G418-Resistance as a Dominant Selectable Marker for Heterogenous Gene Expression in Antagonist Pichia membranefaciens

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.

Haplotype variation and KASP markers for SiPSY1-A key gene controlling yellow kernel pigmentation in foxtail millet

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.

Molecular Marker Assisted Selection for Yield-Enhancing Genes in the Progeny of Minghui63 x O. rufipogon

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.

Development of branchless watermelon near isogenic lines by marker assisted selection

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.

Green Rice Leafhopper Resistance Gene Transferring Through Backcrossing and CAPS Marker Assisted Selection

Grh2, a green rice leafhopper resistant gene from an indica cultivar DV85, was located on chromosome 11, and two RFLP markers C189 and G1465 were found to be linked to this gene. In order to transfer Grh2 into Taichung65, a japonica cultivar with elite characters, backcross method with Taichung65 as the recurrent parent was used and the two RFLP markers were converted into CAPS markers for marker assisted selection (MAS). In the BC6F3 population, both phenotypic evaluation and MAS were conducted to screen the resistant plants with Taichung65 background. The linkage distance between CAPS markers and Grh2 was calculated and the efficiency of MAS was analyzed.

Analysis of short fruiting branch gene and Marker-assisted selection with SNP linked to its trait in upland cotton

Background： With the rapid development of genomics, many functional genes have been targeted. Molecular marker assisted selection can accelerate the breeding process by linking selection to functional genes. Methods： In a study of upland cotton （Gossypium hirsutum L.）, the F2 segregated population was constructed by crossing X1570 （short branches） with Ekangmian 13 （long branches） to identify the short fruiting branch gene and marker assisted selection with SNP（Single Nucleotide Polymorphisms, SNP） linked to its trait. Result： The result demonstrated that linked SSR marker BNL3232 was screened by BSA（Bulked segregant analysis, BSA） method; one SNP locus was found, which was totally separated from the fruiting branches trait in upland cotton. Conclusion： It was verified that this SNP marker could be used for molecular assisted selection of cotton architecture

Application of the New Gene Gm6 Against Rice GallMidge in Resistance Breeding Through PCR-BasedMarker Aided Selection

The research results of marker aided selection(MAS)for resistant varieties and lines against rice gall midge Orseolia oryzae Wood-Mason successfully in 1999 - 2002 were reported in the present paper. The molecular markers linked to the gene Gm6 against rice gall midge were used to select and breed the resistant varieties and lines. The RAPD marker OPM06 was used to verify the existence actually of gene Gm6 in ten developed varieties resistant to gall midge such as Duokang1, Duokang2, Kangwen2, Kangwen3, Kang-wen5, Duokangzaozhan, Kangwenqinzhan, which were derived from Daqiuqi. For resistance breeding through PCRbased marker aided selection(MAS), the polymorphisms in the resistant and susceptible parents were i-dentified by RG476/Alu I and RG476/Sca I respectively. The RAPD marker OPM06(1.4 kb)was used to i-dentify 15 new resistance lines from F3 lines of Fengyinzhan1/Daqiuqi in 1999. 21 and 7 resistance lines were selected from F4 and F6 lines of KWQZ/Gui99(restored line of hybrid rice)using RG476/Alu I in 2000-2001 respectively. The Gm6 gene was transferred into the restored line of hybrid rice. In 2001 - 2002, RG214/ Hha I and G214/Sca I were used for selecting 11 and 5 resistance lines from F3 lines of KWQZ/IR56 and AXZ/KWQZ successfully. The application of the resistance gene through PCR-based marker aided selection is a new and effective approach in resistance breeding.

Rice molecular markers and genetic mapping:Current status and prospects

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.

Search for a Microsatellite Marker Linked with Resistance Gene to Xanthomonas axonopodis pv.malvacearum in Brazilian Cotton

The cotton cultivar DELTAOPAL is resistant under field as well as under glasshouse conditions to the Brazilian isolates of Xanthomonas axonopodis pv. malvacearum (Xam). Segregating populations derived from the cross between this cultivar and one susceptible cv. BRS ITA 90, were utilized to identify molecular marker linked with the resistance gene to Xam by “Bulk Segregant Analysis (BSA)”. Two hundred and twenty microsatellite (Single Sequence Repeat—SSR) primers were tested. The amplification products were visualized in polyacrylamide gels stained with silver nitrate. Only one primer was informative and showed polymorphism between the DNA of the parents and their respective bulks of homozygous F2 populations contrasting for resistance and susceptibility, and hence was used to analyze DNA of 120 F2 populations. The microsatellite primer yielded one band of 80 bp linked with the resistance locus, which was absent in the susceptible parent as well as in the bulk of the homozygous susceptible plants of the cross. The segregation ratio as determined by phenotypic analysis was 3R:1S. It is believed that the microsatellite marker was linked with the resistance locus and hence may offer new perspectives for marker assisted selection against the angular leaf spot disease of cotton. It is however, felt necessary to repeat the microsatellite analysis and make sure that the primer is tightly linked with the resistance locus and at the same time verify the genetic distance between the marker and the resistance locus.

Applications of Molecular Markers in Fruit Crops for Breeding Programs—A Review

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.

Genome Mapping to Enhance Efficient Marker-Assisted Selection and Breeding of the Oil Palm (<i>Elaeis guineensis</i>Jacq.)

The oil palm (Elaeis guineensis Jacq.) is one of the major cultivated crops among the economically important palm species. It is cultivated mainly for its edible oil. For a perennial crop like oil palm, the use of Marker Assisted Selection (MAS) techniques helps to reduce the breeding cycle and improve the economic products. Genetic and physical maps are important for sequencing experiments since they show the exact positions of genes and other distinctive features in the chromosomal DNA. This review focuses on the role of genome mapping in oil palm breeding. It assesses the role of genome mapping in oil palm breeding and discusses the major factors affecting such mapping. Generating a high-density map governed by several factors, for instance, marker type, marker density, number of mapped population, and software used are the major issues treated. The general conclusion is that genome mapping is pivotal in the construction of a genetic linkage map. It helps to detect QTL and identify genes that control quantitative traits in oil palm. In perspective, the use of high-density molecular markers with a large number of markers, a large number mapping population, and up-to-date software is necessary for oil palm genome mapping.

A Personalized Digital Code from Unique Genome Fingerprinting Pattern for Use in Identification and Application on Blockchain

With over 10 million points of genetic variation from person to person, every individual’s genome is unique and provides a highly reliable form of identification. This is because the genetic code is specific to each individual and does not change over time. Genetic information has been used to identify individuals in a variety of contexts, such as criminal investigations, paternity tests, and medical research. In this study, each individual’s genetic makeup has been formatted to create a secure, unique code that incorporates various elements, such as species, gender, and the genetic identification code itself. The combinations of markers required for this code have been derived from common single nucleotide polymorphisms (SNPs), points of variation found in the human genome. The final output is in the form of a 24 numerical code with each number having three possible combinations. The custom code can then be utilized to create various modes of identification on the decentralized blockchain network as well as personalized services and products that offer users a novel way to uniquely identify themselves in ways that were not possible before.

Study on the Factors Influencing the Efficiency of Wheat Transformation

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.

水稻条纹叶枯病抗性基因SSR标记的筛选及应用(英文)

[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.

Large-scale Purification and Acute Toxicity of Hygromycin B PhoSphotransferase

Objective To provide the acute toxicity data of hygromycin B phosphotransferase （HPT） using recombinant protein purified from E. coli. Methods Recombinant HPT protein was expressed and purified from E. coli. To exclude the potential adverse effect of bacteria protein in recombinant HPT protein, bacterial control plasmid was constructed, and bacteria control protein was extracted and prepared as recombinant HPT protein. One hundred mice, randomly assigned to 5 groups, were administrated 10 g/kg, 5 g/kg, or 1 g/kg body weight of HPT or 5 g/kg body weight of bacterial control protein or phosphate-buffered saline （PBS） respectively by oral gavage. Results All animals survived with no significant change in body weight gain throughout the study. Macroscopic necropsy examination on day 15 revealed no gross pathological lesions in any of the animals. The maximum tolerated dose （MTD） of HPT was 10 g/kg body weight in mice and could be regarded as nontoxic. Conclusion HPT protein does not have any safety problems to human health.

Using the Phosphomannose Isomerase (PMI) Gene from Saccharomyces cerevisiae for Selection in Rice Transformation

The phosphomannose isomerase （PMI） gene from Saccharomyces cerevisiae acted as selectable marker and mannose acted as selective agent for the production of transgenic plants of rice （Oryza sativa L.） via Agrobacterium-mediated transformation. The concentration of mannose during the selection was stepwise increased, 5 g L-1 mannose combined with 15 g L-1 sucrose and 500 mg L-1 cefotaxime was used in the initial selection stage, then the concentration of mannose was increased to 11 g L-1, the highest transformation rate was 20.0%. The integration of PMI gene was confirmed by PCR, and the result of RT-PCR assay proved that the intron of PMI gene can be excised correctly during RNA splicing. 13- Glucuronidase （GUS） activity analysis confirmed the expression of GUS gene. All those means the PMI gene from yeast can be used as a selectable marker in rice transformation.

An Efficient Intragenic Vector for Generating Intragenic and Cisgenic Plants in Citrus

Genetic transformation has become a promising tool for improvement of a variety of crop species. However, transferring genes across species, the presence of selectable marker genes, and bacteria-derived vector backbone sequences have raised considerable health and environmental concerns. Intragenic vector system-based intragenesis/cisgenesis is a new method using transgenic approach to achieving traditional breeding objectives but circumventing many of the associated shortcomings. We report here the development of an intragenic vector by assembling a T-DNA-like fragment and a buffering sequence following the left border from Citrus clementina into the backbone of the binary vector pCB302. Recovery of citrus regenerants is performed under non-selective conditions and positive intra-/cisgenic regenerants were identified through PCR analysis. Transformation efficiencies obtained in Arabidopsis and “Duncan” grapefruit were ~3% and ~0.67%, respectively, demonstrating the potential of the system for development of “foreign DNA-free” intra-/cisgenic citrus cultivars.

Meta-Analysis of 100-Seed Weight QTLs in Soybean

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.

qRgls1.06, a major QTL conferring resistance to gray leaf spot disease in maize

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.