A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris

The methylotrophic budding yeast Pichia pastoris has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter(pAOX1).However,it is difficult to use P.pastoris as the chassis cell factory for high-valuable metabolite biosynthesis due to the low homologous recombination(HR)efficiency and the limitation of handy selective markers,especially in the condition of multistep biosynthetic pathways.Hence,we developed a novel CRISPR/Cas9 system with highly editing efficiencies and recyclable auxotrophic selective marker(HiEE-ReSM)to facilitate cell factory in P.pastoris.Firstly,we improved the HR rates of P.pastoris through knocking out the non-homologous-end-joining gene(Δku70)and overexpressing HR-related proteins(RAD52 and RAD59),resulting in higher positive rate compared to the basal strain,achieved 97%.Then,we used the uracil biosynthetic genes PpURA3 as the reverse screening marker,which can improve the recycling efficiency of marker.Meanwhile,the HR rate is still 100%in uracil auxotrophic yeast.Specially,we improved the growth rate of uracil auxotrophic yeast strains by overexpressing the uracil transporter(scFUR4)to increase the uptake of exogenous uracil from medium.Meanwhile,we explored the optimal concentration of uracil(90 mg/L)for strain growth.In the end,the HiEE-ReSM system has been applied for the inositol production(250 mg/L)derived from methanol in P.pastoris.The systems will contribute to P.pastoris as an attractive cell factory for the complex compound biosynthesis through multistep metabolic pathway engineering and will be a useful tool to improve one carbon(C1)bio-utilization.

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.

Screening and Application of SSR Markers of Resistant Gene against Rice Stripe Virus

[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.

Molecular Marker Assisted Selection for Fusarium Wilt Resistance Breeding in Watermelon(Citrullus lanatus)

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.

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.

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.

SCAR Markers Assisted Selection for a Bentazon Susceptible Lethality Gene (ben) in Rice

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.

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.

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.

Molecular Characterization of Cowpea Breeding Lines for Striga Resistance Using SCAR Markers

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..

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

Construction of two selectable markers for integrative/conjugative plasmids in Flavobacterium columnare

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.

Mutant acetolactate synthase （ALS） gene as a selectable marker for Agrobacterium-mediated transformation of soybean

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.

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.

Validation of SSR Markers Linked to Flowering Time QTLs in Sorghum through Progeny Test

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.

Using Marker Assisted Selection to Hasten Screening of Cassava Cultivars Developed through Introgression of Cassava Mosaic Disease （CMD） Resistance into Cassava Landraces in Ghana

Cassava mosaic disease （CMD）, the most important disease of cassava （Manihot esculenta Crantz） is a potential threat to Africa＇s cassava production. The disease is embedded in most landraces resulting in low yields. Host plant resistance has been found to be the best control strategy. A breeding programme using genetic hybridisation and Marker Assisted Selection was initiated in 2007 to improve the resistance levels of farmer-preferred landraces and reduce the long breeding cycle for developing improved cassava varieties. Thirty farmer-preferred landraces were selected and crossed with a high yielding and mosaic resistant cultivar （TMEI l） from International Institute of Tropical Agriculture （IITA）. The resultant progenies found to have a reasonable level of resistance （score, 1-3） to the CMD were AW 18, 273 Nyamebekyere, NK 43, AW 3, NK 26, K 25, Dabodabo, Ahwengyankwa, 674 Debor, Degarti, Agric Bankye, and NK 57. These resultant progenies were backcrossed to the resistant cultivar. The backcross one （BC l） progenies totaling 224 were screened with molecular markers that are associated to the CMD 2 gene. De-oxyribonucleic Acid （DNA） was extracted from leaves of the 224 BC1 progenies and 13 parents. Two Simple Sequence Repeats （SSR） markers （SSY28 and NSl58） and one Sequenced Characterized Amplified Region （SCAR） marker RME1, were used to screen and select for the resistant BC l progenies. Marker Assisted Selection （MAS） revealed that 82% of the genotypes had at least a marker allele for the CMD2 gene, indicating resistance. The study further revealed that by using MAS, the breeding cycle of cassava in the generation of varieties could be reduced from 8 years to 2 years. The resistant genotypes identified will be evaluated for yield and starch cooking quality in future breeding work.

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.