Utilization of Cotton DNA Markers in Cotton Breeding

Informative,portable,and efficient DNA markers have the potential to accelerate genetic gain in cotton breeding.Discovery and widespread application of DNA markers to cotton has

Study Genetic Variation Using DNA Molecular Markers and Identification Physiological Races of Wheat Stripe （yellow） Rust Puccinia striiformis f.sp tritici during 2010-2014 in Some Regions of Syria

Yellow Rust （stripe） rust （Puccinia striiformis West. f. sp. tritici） is one of the most epidemic diseases infect wheat in cold and wet regions. In 1988, this disease caused a loss of seasonal production amounted 70% on wheat variety Mexipak in Syria, and recurrent infection in 2010, caused by a virulent race called Yr27, caused a considerable loss in the production of bread wheat cultivars （Cham 8, Cham 6 particularly） amounted 90%. Recently, 15 races of yellow rust had been addressed in Syria for seasons 2010-2014; 159E256, 166E254, 166E256, 255 E112, 0 E0, 64 E 6, 230 El50, 0 E 18, 198 El30, 166 El50, 102 El60, 128 E0, 126 El50, 214E150, and 6E16. The race 6E16 was the most frequent during the two seasons, while the race 255El12 was the most virulent, followed by the race 230E222 and the race 0E0 was the weakest one. This study revealed the presence of fourteen newly observed races in Syria. Molecular Variance Analysis of Molecular Variance （AMOVA） of 55 yellow rust Puccinia striiformis f.sp tritici isolates examined by Amplify Fragment Length Polymorphism （AFLP） revealed high genetic variation within population, and the dimensional scale analysis （MSD） and tree diagram showed that the Syrian yellow rust isolates were clustered in three groups： the first group contained isolates derived from durum wheat, the second one contained bread wheat isolates, but the third was made of isolates derived from both durum and bread wheat species.

DNA Extraction from a Single Seed for Marker-Assisted Selection in Squash

Marker-assisted selection is an important tool in squash (Cucurbita species) breeding. A seed-based genotyping system would not only allow selection of desirable individuals prior to planting, but also reduce the cost associated with leaf-derived DNA genotyping, such as the need for greenhouse facilities and ultra-low-temperature storage freezers. A robust seed-based genotyping system requires a non-destructive sampling method and DNA of sufficient quantity and quality for marker-assisted selection. In the current study, six cultivars representing Cucurbita pepo (Black Beauty and Yellow Crookneck), C. moschata (Butterbush and Fairytale), and C. maxima (Buttercup and Big Max) were used to develop a suitable seed-based genotyping system for squash. Seed chips for DNA extraction were sampled by removing 1/3 of the distal end, while the remnant seed-embryos were sowed to assess germination potential. Four extraction methods including two column-based commercial kits (CTAB and ENZA) and two detergent-based conventional methods (CTAB and SDS) were assessed for DNA quality and quantity. Utility of extracted DNA for downstream applications was tested by genotyping with SSR and SNP markers. There was no significant difference in germination percentage between whole and cut seeds across the six cultivars. The average DNA concentration across methods ranged from 11.6 ng/μL to 62.6 ng/μl, while the DNA quality (A260/280) ranged from 0.89 to 1.95. Although DNA was obtained for all the extraction methods, only EZNA and Favorgen methods yielded DNA of sufficient quality for marker-assisted selection.

Genetic Structure and Diversity of Parental Cultivars Involved in China Mainland Sugarcane Breeding Programs as Inferred from DNA Microsatellites

To understand genetic structure and diversity of parental cultivars involved in China Mainland sugarcane breeding programs, 92 elite parents and 4 wild relatives were genotyped with 18 microsatellite DNA markers. The genetic similarity （GS） values among the cultivars ranged from 0.346 to 0.960 with an average of 0.533. Among the introduced cultivars, India accessions had the closest genetic distance to China Mainland accessions （0.447）, while Australia accessions have the furthest distance （0.503）. A comparison of allelic diversity among geographical origins showed that there were 22 China Mainland specific alleles, of which 28% were derived from native S. spontaneaum germplasm in China. Model-based genetic structure, clustering, and principal components analyses consistently revealed there were five groups within the 96 accessions. Groups 1, 2, 4, and 5 consisted of all cultivars and group 3 only contained wild germplasm. Group 2 was characterized as the Introduction group with 46 cultivars predominantly introduced from Australia, Taiwan of China, India, and USA. Groups 1, 4, and 5 consisted of cultivars mostly originated from China Mainland, defined as the Complex group, Yacheng lines group, and F134/CP72-1210 group, respectively, upon their pedigree. By understanding the genetic relationships among the parental cultivars, breeders can gain a rational basis for expanding the gene pool and select the best parental accessions for crossing.

Development of high-resolution chloroplast markers for intraspecific phylogeographic studies of Phaeocystis globosa

Phaeocystis globosa is an important harmful algal bloom causative species distributing widely in temperate and tropical coastal waters in the world.The morphological,physiological,and biochemical characteristics are different among geographic strains,which can not be distinguished with nuclear ribosomal DNA markers at present.Therefore,the genetic distance and phylogeographic relationships of nuclear 28S rDNA D1–D2 and ITS regions,and three chloroplast intergenic spacers(petN-trnS1,trnM1-psbA,and rbcS-rpl27)were analyzed and compared among 13 strains of P.globosa isolated from the Pacific Ocean and Atlantic Ocean in this study.In addition,the nucleotide polymorphisms of 28S rDNA D1–D2,ITS,and rbcS-rpl27 regions were evaluated in two P.globosa strains.The various levels of nucleotide polymorphism were in the nuclear 28S rDNA D1–D2 region and ITS region,but no polymorphism was in the chloroplast rbcS-rpl27 intergenic spacer.A reasonable intraspecific phylogeographic relationship was presented by rbcS-rpl27 intergenic spacer,which had the strongest distinction to geographic strains compared to those of 28S rDNA D1–D2 and ITS regions.In the phylogenetic tree of rbcS-rpl27 intergenic spacer,the two strains from the North Sea of the Atlantic Ocean were divided firstly from the species of P.globosa,and then formed an independent clade,while the other Atlantic strains and all of Pacific strains joined up to build the other clade.It was implied that at least two genetically distant populations of P.globosa existed in the Atlantic coastal regions.This study provided a high-resolution chloroplast marker to analyze intraspecific phylogeographic populations of P.globosa,and preliminarily clarified the genetic relationships of the Pacific and Atlantic strains of P.globosa.

Heavy metal induced DNA changes in aquatic macrophytes: Random amplified polymorphic DNA analysis and identification of sequence characterized amplified region marker

Plants have been used as good bio-indicators and genetic toxicity of environmental pollution in recent years. In this study, aquatic plants Hydrilla verticillata and Ceratophyllum demersum treated with 10umol/L Cd, 5 umol/L Hg, and 20 umol/L Cu for 96 h, showed changes in chlorophyll, protein content, and in DNA profiles. The changes in DNA profiles included variation in band intensity, presence or absence of certain bands and even appearance of new bands. Genomic template stability test performed for the qualitative measurement of changes in randomly amplified polymorphic DNA （RAPD） profiles, showed significant effect at the given concentration of metals. Cloning and sequencing of bands suggested that these markers although may not be homologous to any known gene but its conversion as a sequence characterized amplified region （SCAR） marker is useful in detecting the effects of genotoxin agents.

Tagging and Utilization Bruchid Resistance Gene Using PCR Markers in Mungbean

Sixteen mungbean lines were analyzed using 56 random primers. Different DNA bands were detected between Bruchid resistant lines and susceptible lines. According to the cluster results, the 16 lines can be divided into four groups, including brucid resistant wild types, resistant cultivated lines, resistant progenies and a mixed group. BSA method was used to identify DNA markers that related with bruchid resistant gene by using resistant line and susceptible line and their F2 progeny. One codominant marker was identified, which generated a fragment of 1.79 kb in resistant lines and 1.03 kb in susceptible lines. Finally, this codominant marker was considered to be tightly linked with bruchid resistant gene and could be useful in resistant germplasm identification and marker-assisted selection.

Integration of morpho-physico-biochemical traits with SSR and SRAP markers for characterization of castor genotypes of Indian origin

Castor(Ricinus communis L.)is an important tropical oilseed crop,whose oil has versatile,practical value,especially in industries.The present study aimed to estimate the nature and magnitude of variability in the castor germplasm concerning yield and its component traits and physico-biochemical characters.Seed yield per plant and oil content ranged from 80.90 g(ICS-165)to 248.30 g(RG-3216),and 34.7%(ICS-172)to 58.7%(JI-277),respectively.The iodine value of oil ranged from 76.36(JI-370)to 89.84(P2-135)with an average value of 83.02.The mean saponification value of oil was 182.24.The genotypic and phenotypic coefficients of variation were high for acid value,capsules on the main raceme,seed yield per plant,and total length of the main raceme.A positive association of porosity,average unit volume,and total length of the main raceme with seed yield per plant showed that these characters might be directly attributed to seed yield improvement.By Manhattan distances,the 30 genotypes were grouped into 3 clusters.Their genetic diversity was elucidated using SSR and SRAP markers.SRAP marker produced higher mean number of total bands(5.71),polymorphic bands(4.57),percentage polymorphism(83.10%),PIC(1.72),RP(5.90),mean RP(1.02),MR(5.71),EMR(4.57)and MI(1.44)values when compared to SSR(2.89,2.11,79.63%,0.61,1.90,0.72,2.89,2.11 and 0.49,respectively)marker.The highest genetic distance(0.77)was between 48-1 and JI-370,which indicated that these genotypes could be used in biparental mating schemes,QTL map development,and hybridization programmes to increase oil content and quality for industrial purposes.

Identification of Large Deletion of Ccs Responsible for Non-Red Fruit Color in Pepper (Capsicum annuum) and Development of DNA Marker to Distinguish the Deletion

Chili pepper (Capsicum spp.) fruit color is an important agronomical trait. It has been known that a large deletion in the 5' upstream region of the Ccs gene generates non-red fruit color in pepper, but the accurate size and position of the deletion and whether all the non-red cultivars had the same large deletion or not were unclarified. In this study, to identify the Ccs upstream large deletion, we carried out diagnostic PCR using six forward primers at 300 - 900 bp intervals in the 5' untranslated region of Ccs with a fixed reverse primer for a yellow fruit pepper “Sonia Gold”. Then it was revealed that 4430 bp from -3234 bp position in upstream region to 1196 bp position in exon was deleted in Ccs of “Sonia Gold”. The allele having this deletion was named ccs-del. Probably this allele is substantially the same as ccs-p1 having 4879 bp deletion reported previously. Based on the sequence determined, we developed a PCR marker to distinguish ccs-del. Genotyping of 16 cultivars of C. annuum showed that 14 had ccs-del and the remaining two had another mutant allele ccs-3. This result indicates that ccs-del is the most common allele and widely shared in non-red fruit cultivars in C. annuum. Genotyping of 16 cultivars of C. chinense clarified that one cultivar each possessed ccs-del and ccs-3. These results indicate that major alleles responsible for non-red fruit color in C. annuum were shared across species throughout interspecific introgression.

Systematic Evaluation of Pharmacognostic Identification of Polygonum capitatum

[Objectives] To investigate the systematic evaluation of pharmacognostic identification of Polygonum capitatum . [Methods] 10 batches of P. capitatum cultivated in Guizhou were chosen for plant samples. Macroscopical identification was conducted on plant roots, stems, leaves, flowers and fruits. The P. capitatum powder was processed for physical and chemical distinction by FeCl 3 chromogenic reaction, hydrochloric acid magnesium powder reaction, AlCl 3 color development reaction and thin-layer chromatography.Microscope identification was carried out on the powder. Plant genome DNeasy Plant Kit was adopted for DNA molecular marker identification. [Results] The results showed that the stem of P. capitatum was tufted, the leaves were oval, 2 to 5 cm long, and 1 to 2 cm wide;the leaf apex was acute and cuneate at the base, the inflorescence was capitate, paired or solitary;the raceme was erect and nearly spherical, and the perianth was light red. Furthermore, for the chromogenic reaction of FeCl 3 ethanol extract of P. capitatum , appeared blue and turned to dark blue after long time storing at room temperature. For the reaction of hydrochloric acid magnesium powder, the alcohol extract of P. capitatum , exhibited deep red. In the color reaction of AlCl 3, the alcohol extract revealed yellow fluorescence under 360 nm UV lamp. Microscope identification of the powder displayed pollen grains, crystal sheath fibers, cellulose, vessels, starch grains, cork cells, and other characteristic fragments. In addition, DNA barcoding electrophoresis results showed that P. capitatum showed a clear and bright single band near 500 bp, and further sequencing results showed that the sequence differences were mainly concentrated in ITS1 and ITS2 region. [Conclusions] Systematic evaluation for the identification of P. capitatum is established, which combines with macroscopic identification, physicochemical identification, powder microscope identification, and DNA molecular identification. Finally, the original medicinal material is identified as P. capitatum Buch.-Ham. ex D. Don.

Characterization and distribution of novel alleles of the vernalization gene Vrn-A1 in Chinese wheat(Triticum aestivum L.) cultivars

The ability of wheat to adapt to a wide range of environmental conditions is determined mostly by allelic diversity among genes regulating vernalization requirement.Vrn-1 is a major regulator of this requirement.In this study,two novel alleles of Vrn-A1 were discovered in Chinese cultivars:vrn-A1n was identified in two landraces,Jiunong 2 and Ganchun 16,and Vrn-A1o was detected in Duanhongmangmai.Both novel alleles showed a linked duplication in the promoter region.The common copy of these two alleles was identical to the recessive allele vrn-A1.Compared with the recessive allele vrn-A1,the other copy of vrn-A1n contained a 54-bp deletion in the promoter region and the distinct copy of Vrn-A1o contained an11-bp deletion in the promoter region.In segregating populations in the greenhouse under nonvernalizing(20–25°C)and long-day(16 h light)conditions,plants with the novel vrn-A1n allele did not head earlier than those with the recessive vrn-A1 allele.However,plants that were either homozygous or heterozygous for the novel Vrn-A1o allele headed earlier than those with the recessive vrn-A1 allele.To identify the novel allele with the small-sized product and facilitate screening,a DNA marker for the novel dominant allele Vrn-A1o was designed.Analysis of the novel-allele distribution showed that two cultivars carrying the vrn-A1n allele were dispersed in the northwestern spring wheat zone,and 12 cultivars carrying the dominant Vrn-A1o allele were widely distributed in the northwestern spring wheat zone,Xinjiang winter and spring wheat zone,Yellow and Huai River valley winter wheat zone,and QinghaiTibetan Plateau spring and winter wheat zone.Our study identifies useful germplasm and a DNA marker for wheat breeding.

Chloroplast genomic diversity in Bulbophyllum section Macrocaulia(Orchidaceae,Epidendroideae,Malaxideae):Insights into species divergence and adaptive evolution

Bulbophyllum is the largest genus in Orchidaceae with a pan tropical distribution.Due to highly significant diversifications,it is considered to be one of the most taxonomically and phylogenetically complex taxa.The diversification pattern and evolutionary adaptation of chloroplast genomes are poorly understood in this species-rich genus,and suitable molecular markers are necessary for species determination and phylogenetic analysis.A natural Asian section Macrocaulia was selected to estimate the interspecific divergence of chloroplast genomes in this study.Here,we sequenced the complete chloroplast genome of four Bulbophyllum species,including three species from section Macrocaulia.The four chloroplast genomes had a typical quadripartite structure with a genome size ranged from 156,182 to 158,524 bp.The chloroplast genomes included 113 unique genes encoding 79 proteins,30 tRNAs and 4 rRNAs.Comparison of the four chloroplast genomes showed that the three species from section Macrocaulia had similar structure and gene contents,and shared a number of indels,which mainly contribute to its monophyly.In addition,interspecific divergence level was also great.Several exclusive indels and polymorphism SSR loci might be used for taxonomical identification and determining interspecific polymorphisms.A total of 20 intergenic regions and three coding genes of the most variable hotspot regions were proposed as candidate effective molecular markers for future phylogenetic relationships at different taxonomical levels and species divergence in Bulbophyllum.All of chloroplast genes in four Bulbophyllum species were under purifying selection,while 13 sites within six genes exhibited sitespecific selection.A whole chloroplast genome phylogenetic analysis based on Maximum Likelihood,Bayesian and Parsimony methods all supported the monophyly of section Macrocaulia and the genus of Bulbophyllum.Our findings provide valuable molecular markers to use in accurately identifying species,clarifying taxonomy,and resolving the phylogeny and evolution of the genus Bulbophyllum.The molecular markers developed in this study will also contribute to further research of conservation of Bulbophyllum species.

Status of genetic studies and breeding of Saccharina japonica in China

Saccharina japonica is one of the most important economic brown seaweeds.It is intensively cultivated on large scales in a number of Asian countries.The current annual,global production is about 8 million tons valued as about 4 million US dollars.Considerable efforts have been made to S.japonica in China since the 1950s on its cultivation.To further advance the cultivation of this species,detailed research of genetics and breeding studies are required.Recently,with the advancement of sequencing techniques,the genomics and comparative transcriptomics data were yielded,and quantitative trait locus(QTL)mapping has been conducted,along with genetic linkage maps constructed to this species.New strains have been bred and selected,with better characteristics,e.g.higher seawater temperature resistances and higher yields.In this review,we present the current status of genetic and breeding studies that have been performed to S.japonica in China,and provide guidelines for future developments in the areas of genetic selection and breeding for this species.

Brazilian Buffalo Genetic Variability by Cross-Specific Microsatellite Set

Buffaloes （Bubalus bubalis） are widely distributed and were introduced to Brazil in 1895. Most of the molecular genetic characterization of buffaloes has been done with cross-specific （cattle） markers, but few of them include Brazilian populations. Nineteen commonly used cattle microsatellites were tested to develop a multiplexed set of microsatellites and characterize Brazilian buffalo. Three PCR mixes were finally developed with the 11 markers that succeed in amplify and were polymorphic （58%）. The average number of alleles was 5.42, with an average observed and expected heterocigozity of 0.441 and 0.695, respectively. As it was expected, Brazilian buffalo variability was lower than the previously reported from the domestication centres （China and India）, but higher than the seriously selected European populations. The exclusion power calculated for the eleven markers in Brazilian buffalo was 0.9999999996, this allows its use in DNA based traceability.

Development of Chromosomal Segment Substitution Lines from a Backcross Recombinant Inbred Population of Interspecific Rice Cross

A backcross recombinant inbred line population consisting of 202 lines was developed from Xieqingzao B//Xieqingzao B / Dongxiang wild rice. The population was assayed with DNA markers and phenotyped on planthopper resistance and yield traits. A linkage map consisting of 119 DNA markers and spanned for 1188 cM over the 12 rice chromosomes was constructed. Thirty-two chromosomal segment substitution lines were selected based on the percentage of Xieqingzao B allele at marker loci. These lines are of great potential for gene mapping and alien gene introgression.

Analysis of the Genetic Diversity and Origin of Some Chinese Domestic Duck Breeds

Twelve fluorescence-labeled microsatellite markers were used to analyze the genetic diversity of 12 domestic duck breeds and 2 wild duck breeds to determine the relationship and origin of Chinese domestic duck breeds. Gene frequency, effective number of alleles （Ne）, expected heterozygosity （He）, polymorphism information contents （PIC）, inbreeding coefficient in population （Fis）, standard genetic distance （Ds）, and genetic distance （DA） were calculated by FSTAT and distance and phylogenetic analysis after the dates which were output from the Microsatellite-Toolkit software. Genetic distances between 12 domestic duck breeds and 2 wild duck breeds were analyzed by variance analysis. Unweighted pair group method with arithmetic mean （UPGMA） and phylogenetic trees used for cluster analysis were structured. The results indicated that 11 loci had medium- or high-level genetic diversity among the 12 loci, which could be efficiently used in the detection of the genetic parameters of each population. The values of He were 0.5414 to 0.7343, those of PIC proved similar, and those of Fis were 0.1101 to 0.3381 among all populations. All breeds were clustered into three groups by UPGMA phylogenetic trees. Banzui duck was clustered into a separate group. Differences of the DA were analysed by t-test. The results showed that difference in DA between the 12 domestic duck breeds and Lvtou duck and the Banzui duck were very significant （P〈0.01）, indicating that these 12 domestic duck breeds originated from Lvtou wild duck, but not Banzui duck.

Tagging of Brown Planthopper Resistance Genes in F_2s of IR50 × Ptb33 of Rice by Using Bulked Segregant Analysis

Brown planthopper （Nilaparvata lugens Stal） is one of the most damaging pests causing hopper burn in rice, and thereby reducing the productivity and also the quality of the product. The effective management strategy to control this pest is the identification and transfer of desirable genes to local rice cultivars. The most important approach for developing resistant cultivars is the identification of markers, which can help in marker-assisted selection of more durable resistant genotype. The susceptible parent IR50 and the resistant parent Ptb33, and their F2 populations were used in bulked segregant analysis for identification of resistant genes with random amplified polymorphic DNA marker （RAPD） primers. The primers OPC7 and OPAG14 showed both dominant and susceptible specific banding pattern so called co-dominant markers. Moreover, OPC7697 and OPAG14680 showed resistant specific bands and thus being in coupling phase, whereas OPC7846 and OPAG14650 showed susceptible specific genotypic bands in bulked segregant analysis. Therefore, the coupling phase markers, OPC7697 and OPAG14680, are considered to be more useful in marker-assisted selection of rice genotypes in crop improvement.

QTL Detection on Chromosome 6 in Landrace×Lantang Pig Resource Population

A resource population constructed by F2 design with Landrace and Chinese indigenous Lantangpigs was used in this study. Seven microsatellite DNA markers on chromosome 6 and USDA2.6 piggenetic linkage map were used for interval QTL mapping. The results revealed that at theposition of 38-41 cM there was a chromosome-wide highly significant QTL affecting carcassbackfat A thickness (P < 0.01), which was closely linked with MN007 and the ratio of QTL addi-tive variance to F2 phenotypic variance was 5.90%. At the position of 60-70 cM there were twochromosome-wide significant QTLs affecting carcass lean percentage (P < 0.01) and skin and fatpercentage (P < 0.05), which were closely linked with MN003 and the ratio of QTL additivevariance to F2 phenotypic variance were 18.44 and 3.75%, respectively. At the same position,there was a single-point QTL also closely linked with MN003 and highly significantly (P < 0.01)affecting carcass lean. In addition, there were two chromosome-wide highly significant (P < 0.01)QTLs affecting meat color and marbling, which were closely linked with MN13 at the positionof 70-75 cM and the ratio of QTL additive variance to F2 phenotypic variance were 14.05and 1.77%, respectively.

DNA琼脂糖凝胶电泳相关问题探讨

针对人教版高中《生物学·选择性必修3·生物技术与工程》教材中DNA琼脂糖凝胶电泳的教学实践中,学生容易产生的几个疑惑“为什么要用电泳缓冲液”“为什么要用琼脂糖配置凝胶”“为什么要加核酸染料”“为什么要用凝胶载样缓冲液”“DNA marker是什么”以及操作过程中的注意事项等进行探讨,帮助学生更好地掌握琼脂糖凝胶电泳这项常规的分子生物学实验方法。