Molecular characterization of sweet potato(Ipomoea batatas[L.]Lam)germplasms for desirable traits by using simple sequence repeats markers

Every breeding program that aims to create new and improved cultivars with desired traits mostly relies on information related to genetic diversity.Therefore,molecular characterization of germplasms is important to obtain target cultivars with desirable traits.Sweet potato[Ipomoea batatas(L.)Lam]is widely considered the world’s most important crop,with great diversity in morphological and phenotypic traits.The genetic diversity of 20 sweet potato germplasms originating from Bangladesh,CIP,Philippines,Taiwan,and Malaysia were compared,which was accomplished by genetic diversity analysis by exploring 20 microsatellite DNA markers for germplasm characterization and utilization.This information was effective in differentiating or clustering the sweet potato genotypes.A total of 64 alleles were generated using the 20 primers throughout the 20 germplasm samples,with locus IBS97 having the highest number of alleles(5),whereas locus IbU33 had the fewest alleles(2).The alleles varied in size from 105(IbU31)to 213 base pairs(IBS34).The Polymorphism Information Content(PIC)values for the loci IbL46 and IBS97 varied from 0.445 to 0.730.IBS97 has the highest number of effective alleles(3.704),compared to an average of 2.520.The average Shannon’s diversity index(H)was 1.003,ranging from 0.673 in IbU3 to 1.432 in IBS97.The value of gene flow(Nm)varied between 0.000 and 0.005,with an average of 0.003,whereas genetic differentiation(FST-values)ranged between 0.901 and 1.000.The sweet potato germplasm included in this study had a broad genetic base.SP1 vs.SP9 and SP12 vs.SP18 germplasm pairings had the greatest genetic distance(GD=0.965),while SP1 vs.SP2 germplasm couples had the least genetic diversity(GD=0.093).Twenty genotypes were classified into two groups in the UPGMA dendrogram,with 16 genotypes classified as group“A”and the remaining four genotypes,SP10,SP18,SP19,and SP20,classified as group“B.”According to cluster analysis,the anticipated heterozygosity(gene diversity)of Nei(1973)was 0.591 on average.In summary,SSR markers successfully evaluated the genetic relationships among the sweet potato accessions used and generated a high level of polymorphism.The results of the present study will be useful for the management of germplasm,improvement of the current breeding strategies,and the release of new cultivars as varieties.

Enhancing rice grain production by manipulating the naturally evolved cis-regulatory element-containing inverted repeat sequence of OsREM20

Grain number per panicle(GNP)is an important agronomic trait that contributes to rice grain yield.Despite its importance in rice breeding,the molecular mechanism underlying GNP regulation remains largely unknown.In this study,we identified a previously unrecognized regulatory gene that controls GNP in rice,Oryza sativa REPRODUCTIVE MERISTEM 20(OsREM20),which encodes a B3 domain transcription factor.Through genetic analysis and transgenic validation we found that genetic variation in the CArG box-containing inverted repeat(IR)sequence of the OsREM20 promoter alters its expression level and contributes to GNP variation among rice varieties.Furthermore,we revealed that the IR sequence regulates OsREM20 expression by affecting the direct binding of OsMADS34 to the CArG box within the IR sequence.Interestingly,the divergent pOsREM20IR and pOsREM20ΔIR alleles were found to originate from different Oryza rufipogon accessions,and were independently inherited into the japonica and indica subspecies,respectively,during domestication.Importantly,we demonstrated that IR sequence variations in the OsREM20 promoter can be utilized for germplasm improvement through either genome editing or traditional breeding.Taken together,our study characterizes novel genetic variations responsible for GNP diversity in rice,reveals the underlying molecular mechanism in the regulation of agronomically important gene expression,and provides a promising strategy for improving rice production by manipulating the cis-regulatory element-containing IR sequence.

Genetic Diversity of Chinese and Swedish Rapeseed (Brassica napus L.) Analyzed by Inter-Simple Sequence Repeats (ISSRs)

We have compared genetic diversity of 24 Chinese weak-winter, Swedish winter and spring B. napus accessions by inter-simple sequence repeats (ISSRs). By cluster analysis (UPGMA) based on 125 polymorphism bands amplified with 20 primers, the 24 accessions were divided into three groups. Six Swedish winter lines and eight Chinese weak-winter lines were in the group I and the groupⅡwere two Chinese weak-winter lines XiangyoulS and Bao81. The third group contained eight Swedish spring lines. Principal co-ordinates analysis (PCO) showed similar groupings to cluster analysis. Results from cluster analysis and PCO analysis showed very clearly that Chinese weak-winter, Swedish spring and winter accessions were distinguished from each other and Chinese weak-winter accessions in this study were genetically closer to Swedish winter accessions than to Swedish spring accessions. The Chinese weak-winter accessions had larger diversity than Swedish spring or winter accessions did. This study indicated that ISSR is a suitable and effective tool to evaluate genetic diversity among rapeseed germplasm.

Mapping a Rice Glabrous Gene Using Simple Sequence Repeat Markers

Inheritance analysis and gene mapping of the glabrous trait were conducted using the crosses between a pubescent rice material Chuanxiang 29B （an aromatic elite maintainer line of hybrid rice）, and two glabrous rice materials, Lemont and R2 （R2 is a new restorer line of hybrid rice）. All F1 plants from the two crosses had pubescent leaves, confirming that the pubescence trait in Chuanxiang 29B is dominant over the glabrous trait. In F2 individuals, the segregation ratio of three pubescent to one glabrous plant suggests that a single glabrous gene was present in Lemont and R2. Using the bulked segregant analysis, the linkage analysis of the simple sequence repeat markers showed that the glabrous gene, gl-1, was flanked by GL311 and GL8 with the genetic distances of 0.19 and 0.92 cM on chromosome 5, respectively.

Haplotyping of Rice Genotypes Using Simple Sequence Repeat Markers Associated with Salt Tolerance

Salt stress is a major problem in most of the rice growing areas in the world. A major QTLSaltol associated with salt tolerance at the seedling stage has been mapped on chromosome 1 in rice.This study aimed to characterize the haplotype diversity at Saltol and additional QTLs associated withsalt tolerance. Salt tolerance at the seedling stage was assessed in 54 rice genotypes in the scale of 1to 9 score at EC = 10 dSm^-1 under controlled environmental conditions. Seven new breeding linesincluding three KMR3/O. rufipogon introgression lines showed similar salt tolerant ability as FL478 andcan be good sources of new genes/alleles for salt tolerance. Simple sequence repeat （SSR） markerRM289 showed only two alleles and RM8094 showed seven alleles. Polymorphic information contentvalue varied from 0.55 for RM289 to 0.99 for RM8094 and RM493. Based on 14 SSR markers, the 54lines were clearly separated into two major clusters. Fourteen haplotypes were identified based onSaltol linked markers with FL478 as the reference. Alleles of RM8094 and RM3412 can discriminatebetween the salt tolerant and susceptible genotypes clearly and hence can be useful in marker-assistedselection at the seedling stage. Other markers RM10720 on chromosome 1 and RM149 and RM264 onchromosome 8 can also distinguish tolerant and susceptible lines but with lesser stringency.

Genetic Diversity among Parents of Hybrid Rice Based on Cluster Analysis of Morphological Traits and Simple Sequence Repeat Markers

The genetic diversity of 41 parental lines popularized in commercial hybrid rice production in China was studied by using cluster analysis of morphological traits and simple sequence repeat （SSR） markers. Forty-one entries were assigned into two clusters （i.e. early or medium-maturing cluster; medium or late-maturing cluster） and further assigned into six sub-clusters based on morphological trait cluster analysis, The early or medium-maturing cluster was composed of 15 maintainer lines, four early-maturing restorer lines and two thermo-sensitive genic male sterile lines, and the medium or late-maturing cluster included 16 restorer lines and 4 medium or late-maturing maintainer lines. Moreover, the SSR cluster analysis classified 41 entries into two groups （i.e, maintainer line group and restorer line group） and seven sub-groups. The maintainer line group consisted of all 19 maintainer lines, two thermo-sensitive genic male sterile lines, while the restorer line group was composed of all 20 restorer lines. The SSR analysis fitted better with the pedigree information. From the views on hybrid rice breeding, the results suggested that SSR analysis might be a better method to study the diversity of parental lines in indica hybrid rice.

Evaluation of Population Structure, Genetic Diversity and Origin of Northeast Asia Weedy Rice Based on Simple Sequence Repeat Markers

Weedy rice exerts a severe impact on rice production by competing for sunlight, water and nutrients. This study assayed the population structure, genetic diversity and origin of Northeast Asia weedy rice by using 48 simple sequence repeat markers. The results showed that weedy rice in Northeast Asia had a high genetic diversity, with Shannon＇s diversity index （I） of 0.748 and the heterozygosity （He） of 0.434. In each regional population, I value varied widely. The widest range of I （0.228-0.489） was observed in the weedy rice of Eastern China, which was larger than that of Northeast China and Korea （0.168-0.270）. The F-statistics of regional populations （Fis, Fit and Fst） also showed higher values in the weedy rice of Eastern China than those of Northeast China and Korea All weedy rice accessions were grouped into two clusters in the unweighted pair group method with arithmetic mean cluster analysis dendrogram, namely Eastern China branch and Northeastern China plus Korea branch. There was significant differentiation in genetic characteristics in weedy rice of northeastern and eastern Asia, especially in Eastern China.

THE ABNORMAL EXPRESSION OF CLONED REPEATED SEQUENCE DNA, L5B-4, IN RAT HEPATOMA BERH-2

A repeated sequence DNA fragment, L5B-4, was cloned from the 5 kb BamHI DNA fragments of rat genomic DNA. The expressions of the L5B-4 DNA fragment are different in liver and hepatoma cells. The amounts of transcripts in hepatoma cells are lower in nucleus and higher in cytoplasm, especially in polysomal RNA, as compared with that in liver cells. The alteration shown in polysomal RNA of hepatoma cells seems to be specific. These results are discussed with respect to the possible function of this repeated DNA and its variation in hepatoma cells.

Genetic Differentiation Among Populations of Octopus minor Based on Simple Sequence Repeats Mined from Transcriptome Data

Octopus minor(Sasaki 1920)is an important commercial cephalopod species in China.This species has declined sharply in recent years.Hence,genetic studies of O.minor are imperative to exploit and manage the wild resource.In this study,46192 microsatellite loci were discovered in 70174 unigenes from the transcriptomic data.Among all of the simple sequence repeat(SSR)unit categories,di-nucleotide and tri-nucleotide SSRs accounted for 45.26%and 14.49%,respectively.A total of 108 SSRs were tested,of which 21 were neutral and polymorphic.Seven SSRs were selected for genetics analyses of the O.minor populations in the Bohai Sea,the Yellow Sea,and the southwest coast of the Taiwan Strait region.Significant pairwise F_(st) values were detected among the samples.The UPGMA tree based on genetic distances suggested that the sampling locations could be arranged in three clusters.These markers are evidence that the populations in this region may be structured,with samples from Penghu differing remarkably from those in northern China.The present study characterized genetic markers for population assessment,management,and conservation of O.minor.

Analysis of Simple Sequence Repeats in Genomes of Rhizobia

Simple sequence repeats （SSRs） or microsatellites, as genetic markers, are ubiquitous in genomes of various organisms. The analysis of SSR in rhizobia genome provides useful information for a variety of applications in population genetics of rhizobia. We analyzed the occurrences, relative abundance, and relative density of SSRs, the most common in Bradyrhizobium japonicum, Mesorhizobium loti, and Sinorhizobium meliloti genomes se- quenced in the microorganisms tandem repeats database, and SSRs in the three species genomes were compared with each other. The result showed that there were 1 410, 859, and 638 SSRs in B. japonicum, M. loti, and S. meliloti genomes, respectively. In the genomes of B. japonicum, M. loti, and S. meliloti, tetranucleotide, pentanucleotide, and hexanucleotide repeats were more abundant and indicated higher mutation rates in these species. The least abundance was mononucleotide repeat. The SSRs type and distribution were similar among these species.

Identification and Characterization of Simple Sequence Repeats (SSR) Markers Associated with Downy Mildew Resistance Locus “<i>RPV1</i>” in Grapes

The cultivation of grapes is severely impacted by the emergence of downy mildew (DM) disease which negatively affects quality and yield possibly resulting in heavy losses. Due to certain shortcomings in the usage of fungicides and the development of new cultivars by plant breeding, marker assisted selection (MAS) will be an efficient alternative method to introduce desired genes into the cultivated varieties in a short time period. The Simple sequence repeats (SSR) markers seem to be the most popular genetic marker of choice for MAS. In the present study, we identified 14 new SSR markers in RPV1 locus that are associated with downy mildew resistance in grapes. The characterization of the identified markers was carried out on the basis of various parameters such as types of repeat motifs, number of repeats, different classes and structure of microsatellites. Additionally, SSR genotyping in 56 different grape accessions was done to determine the susceptibility or resistance of these accessions to DM.

Full-length transcriptome sequence and SSR marker development for genetic diversity research in yellowfin seabream Acanthopagrus latus

Yellowfin seabream Acanthopagrus latus is an important economic fish in Chinese coastal areas.Given its narrow distribution and overfishing,the genetic diversity of yellowfin seabream has been restricted for artificial breeding and reproduction.We performed full-length transcriptome sequencing and assembly of the genome of yellowfin seabream.A total of 68086 unigenes were obtained,with an N50 of 3391 bp on average length of 2933 bp.A total number of 50593 expressed sequence tags linked to simple sequence repeats(EST-SSR)were identified,among them dinucleotide repeats(40.6%)and AC/GT motifs(38.5%)were the most frequent.Of the 190 EST-SSRs for which PCR primer pairs were designed,150 primer pairs successfully amplified target loci and 15 SSRs showed high polymorphism.The alleles per locus ranged 6-50 on average of 25.3.The expected and observed heterozygosity varied from 0.632 to 0.969 and from 0.519 to 0.953,respectively.The polymorphic index content(PIC)values of each locus ranged 0.587-0.966 on average of 0.851.Among six yellowfin seabream population samples preliminarily tested for genetic diversity and differentiation,the Fangchenggang(FCG)population in Guangxi Province had the highest mean observed heterozygosity(H_(o))value(0.786),whereas the Zhangzhou(ZZ)population in Fujian Province had the lowest(0.678).The pairwise fixation index(Fst)values indicated significant population differentiation among six yellowfin seabream populations.This study provided evidence for the usefulness of the transcriptomic resource information and EST-SSR markers for natural resource conservation,population genetics,and breeding studies of yellowfin seabream in South China.

基于SCAR标记和DNA条形码技术的苍术基原鉴别研究

目的开发出能同时鉴别北苍术和关苍术的分子标记方法,并探究不同种质资源苍术的遗传进化关系。方法对不同地区北苍术Atractylodes chinensis(Bunge)Koidz及关苍术A.japonica Koidz.ex Kitam基因组DNA的差异片段进行测序,结合SRAP、ISSR、DAMD分子标记方法,优化PCR反应体系,筛选并转换成特异性标记,同时,采用条形码方法分析种间序列差异。结果通过SRAP、ISSR、DAMD三种分子标记方法的PCR扩增,共筛选出198对能稳定扩增且重现性好的引物,转换出7对能稳定、快速鉴别北苍术和关苍术的SCAR引物。条形码方法检测出北苍术ITS2序列长度为454 bp,关苍术ITS2序列长度为453 bp,与其他苍术属植物之间遗传距离较远。NJ树结果显示,北苍术、关苍术及其他苍术属植物均各自聚为一支,表现出良好的单系性。依据ITS2二级结构,4种苍术属植物在螺旋区的茎环数目、大小、位置均有明显差异,可以直观地进行区分。结论所开发的特异性SCAR标记为苍术属植物优良品种的筛选提供了新方法,DNA条形码能稳定、准确鉴别北苍术。

Parent-offspring relationship recognition based on SSR and mtDNA confirmed resource supplement effect of Fenneropenaeus chinensis release

The resource of Fenneropenaeus chinensis has declined sharply due to excessive fishing intensity,ecological changes and diseases.In order to supplement the fishing yield and restore resources of F.chinensis,the relevant authorities have carried out the activities of stock enhancement and releasing.It can increase biomass and recover resources.However,compared with increasing biomass,there were still few reports on its effect on the recovery of resources.Resource recovery is a process related to whether the released individuals can form a reproductive population.Up to now,there has been a lack of evidence whether the released F.chinensis can complete the entire life history,and form reproduction population.In this study,gravid female shrimp after spawning migration were captured from coastal waters of Haiyang,Qingdao,and Yellow Sea.After identifying parentage relationships using simple sequence repeat(SSR)and mtDNA haplotype,it was finally confirmed that there were eight released individuals in the recapture samples.It was confirmed for the first time that at least part of the released F.chinensis can complete overwintering and reproductive migration,and maintain the migration habits as their wild counterparts.Therefore,we infered that the released shrimp can reproduce under natural conditions,these F.chinensis can form reproductive populations theoretically if without human intervention.These results indicated that enhancenment and release activities have a positive effect on resource recovery.

Analysis of SSR in Citrus Sequences from EMBL Database

Abundance of simple sequence repeat (SSR) in Citrus sequences from EMBL database was investigated by usingcomputer program MISA (MIcroSAtellite), which aimed to provide useful information for the development of SSR markers.Among 32 896 sequences of Citrus, 4 987 SSRs were found in 4 167 sequences and the average distance between SSRs wasapproximately 3.5 kb. Mononucleotide repeats (50.6%) were the most abundant repeats. And di-, tri-, tetra-, penta- andhexa-nucleotide repeats were 22.8, 25.2, 1, 0.08, and 0.36%, respectively. The most abundant motif was A/T followed indescending order by AG/CT, AC/GT, AT/TA. AAT/ATT, AAG/CTT, AGC/CGT, ACG/CTG and C/G. They comprised about90% of all microsatellites. Ten primer pairs were designed, and three of them produced clear visible bands among Citrusand its related genera.

A hybrid swarm population of Pinus densiflora × P. sylvestris inferred from sequence analysis of chloroplast DNA and morphological characters

To confirm a hybrid swarm population of Pinus densiflora × P. sylvestris in Jilin, China, we used needles and seeds from P. densiflora, P. sylvestris, and P. densiflora × P. sylvestris collected from natural stands or experimental stations to study whether shoot apex morphology of 4-year old seedlings can be correlated with the sequence of a chloroplast DNA simple sequence repeat marker （cpDNA SSRs）. Total genomic DNA was extracted and subjected to sequence analysis of the pine cpDNA SSR marker Pt15169. Results show that morphological characters from 4-year old seedlings did not correlate with sequence variants of this marker. Marker haplotypes from all P. sylvestris trees had a CTAT element that was absent from all sampled P. densiflora trees. However, both haplotype classes involving this insertion/deletion element were found in a P. densiflora × P. sylvestris population and its seedling progeny. It was concluded that the P. densiflora × P. sylvestris accessions sampled from Jilin, China resulted from bi-directional crosses, as evidenced by both species’ cpDNA haplotypes within the hybrid swarm population.

RSMD-repeat searcher and motif detector

The functionality of a gene or a protein depends on codon repeats occurring in it.As a consequence of their vitality in protein function and apparent involvement in causing diseases,an interest in these repeats has developed in recent years.The analysis of genomic and proteomic sequences to identify such repeats requires some algorithmic support from informatics level.Here,we proposed an offline stand-alone toolkit Repeat Searcher and Motif Detector（RSMD）,which uncovers and employs few novel approaches in identification of sequence repeats and motifs to understand their functionality in sequence level and their disease causing tendency.The tool offers various features such as identifying motifs,repeats and identification of disease causing repeats.RSMD was designed to provide an easily understandable graphical user interface（GUI）,for the tool will be predominantly accessed by biologists and various researchers in all platforms of life science.GUI was developed using the scripting language Perl and its graphical module PerlTK.RSMD covers algorithmic foundations of computational biology by combining theory with practice.

Structural characterization of four Rhododendron spp.chloroplast genomes and comparative analyses with other azaleas

Azalea is a general designation of Rhododendron in the Ericaceae family.Rhododendron not only has high ornamental value but also has application value in ecological protection,medicine,and scientific research.In this study,we used Illumina and PacBio sequencing to assemble and annotate the entire chloroplast genomes(cp genomes)of four Rhododendron species.The chloroplast genomes of R.concinnum,R.henanense subsp.lingbaoense,R.micranthum,and R.simsii were assembled into 207,236,208,015,207,233,and 206,912 bp,respectively.All chloroplast genomes contain eight rRNA genes,with either 88 or 89 protein-coding genes.The four cp genomes were compared and analyzed by bioinformatics,and the phylogenetic analysis based on chloroplast genomes of 26 species of Ericaceae,Actinidiaceae,and Primulaceae under Ericales was conducted.A comparison of the linear structure of cp genomes of four Rhododendron showed that there were substantial sequence similarities in coding regions,but high differences in non-coding regions.A phylogenetic analysis,based on chloroplast whole genome sequences,showed that all Rhododendron species are in the clade Ericaceae.This study provides valuable genetic information for the study of population genetics and evolutionary relationships in Rhododendron and other azalea species.

简单重复序列标记荧光染料检测和银染显影技术的比较

简单重复序列(Simple Sequence Repeat,SSR)标记具有数量丰富、多态性、共显性等优点,在遗传育种研究中被广泛应用。传统的SSR标记检测为聚丙烯酰胺凝胶电泳后进行银染显影,该方法操作步骤繁琐且对环境造成较大污染,已逐渐被淘汰。开发新的银染显影替代技术具有很好的实用价值。利用抗稻瘟病SSR标记Pi2检测48份水稻材料,分别采用荧光染料和银染显影技术显示SSR结果,基于相同的材料和引物对2种方法的检测结果进行比较,建立并优化了荧光染料检测技术。结果表明,与传统的银染显影法相比,荧光染料检测的灵敏度与银染显影法相当,但荧光染料检测技术克服了银染显影技术的不足,具有高效率、综合成本低、操作简便、可靠及环境友好等优点,减小了对人体、环境的毒害。与传统银染法相比,荧光染料检测技术在不危害人体健康,不威胁环境的情况下,应用更广泛。

Quantitative Trait Loci(QTL)Mapping and Marker Analysis of Fatty Acids in Peanut

Peanut,with high oil content,has been a major oil and food crop globally.The compositions of the fatty acids are the common factors in determining the oil quality.In the present study,an F2 segregated population with 140 individuals derived from the cross of Weihua8(a cultivar)and 12L49(a line with high oleic acid concentration)was used to construct a genetic map and conduct QTL mapping analysis.A total of 103 polymorphic SSR primers were utilized for genotyping the RILs and finally generating the SSR loci.Within the 103 SSR loci,a genetic linkage map,covering a total length of 3592.35 cM of the whole peanut genome,was constructed.Based on the genetic map,sixteen QTLs located on nine linkage groups related to peanut fatty acids were finally identified.Among them,four QTLs were detected associated with various traits simultaneously,which showed genetic stability in relation to fatty acids of peanut.Except for the QTLs for oleic acid,linoleic acid,and linolenic acid,three novel QTLs for arachidic acid and behenic acid were also detected.These QTLs might be helpful for further fine mapping analysis and marker-assisted selection of fatty acids in peanut.