foxtail millet grains were identified by genome-wide association analysis(GWAS),and SiPSY1(Phytoene synthase 1 which regulates formation of the 40-carbon backbone of carotenoids)was confirmed as the main contributor to all three components by knockout and overexpression analysis.SiPSY1 was expressed in seedlings,leaves,panicles,and mature seeds,and was subcellularly localized to chloroplasts.Transcription of SiPSY1 in 15 DAP immature grains was responsible for YPC in mature seeds.Selection of SiPSY1 combined with increased YPC in mature grains during domestication of foxtail millet was confirmed.Haplotype analysis suggested that expression level of SiPSY1 could be a selection target for future breeding programs,and a KASP marker was developed for selection of favorable SiPSY1 alleles in breeding.The results of this work will benefit nutritional and commercial improvement of foxtail millet varieties,as well as other cereal crops.展开更多
Selection and use of molecular markers for evaluation of DNA polymorphism in plants are couple of the most important approaches in the field of molecular genetics.The assessment of genetic diversity using morphologica...Selection and use of molecular markers for evaluation of DNA polymorphism in plants are couple of the most important approaches in the field of molecular genetics.The assessment of genetic diversity using morphological markers is not sufficient due to little differentiating traits among the species,genera or their individuals.Morphological markers are not only highly influenced by environmental factors but skilled assessment is also prerequisite to find the variations in plant genetic resources.Therefore,molecular markers are considered as efficient tools for detailed DNA based characterization of fruit crops.Molecular markers provide new directions to the efforts of plant breeders particularly in genetic variability,gene tags,gene localization,taxonomy,genetic diversity,phylogenetic analysis and also play an important role to decrease the time required for development of new and excellent cultivars.The success of molecular markers technology in genetic improvement programs depends on the close relationship among the plant breeders,biotechnologists,skilled manpower and good financial support.The present review describes application and success of molecular markers technology used for genetic improvement in different fruit crops.展开更多
In progenies resulting from crosses involving rice cultivar Norin 8m susceptible to bentazon as the donor of ben gene, SCARs tightly linked to ben were utilized for selection of ben. The homozygous and heterozygous ge...In progenies resulting from crosses involving rice cultivar Norin 8m susceptible to bentazon as the donor of ben gene, SCARs tightly linked to ben were utilized for selection of ben. The homozygous and heterozygous genotypes with ben could be identified with the SCARs. The molecular markers offer a powerful tool for indirect selection of ben and can accelerate the introgression of ben into current rice cultivars.展开更多
Dramatic changes in climatic conditions that supplement the biotic and abiotic stresses pose severe threat to the sustainable rice production and have made it a difficult task for rice molecular breeders to enhance pr...Dramatic changes in climatic conditions that supplement the biotic and abiotic stresses pose severe threat to the sustainable rice production and have made it a difficult task for rice molecular breeders to enhance production and productivity under these stress factors. The main focus of rice molecular breeders is to understand the fundamentals of molecular pathways involved in complex agronomic traits to increase the yield. The availability of complete rice genome sequence and recent improvements in rice genomics research has made it possible to detect and map accurately a large number of genes by using linkage to DNA markers. Linkage mapping is an effective approach to identify the genetic markers which are co-segregating with target traits within the family. The ideas of genetic diversity, quantitative trait locus（QTL） mapping, and marker-assisted selection（MAS） are evolving into more efficient concepts of linkage disequilibrium（LD） also called association mapping and genomic selection（GS）, respectively. The use of cost-effective DNA markers derived from the fine mapped position of the genes for important agronomic traits will provide opportunities for breeders to develop high-yielding, stress-resistant, and better quality rice cultivars. Here we focus on the progress of molecular marker technologies, their application in genetic mapping and evolution of association mapping techniques in rice.展开更多
S. gesnerioides （Willd） Vatke is a major biological constraint to cowpea production in the dry savanna of sub-Saharan Africa. Yield losses caused by S. gesnerioides in these regions are estimated in millions of tons...S. gesnerioides （Willd） Vatke is a major biological constraint to cowpea production in the dry savanna of sub-Saharan Africa. Yield losses caused by S. gesnerioides in these regions are estimated in millions of tons annually, and prevalence of Striga soil infestation is steadily increasing. The availability of molecular markers tightly linked to S. gesnerioides resistance genes opens up the possibility of applying Marker-Assisted Selection （MAS） to cowpea and would fast track the process of developing resistance varieties to the parasite. In the present study, we report the use of Fast Technology for Analysis （FTA） also known as PlantSaver Cards （Whatman~ FTA）, developed by Flinder Technology associate to retrieve DNA from plant tissue for molecular analysis. A total of 100 F2 individual plants derived from two crosses were validated for SG3 resistance using two different SCAR markers （MahSe2 and C42B） linked to Striga race 3 （SG3） and 5 （SG5） resistance in other segregating populations. Genomic DNA was successfully recovered from leaf tissues of cowpea pressed onto FTA classic card and the DNA obtained from the FTA papers was found to be suitable for molecular analysis by PCR-based techniques. The marker efficiency of SCAR MahSe2 and C42B in detecting SG3 resistance was 98.5% and 93% respectively. This result revealed the utility of SCAR markers in cowpea breeding programme. Therefore, the application of MAS using FTA technology has the potential to increase efficiency of selection and for molecular characterization of cowpea lines for Striga resistance..展开更多
Soybean is one of the crops most difficult to be manipulated in vitro. Although several soybean marker genes, all the selectable markers used were from bacteria origin. To find suitable selectable marker gene from pla...Soybean is one of the crops most difficult to be manipulated in vitro. Although several soybean marker genes, all the selectable markers used were from bacteria origin. To find suitable selectable marker gene from plant origin for soybean transformation, a mutant acetolactate synthase （ALS） gene from Arabidopsis thaliana was tested for Agrobacterium-mediated soybean embryo axis transformation with the herbicide Arsenal as the selective agent. Transgenic soybean plants were obtained after the herbicide se- lection and the To transgenic lines showed resistance to the herbicide at a concentration of 100 g/ha. ALS enzyme assay of To transgenic line also showed higher activity compared to the wild type control plant. PCR analysis of the T1 transgenic lines confirmed the integration and segregation of the transgene. Taken together, our results showed that the mutant ALS gene is a suitable selectable marker for soybean transformation.展开更多
Flowering time is critically important for crop yield, and detection of its genetic factors with strongly associated DNA markers is necessary in breeding programs. This study was undertaken to validate the quantitativ...Flowering time is critically important for crop yield, and detection of its genetic factors with strongly associated DNA markers is necessary in breeding programs. This study was undertaken to validate the quantitative trait loci （QTLs） underlying flowering time of sorghum based on the association between genotypes at SSR marker loci and flowering time in F3 family lines from self-pollinated heterozygous F2 plants developed by crossing between ＂SC112＂---an early flowering variety from Ethiopia and ＂Kikuchi Zairai＂--a late flowering variety from Japan. The results showed that the SSR markers linked to the QTLs on sorghum chromosomes 1, 2, 3, 5b, 7 and 8b were significantly （P 〈 0.05） associated with flowering time, and these markers and the QTLs reported previously are valid. On the other hand, the genotypes at the marker locus SB596 of qFT1-2 on chromosome 1 was not significantly associated with flowering time. The valid DNA markers, SB258 in qFTI-1, SB 1512 in qFT2, SB 1839 in qFT3, SB3369 in qFT5b, SB4096 in qFT7 and SB4540 and SB4660 in qFT8b, might be useful for DNA-marker assisted breeding.展开更多
Psathyrostachys huashanica Keng ex P.C.Kuo(2n=2x=14,NsNs),a wild relative of wheat,represents a valuable germplasm resource for genetic improvement of wheat.We previously confirmed that a chromosome 7Ns from P.huashan...Psathyrostachys huashanica Keng ex P.C.Kuo(2n=2x=14,NsNs),a wild relative of wheat,represents a valuable germplasm resource for genetic improvement of wheat.We previously confirmed that a chromosome 7Ns from P.huashanica carries genes that accelerate heading and maturity in wheat.Here,we developed three small segment translocation lines(T7NsS-2BL 2BS,T7NsS-1AS 1AL#1,and T7NsS-1AS 1AL#2)along with one additional small segment translocation line(T7NsS-7BS 7BL)through^(60)Co-γ irradiation,identified using genomic in situ hybridization(GISH),fluorescence in situ hybridization(FISH),and liquid chip array analyses.Our findings demonstrated that chromosome 7NsS contained a major early heading date gene,tentatively designated Ehd-7Ns,which was mapped to an approximate31.45 Mb region,corresponding to the short arm of wheat chromosome 7A(IWGSC RefSeq v1.0).The T7NsS-1AS 1AL#2 line exhibited no significant yield penalty and possessed superior agronomic traits relative to the other translocation lines in the field,making it a promising pre-breeding donor for breeding early maturing wheat.Furthermore,21 specific Kompetitive Allele Specific PCR(KASP)markers were developed based on transcriptome data,enabling effective tracing of alien chromosomal segments carrying this source of Ehd-7Ns in marker-assisted breeding.Collectively,these newly developed translocation lines and specific KASP markers will facilitate the transfer and utilization of favorable genes from P.huashanica chromosome 7Ns in future wheat breeding programs.展开更多
Molecular identification on diploid and tetraploid watermelon breeding lines which were resistant to Fusarium wilt was carried out with the published dCAPS marker ＂4451_fon＂ which was closely linked with resistance ...Molecular identification on diploid and tetraploid watermelon breeding lines which were resistant to Fusarium wilt was carried out with the published dCAPS marker ＂4451_fon＂ which was closely linked with resistance gene of Fusarium wilt race 1. The results showed that all the diploid and tetraploid lines expressed as re- sistant genotype, which were defined as Fusarium wilt-resistant materials. The re- sults were consistent with that of artificial inoculation identification. Molecular identifi- cation results also indicated that the resistant lines were homozygote, and the Fusarium wilt-resistant gene would not separate or lose during the future self- crossed purification. Therefore, resistance selection would not be necessary in their progeny populations. The study results thought that dCAPS marker ＂4451_fon＂ could be applied on molecular marker assisted selection for Fusarium wilt resistance breeding in watermelon to increase breeding selection efficiency and accelerate breeding progress.展开更多
The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi...The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase （GALl） and phosphoglycerate kinase 1 （PGK1） promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and ex- pressed under the control of the GALl promoter, α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was en- hanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.展开更多
1000-Grain weight and spikelet number per panicle are two important components for rice grain yield. In our previous study, eight quantitative trait loci （QTLs） conferring spikelet number per panicle and 1000-grain ...1000-Grain weight and spikelet number per panicle are two important components for rice grain yield. In our previous study, eight quantitative trait loci （QTLs） conferring spikelet number per panicle and 1000-grain weight were mapped through sequencing-based genotyping of 150 rice recombinant inbred lines （RILs）. In this study, we validated the effects of four QTLs from Nipponbare using chromosome segment substitution lines （CSSLs）, and pyramided eight grain yield related QTLs. The new lines containing the eight QTLs with positive effects showed increased panicle and spikelet size as compared with the parent variety 93-11. We further proposed a novel pyramid breeding scheme based on marker-assistant and phenotype selection （MAPS）. This scheme allowed pyramiding of as many as 24 QTLs at a single hybridization without massive cross work. This study provided insights into the molecular basis of rice grain yield for direct wealth for high-yielding rice breeding.展开更多
Two yield-enhancing genes (yld1.1 and yld2.1) are located on chromosomes 1 and 2 respectivelyin a weedy relative of cultivated rice, Oryza rufipogon. SSR markers RM9 and RM166 are closelylinked with the two loci respe...Two yield-enhancing genes (yld1.1 and yld2.1) are located on chromosomes 1 and 2 respectivelyin a weedy relative of cultivated rice, Oryza rufipogon. SSR markers RM9 and RM166 are closelylinked with the two loci respectively. Minghui63 (MH63) has been a widely used restorationline in hybrid rice production in China during the past two decades. The F1 of cross 'MH63O.rufipogon' was backcrossed with MH63 generation by generation. RM9 and RM166 were used toselect the plants from the progeny of the backcross populations. The results were as follows:(1) In BC2F1 population, the percentage of the individuals which have RM9 and RM166 amplifiedbands simultaneously was 12.2%, while in the BC3F1 population, that was 16.3%. (2) Among 400individuals of BC3F1, four yield-promising plants were obtained, with yield being 30% more thanthat of MH63. (3) The products amplified by primer RM166 in O. rufipogon and MH63 weresequenced. It was found that the DNA fragment sequence amplified by RM166 from MH63 was 101 bpshorter than that from O. rufipogon. The 101bp sequence is a part of an intron of the PCNA(proliferating cell nuclear antigen) gene.展开更多
Gray leaf spot(GLS)caused by Cercospora zeae-maydis and C.zeina is an extremely devastating leaf disease that limits maize production annually.The use of GLS-resistant maize hybrids is the most cost-effective approach...Gray leaf spot(GLS)caused by Cercospora zeae-maydis and C.zeina is an extremely devastating leaf disease that limits maize production annually.The use of GLS-resistant maize hybrids is the most cost-effective approach for reducing losses.Resistance to GLS is quantitatively inherited in maize(Zea mays L.)and further sources of resistance remain to be analyzed.Here,we detected qRgls1.06,a major quantitative trait locus for GLS resistance in bin 1.06 that explained approximately 55%of the phenotype variance.Fine mapping over 2 consecutive years localized qRgls1.06 to a 2.38-Mb region.Homozygous qRgls1.06^(WGR/WGR) plants in DZ01 background displayed higher GLS resistance and 100-grain weight than DZ01 plants.The GLS responses of several susceptible elite inbred lines were improved by the introduction of qRgls1.06 by marker-assisted backcrossing.Our findings extend the understanding of the genetic basis of resistance to GLS and provide a set of resistant germplasm for genetic improvement of resistance to GLS in maize.展开更多
Pruning is time-consuming and laborious in watermelon cultivation,which can not meet the needs for simplified cultivation in the future.The development of branchless lines will provide important germplasms for breedin...Pruning is time-consuming and laborious in watermelon cultivation,which can not meet the needs for simplified cultivation in the future.The development of branchless lines will provide important germplasms for breeding watermelon varieties and is an important method for genetic improvement.In this study,the watermelon accession,Wu Cha Zao(WCZ)is a branchless inbred line that carries the branchless gene Clbl,which was used as the donor parent to develop branchless near isogenic lines(NILs).To construct the NILs of Clbl,WCZ crossed with the normal branching watermelon inbred line WT20 which was used as the recurrent parent.The co-segregating markers dCAPS10 and Indel1 with Clbl were used for foreground selection,and a total of 108 SSR markers was selected with good polymorphism between two parental lines for background selection which had relatively uniform distribution across 11 chromosomes.Using these markers to select individuals from the BC_(1)F_(1),BC_(2)F_(1),and BC_(2)F_(2) generations,three NILs with a proportion of recurrent parent genome(PRPG)>99%were finally obtained.The lateral branch and plant height phenotypes did not significantly differ between the NILs and WCZ,indicating that the NILs of Clbl under the genetic background of WT20 has been successfully developed.These results provide ideal materials for further in-depth analysis of the genetic mechanisms of lateral branch development and ideal plant architecture breeding in watermelon.展开更多
Wheat transformation efficiency is closely related to several factors such as receptor genotype, constructed plasmid and selection procedure after bombardment or co-cultivation. In our study, several kinds of antibiot...Wheat transformation efficiency is closely related to several factors such as receptor genotype, constructed plasmid and selection procedure after bombardment or co-cultivation. In our study, several kinds of antibiotics, which were normally used in plant transformation to the selection genes of nptII, bar and hpt, were tested for the optimal concentrations for wheat transformation. The results showed that 25 - 50mg/L of geneticin (G418) was suitable for the selection of nptll, kanamycin or neomycin was not suitable for use. 3 -5mg/L of phosphinothricin (PPT) or biolaphos could be used for the selection of bar, 100 - 150mg/L of hygromycin for the selection of hpt. Yangmai 158 and Yangmai 10 with high tissue culture response and good agronomic characteristics were screened from 25 potential Chinese wheat cultivars. The concentration changing of selectable agent in selection medium was helpful to obtain enough regeneration plantlets with strong root system.展开更多
The extraction of DNA is often the most time consuming and laborious step in high-throughput molecular genetic analysis and marker assisted selection （MAS） programs. A simple method for preparation of rice genomic D...The extraction of DNA is often the most time consuming and laborious step in high-throughput molecular genetic analysis and marker assisted selection （MAS） programs. A simple method for preparation of rice genomic DNA was developed. A small amount （1~50 mg） of leaf tissue of rice seedling, 500 pL of extraction buffer, and one steel bead were put into a 2-mL microcentrifuge tube. After vigorously mashing for 2 min, 5 μL of supernatant was directly applied to PCR amplification. Otherwise, the supematant was precipitated with two times volume of ethanol to obtain high quality genomic DNA. This method is simple, rapid, low cost, and reliable for PCR analysis. One person can manipulate as many as 96 samples for PCR in 10 min. It is especially suitable for genotyping of large number of samples.展开更多
100-seed weight is a very complicated quantitative trait of yield. The study of gene mapping for yield trait in soybean is very important for application. However, the mapping result of 100-seed weight was dispersed, ...100-seed weight is a very complicated quantitative trait of yield. The study of gene mapping for yield trait in soybean is very important for application. However, the mapping result of 100-seed weight was dispersed, the public map should be chosen which was suitable for the published results integrated, and to improve yield. In this research, an integrated map of 100-seed weight QTLs in soybean had been established with soymap2 published in 2004 as a reference map. QTLs of 100-seed weight in soybean were collected in recent 20 yr. With the software BioMercator 2.1, QTLs from their own maps were projected to the reference map. From published papers, 65 QTLs of 100-seed weight were collected and 53 QTLs were integrated, including 17 reductive effect QTLs and 36 additive effect QTLs. 12 clusters of QTLs were found in the integrated map. A method of meta-analysis was used to narrow down the confidence interval, and 6 additive QTLs and 6 reductive QTLs and their corresponding markers were obtained respectively. The minimum confidence interval （C.I.） was shrunk to 1.52 cM. These results would lay the foundation for marker-assisted selection and mapping QTL precisely, as well as QTL gene cloning in soybean.展开更多
基金Supported by the National Basic Research Program of China(973Program)(No.2009CB118703)
文摘Flavobacterium columnare, the etiological agent of colunmaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers （chloramphenicol and spectinomycin resistance） for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G4 via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.
基金Supported by Major Projects in Zhejiang Province(2006E10053)National Science and Technology Support Program (2006BAD01A01-5)~~
文摘[Objective] New SSR primers were designed and screened to apply in the backcross breeding for modified resistance against rice stripe virus.[Method] The conventional late japonica rice varieties including 502 with high resistance to stripe virus,Xiushui 09 with high susceptibility to stripe virus and their derived strains were adopted as the test materials,SSR and SAPR markers were used to locate RSV1 gene with high resistance against stripe virus,and three pairs of SSR markers （M-11-1,M-11-2,M-11-3） were further designed.Through screening and analysis,M-11-3 was selected as the RSV1 detection marker gene for tracking RSV1 gene,thus RSV1 gene was successfully introduced to the backcross breeding of late japonica rice varieties such as Xiushui 09,and the resistance expression of different strains was identified.[Result]The resistance of improved strains against stripe virus was significantly higher than Xiushui 09,the resistance of most strains was close to the level of donor,and the expression of resistance among years was stable.Therefore,the resistance effect of RSV1 gene used in the test was very obvious,which was accurate with the assisted selection of RSV1 gene linked markers M-11-3.[Conclusion]The study certified the feasibility of molecular markers application in resistance improvement against rice stripe virus,which also showed that optimization and development of new marker genes could effectively improve the efficiency of marker-assisted selection.
基金supported by the National Science Fund for Distinguished Young Scholars of China(30225030)the National Natural Science Foundation of China(30430480).
文摘Pichia membranefaciens, which was isolated from the surface of peach fruits, showed effective biocontrol capabilityagainst rhizopus rot of peach fruits. Aminoglycoside antibiotic G418 can inhibit the growth of P. membranefaciens. Theminimal inhibitory concentration of G418 to P. membranefaciens in YPD medium was 100g mL-1. We constructed aphosphoglycerate kinase (PGK) promoter-driven neoR expression cassette, which was called pFL61-neo. The biocontrolyeast P. membranefaciens was transformed with pFL61-neo by lithium acetate method. Expression vector pFL61-neoconferred P. membranefaciens drug resistance to 100g mL-1 G418. The transformant could keep a high percentage ofplasmid-containing of transformant with 67.87% after 50 generations in non-selective medium. The result showed that P.membranefaciens could recognize the promoter and terminator of PGK and direct the expression of heterologous neoRgene. Expression vector pFL61-neo could exist stably in P. membranefaciens. Therefore, it is feasible to utilize G418-resistance as a dominant selectable marker for heterogenous gene expression in antagonist P. membranefaciens.
基金the National Natural Science Foundation of China(U21A20216)the National Key Research and Development Program of China(2018YFD1000705/2018YFD1000700)+2 种基金Fundamental Research Funds of the Chinese Academy of Agricultural Sciences(Grant to Guanqing Jia,1610092016116,Y2017JC15)China Agricultural Research System(CARS06-14.5-A04)State Key Laboratory of Crop Gene Resources and Breeding,Key Laboratory of Grain Crop Genetic Resources Evaluation and Utilization,Key Laboratory of Crop Gene Resource and Germplasm Enhancement(MOA),and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.
文摘Carotenoid biosynthesis and accumulation are important in determining nutritional and commercial value of crop products.Yellow pigmentation of mature kernels caused by carotenoids is considered a vital quality trait in foxtail millet,an ancient and widely cultivated cereal crop across the world.Genomic regions associated with yellow pigment content(YPC),lutein and zeaxanthin in foxtail millet grains were identified by genome-wide association analysis(GWAS),and SiPSY1(Phytoene synthase 1 which regulates formation of the 40-carbon backbone of carotenoids)was confirmed as the main contributor to all three components by knockout and overexpression analysis.SiPSY1 was expressed in seedlings,leaves,panicles,and mature seeds,and was subcellularly localized to chloroplasts.Transcription of SiPSY1 in 15 DAP immature grains was responsible for YPC in mature seeds.Selection of SiPSY1 combined with increased YPC in mature grains during domestication of foxtail millet was confirmed.Haplotype analysis suggested that expression level of SiPSY1 could be a selection target for future breeding programs,and a KASP marker was developed for selection of favorable SiPSY1 alleles in breeding.The results of this work will benefit nutritional and commercial improvement of foxtail millet varieties,as well as other cereal crops.
文摘Selection and use of molecular markers for evaluation of DNA polymorphism in plants are couple of the most important approaches in the field of molecular genetics.The assessment of genetic diversity using morphological markers is not sufficient due to little differentiating traits among the species,genera or their individuals.Morphological markers are not only highly influenced by environmental factors but skilled assessment is also prerequisite to find the variations in plant genetic resources.Therefore,molecular markers are considered as efficient tools for detailed DNA based characterization of fruit crops.Molecular markers provide new directions to the efforts of plant breeders particularly in genetic variability,gene tags,gene localization,taxonomy,genetic diversity,phylogenetic analysis and also play an important role to decrease the time required for development of new and excellent cultivars.The success of molecular markers technology in genetic improvement programs depends on the close relationship among the plant breeders,biotechnologists,skilled manpower and good financial support.The present review describes application and success of molecular markers technology used for genetic improvement in different fruit crops.
基金This work was supported by grants from Anhui Province Natura1 Science Foundation(0004111O).
文摘In progenies resulting from crosses involving rice cultivar Norin 8m susceptible to bentazon as the donor of ben gene, SCARs tightly linked to ben were utilized for selection of ben. The homozygous and heterozygous genotypes with ben could be identified with the SCARs. The molecular markers offer a powerful tool for indirect selection of ben and can accelerate the introgression of ben into current rice cultivars.
文摘Dramatic changes in climatic conditions that supplement the biotic and abiotic stresses pose severe threat to the sustainable rice production and have made it a difficult task for rice molecular breeders to enhance production and productivity under these stress factors. The main focus of rice molecular breeders is to understand the fundamentals of molecular pathways involved in complex agronomic traits to increase the yield. The availability of complete rice genome sequence and recent improvements in rice genomics research has made it possible to detect and map accurately a large number of genes by using linkage to DNA markers. Linkage mapping is an effective approach to identify the genetic markers which are co-segregating with target traits within the family. The ideas of genetic diversity, quantitative trait locus（QTL） mapping, and marker-assisted selection（MAS） are evolving into more efficient concepts of linkage disequilibrium（LD） also called association mapping and genomic selection（GS）, respectively. The use of cost-effective DNA markers derived from the fine mapped position of the genes for important agronomic traits will provide opportunities for breeders to develop high-yielding, stress-resistant, and better quality rice cultivars. Here we focus on the progress of molecular marker technologies, their application in genetic mapping and evolution of association mapping techniques in rice.
文摘S. gesnerioides （Willd） Vatke is a major biological constraint to cowpea production in the dry savanna of sub-Saharan Africa. Yield losses caused by S. gesnerioides in these regions are estimated in millions of tons annually, and prevalence of Striga soil infestation is steadily increasing. The availability of molecular markers tightly linked to S. gesnerioides resistance genes opens up the possibility of applying Marker-Assisted Selection （MAS） to cowpea and would fast track the process of developing resistance varieties to the parasite. In the present study, we report the use of Fast Technology for Analysis （FTA） also known as PlantSaver Cards （Whatman~ FTA）, developed by Flinder Technology associate to retrieve DNA from plant tissue for molecular analysis. A total of 100 F2 individual plants derived from two crosses were validated for SG3 resistance using two different SCAR markers （MahSe2 and C42B） linked to Striga race 3 （SG3） and 5 （SG5） resistance in other segregating populations. Genomic DNA was successfully recovered from leaf tissues of cowpea pressed onto FTA classic card and the DNA obtained from the FTA papers was found to be suitable for molecular analysis by PCR-based techniques. The marker efficiency of SCAR MahSe2 and C42B in detecting SG3 resistance was 98.5% and 93% respectively. This result revealed the utility of SCAR markers in cowpea breeding programme. Therefore, the application of MAS using FTA technology has the potential to increase efficiency of selection and for molecular characterization of cowpea lines for Striga resistance..
基金Supported by the National Natural Science Foundation of China （No. 30370133）.
文摘Soybean is one of the crops most difficult to be manipulated in vitro. Although several soybean marker genes, all the selectable markers used were from bacteria origin. To find suitable selectable marker gene from plant origin for soybean transformation, a mutant acetolactate synthase （ALS） gene from Arabidopsis thaliana was tested for Agrobacterium-mediated soybean embryo axis transformation with the herbicide Arsenal as the selective agent. Transgenic soybean plants were obtained after the herbicide se- lection and the To transgenic lines showed resistance to the herbicide at a concentration of 100 g/ha. ALS enzyme assay of To transgenic line also showed higher activity compared to the wild type control plant. PCR analysis of the T1 transgenic lines confirmed the integration and segregation of the transgene. Taken together, our results showed that the mutant ALS gene is a suitable selectable marker for soybean transformation.
文摘Flowering time is critically important for crop yield, and detection of its genetic factors with strongly associated DNA markers is necessary in breeding programs. This study was undertaken to validate the quantitative trait loci （QTLs） underlying flowering time of sorghum based on the association between genotypes at SSR marker loci and flowering time in F3 family lines from self-pollinated heterozygous F2 plants developed by crossing between ＂SC112＂---an early flowering variety from Ethiopia and ＂Kikuchi Zairai＂--a late flowering variety from Japan. The results showed that the SSR markers linked to the QTLs on sorghum chromosomes 1, 2, 3, 5b, 7 and 8b were significantly （P 〈 0.05） associated with flowering time, and these markers and the QTLs reported previously are valid. On the other hand, the genotypes at the marker locus SB596 of qFT1-2 on chromosome 1 was not significantly associated with flowering time. The valid DNA markers, SB258 in qFTI-1, SB 1512 in qFT2, SB 1839 in qFT3, SB3369 in qFT5b, SB4096 in qFT7 and SB4540 and SB4660 in qFT8b, might be useful for DNA-marker assisted breeding.
基金funded by the National Key Research and Development Program of China(2024YFD1201202)the Major Program of National Agricultural Science and Technology of China(NK20220607)+1 种基金the Science and Technology Bureau of Sichuan Province(2023NSFSC1995,2024NSFSC1968,and 2025YFHZ0184)the Science and Technology Bureau of Chengdu City(2024-YF05-00368-SN)。
文摘Psathyrostachys huashanica Keng ex P.C.Kuo(2n=2x=14,NsNs),a wild relative of wheat,represents a valuable germplasm resource for genetic improvement of wheat.We previously confirmed that a chromosome 7Ns from P.huashanica carries genes that accelerate heading and maturity in wheat.Here,we developed three small segment translocation lines(T7NsS-2BL 2BS,T7NsS-1AS 1AL#1,and T7NsS-1AS 1AL#2)along with one additional small segment translocation line(T7NsS-7BS 7BL)through^(60)Co-γ irradiation,identified using genomic in situ hybridization(GISH),fluorescence in situ hybridization(FISH),and liquid chip array analyses.Our findings demonstrated that chromosome 7NsS contained a major early heading date gene,tentatively designated Ehd-7Ns,which was mapped to an approximate31.45 Mb region,corresponding to the short arm of wheat chromosome 7A(IWGSC RefSeq v1.0).The T7NsS-1AS 1AL#2 line exhibited no significant yield penalty and possessed superior agronomic traits relative to the other translocation lines in the field,making it a promising pre-breeding donor for breeding early maturing wheat.Furthermore,21 specific Kompetitive Allele Specific PCR(KASP)markers were developed based on transcriptome data,enabling effective tracing of alien chromosomal segments carrying this source of Ehd-7Ns in marker-assisted breeding.Collectively,these newly developed translocation lines and specific KASP markers will facilitate the transfer and utilization of favorable genes from P.huashanica chromosome 7Ns in future wheat breeding programs.
基金Supported by"12th Five-Year Plan"of National Science and Technology Plan Project in Rural Areas Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences(CAAS-ASTIP-2015-ZFRI-06)~~
文摘Molecular identification on diploid and tetraploid watermelon breeding lines which were resistant to Fusarium wilt was carried out with the published dCAPS marker ＂4451_fon＂ which was closely linked with resistance gene of Fusarium wilt race 1. The results showed that all the diploid and tetraploid lines expressed as re- sistant genotype, which were defined as Fusarium wilt-resistant materials. The re- sults were consistent with that of artificial inoculation identification. Molecular identifi- cation results also indicated that the resistant lines were homozygote, and the Fusarium wilt-resistant gene would not separate or lose during the future self- crossed purification. Therefore, resistance selection would not be necessary in their progeny populations. The study results thought that dCAPS marker ＂4451_fon＂ could be applied on molecular marker assisted selection for Fusarium wilt resistance breeding in watermelon to increase breeding selection efficiency and accelerate breeding progress.
基金Project (No.2006AA10Z316) supported by the Hi-Tech Research and Development Program (863) of China
文摘The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase （GALl） and phosphoglycerate kinase 1 （PGK1） promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and ex- pressed under the control of the GALl promoter, α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was en- hanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.
基金supported by the Ministry of Science and Technology(Grant No. 2011 CB 100205)the Ministry of Agriculture of China (Grant Nos.2011ZX08001-004 and 2011ZX08009-002)the National Natural Science Foundation of China(Grant No. 31121063)
文摘1000-Grain weight and spikelet number per panicle are two important components for rice grain yield. In our previous study, eight quantitative trait loci （QTLs） conferring spikelet number per panicle and 1000-grain weight were mapped through sequencing-based genotyping of 150 rice recombinant inbred lines （RILs）. In this study, we validated the effects of four QTLs from Nipponbare using chromosome segment substitution lines （CSSLs）, and pyramided eight grain yield related QTLs. The new lines containing the eight QTLs with positive effects showed increased panicle and spikelet size as compared with the parent variety 93-11. We further proposed a novel pyramid breeding scheme based on marker-assistant and phenotype selection （MAPS）. This scheme allowed pyramiding of as many as 24 QTLs at a single hybridization without massive cross work. This study provided insights into the molecular basis of rice grain yield for direct wealth for high-yielding rice breeding.
文摘Two yield-enhancing genes (yld1.1 and yld2.1) are located on chromosomes 1 and 2 respectivelyin a weedy relative of cultivated rice, Oryza rufipogon. SSR markers RM9 and RM166 are closelylinked with the two loci respectively. Minghui63 (MH63) has been a widely used restorationline in hybrid rice production in China during the past two decades. The F1 of cross 'MH63O.rufipogon' was backcrossed with MH63 generation by generation. RM9 and RM166 were used toselect the plants from the progeny of the backcross populations. The results were as follows:(1) In BC2F1 population, the percentage of the individuals which have RM9 and RM166 amplifiedbands simultaneously was 12.2%, while in the BC3F1 population, that was 16.3%. (2) Among 400individuals of BC3F1, four yield-promising plants were obtained, with yield being 30% more thanthat of MH63. (3) The products amplified by primer RM166 in O. rufipogon and MH63 weresequenced. It was found that the DNA fragment sequence amplified by RM166 from MH63 was 101 bpshorter than that from O. rufipogon. The 101bp sequence is a part of an intron of the PCNA(proliferating cell nuclear antigen) gene.
基金This work was supported by the National Key Laboratory of Crop Genetic Improvement Self-Research Program(ZW18B0101)the China Scholarship Council(201908420122)+1 种基金the Teachers’Scientific Ability Cultivation Foundation of Hubei University of Arts and Science(PYSB20201001)the Xiangyang Youth Science and Technology Talent Development Plan.
文摘Gray leaf spot(GLS)caused by Cercospora zeae-maydis and C.zeina is an extremely devastating leaf disease that limits maize production annually.The use of GLS-resistant maize hybrids is the most cost-effective approach for reducing losses.Resistance to GLS is quantitatively inherited in maize(Zea mays L.)and further sources of resistance remain to be analyzed.Here,we detected qRgls1.06,a major quantitative trait locus for GLS resistance in bin 1.06 that explained approximately 55%of the phenotype variance.Fine mapping over 2 consecutive years localized qRgls1.06 to a 2.38-Mb region.Homozygous qRgls1.06^(WGR/WGR) plants in DZ01 background displayed higher GLS resistance and 100-grain weight than DZ01 plants.The GLS responses of several susceptible elite inbred lines were improved by the introduction of qRgls1.06 by marker-assisted backcrossing.Our findings extend the understanding of the genetic basis of resistance to GLS and provide a set of resistant germplasm for genetic improvement of resistance to GLS in maize.
基金supported by the National Natural Science Foundation of China(Grant Nos.32102389,32172602,32172574)the Funding of Joint Research on Agricultural Varietie Improvement of Henan Province(Grant No.2022010503)+4 种基金the Zhongyuan Youth Talent Support Program(Grant No.ZYQR201912161)the Program for Science&Technology Innovation Talents in Universities of Henan Province(Grant No.21HASTIT038)the Key Scientific and Technological Project of Henan Province(Grant No.202102110045)the Major Science and Technology Project of Henan Province(Grant No.221100110400)the Science and Technology Innovation Fund of Henan Agricultural University(Grant No.KJCX2021A14).
文摘Pruning is time-consuming and laborious in watermelon cultivation,which can not meet the needs for simplified cultivation in the future.The development of branchless lines will provide important germplasms for breeding watermelon varieties and is an important method for genetic improvement.In this study,the watermelon accession,Wu Cha Zao(WCZ)is a branchless inbred line that carries the branchless gene Clbl,which was used as the donor parent to develop branchless near isogenic lines(NILs).To construct the NILs of Clbl,WCZ crossed with the normal branching watermelon inbred line WT20 which was used as the recurrent parent.The co-segregating markers dCAPS10 and Indel1 with Clbl were used for foreground selection,and a total of 108 SSR markers was selected with good polymorphism between two parental lines for background selection which had relatively uniform distribution across 11 chromosomes.Using these markers to select individuals from the BC_(1)F_(1),BC_(2)F_(1),and BC_(2)F_(2) generations,three NILs with a proportion of recurrent parent genome(PRPG)>99%were finally obtained.The lateral branch and plant height phenotypes did not significantly differ between the NILs and WCZ,indicating that the NILs of Clbl under the genetic background of WT20 has been successfully developed.These results provide ideal materials for further in-depth analysis of the genetic mechanisms of lateral branch development and ideal plant architecture breeding in watermelon.
文摘Wheat transformation efficiency is closely related to several factors such as receptor genotype, constructed plasmid and selection procedure after bombardment or co-cultivation. In our study, several kinds of antibiotics, which were normally used in plant transformation to the selection genes of nptII, bar and hpt, were tested for the optimal concentrations for wheat transformation. The results showed that 25 - 50mg/L of geneticin (G418) was suitable for the selection of nptll, kanamycin or neomycin was not suitable for use. 3 -5mg/L of phosphinothricin (PPT) or biolaphos could be used for the selection of bar, 100 - 150mg/L of hygromycin for the selection of hpt. Yangmai 158 and Yangmai 10 with high tissue culture response and good agronomic characteristics were screened from 25 potential Chinese wheat cultivars. The concentration changing of selectable agent in selection medium was helpful to obtain enough regeneration plantlets with strong root system.
基金supported by grants from the Ministry of Agriculture of China for transgenic research(Grant No.2008ZX08009-003)the National Natural Science Foundation of China(Grant Nos.30710103903 and 30771160)the Natural Science Foundation of Zhejiang Province,China(Grant No.R3090023)
文摘The extraction of DNA is often the most time consuming and laborious step in high-throughput molecular genetic analysis and marker assisted selection （MAS） programs. A simple method for preparation of rice genomic DNA was developed. A small amount （1~50 mg） of leaf tissue of rice seedling, 500 pL of extraction buffer, and one steel bead were put into a 2-mL microcentrifuge tube. After vigorously mashing for 2 min, 5 μL of supernatant was directly applied to PCR amplification. Otherwise, the supematant was precipitated with two times volume of ethanol to obtain high quality genomic DNA. This method is simple, rapid, low cost, and reliable for PCR analysis. One person can manipulate as many as 96 samples for PCR in 10 min. It is especially suitable for genotyping of large number of samples.
基金supported by the Chinese Transgenic Specific Technology Programs (2009ZX08009-013B)
文摘100-seed weight is a very complicated quantitative trait of yield. The study of gene mapping for yield trait in soybean is very important for application. However, the mapping result of 100-seed weight was dispersed, the public map should be chosen which was suitable for the published results integrated, and to improve yield. In this research, an integrated map of 100-seed weight QTLs in soybean had been established with soymap2 published in 2004 as a reference map. QTLs of 100-seed weight in soybean were collected in recent 20 yr. With the software BioMercator 2.1, QTLs from their own maps were projected to the reference map. From published papers, 65 QTLs of 100-seed weight were collected and 53 QTLs were integrated, including 17 reductive effect QTLs and 36 additive effect QTLs. 12 clusters of QTLs were found in the integrated map. A method of meta-analysis was used to narrow down the confidence interval, and 6 additive QTLs and 6 reductive QTLs and their corresponding markers were obtained respectively. The minimum confidence interval （C.I.） was shrunk to 1.52 cM. These results would lay the foundation for marker-assisted selection and mapping QTL precisely, as well as QTL gene cloning in soybean.
