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.

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.

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。

Genetic diversity and population structure of the sea star Asterias amurensis in the northern coast of China

The sea star Asterias amurensis is widely viewed as a severe“marine pest”because of its broad feeding habits.Over the past few decades,A.amurensis undergoes massive and sporadic population outbreaks worldwide,causing extensive economic and ecological losses to the local aquaculture industry and marine ecosystem.Understanding the genetic diversity and population structure of A.amurensis can provide vital information for resource management.By analyzing the polymorphism of the mitochondrial cytochrome C oxidase subunit I(COI)gene and ten simple sequence repeat(SSR)microsatellites markers,the genetic diversity and population structure of A.amurensis of four populations along the northern coast of China was uncovered.A total of 36 haplotypes were identified,and a main haplotype was found in four populations.The Qingdao(QD)population displayed the highest genetic diversity among all the populations.The AMOVA and pairwise F_(st)showed that there was small but statistically significant population differentiation among the four populations,especially between QD and Weihai(WH).Moreover,the principal component analysis(PCA)and admixture analysis showed that several individuals in Yantai(YT)and Dalian(DL)had little genetic association with other individuals.Overall,this study provided useful information of the genetic diversity and population structure of A.amurensis and will contribute to the resource management of A.amurensis in China.

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.

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.

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.

Molecular diversity and genetic structure of 380 sweetpotato accessions as revealed by SSR markers

Sweetpotato, Ipomoea batatas（L.） Lam., is an important food crop widely cultivated in the world. Evaluation of genetic relationships among diverse cultivars and landraces is necessary for efficient exploitation of genetic diversity in the existing germplasm resources. In the present study, a collection of 380 sweetpotato accessions assembled from different agro-climatic zones of China and other countries were genotyped using 30 SSR primer pairs. Model-based structure analysis separated the germplasm into three populations, P1, P2 and P3, containing 228, 133 and 19 accessions, respectively, which was consistent with the results of phylogenic and principal component analysis（PCA）. Analysis of molecular variance（AMOVA） revealed significant genetic differentiation among inferred populations, accounting for 16.47% of the total molecular variance, however, the differences between the regions were not significant, the total variation were due to the differences between the genotypes within the population. Pairwise fixation index（F ST） suggested that populations P1 and P3 had the highest differentiation, while populations P1 and P2 had the lowest differentiation. The diversity among populations was wide, which confirmed the genetic distinction of populations. Through comparing model-based structure and domestication-based classification, it was found that the accessions of population P1 mainly belonged to modern cultivars, and the accessions of populations P2 and P3 basically corresponded to landraces, by which we suggest that modern cultivars maybe had experienced a two-step domestication history. Our results illustrated clear genetic relationships among 380 sweetpotato accessions, exhibiting the potential of accelerating the process of future sweetpotato breeding program by molecular marker based parental selection.

Comparative Analysis of Genetic Diversity in Landraces of Waxy Maize from Yunnan and Guizhou Using SSR Markers

Waxy maize landraces are abundant inYunnan and Guizhou of China. Genetic diversity of waxy maize landraces from Yunnan and Guizhou were analyzed using SSR markers. We screened 38 landraces with 50 primers that generated 3 to 6 polymorphic bands, with an average of 4.13 bands. Shannon＇s information indices for genetic diversity of the 14 waxy maize landraces from Yunnan varied from 4.9571 to 42.1138 and averaged 26.5252; Shannon＇s information indices for genetic diversity of the 24 waxy maize landraces from Guizhou varied from 22.0066 to 40.6320 and averaged 32.3156. For the 14 waxy maize landraces from Yunnan, the within-landrace genetic diversity accounted for 45.40% and the among-landrace genetic diversity accounted for 54.60% of the total genetic diversity observed. For the 24 waxy maize landraces from Guizhou, the within-landrace genetic diversity accounted for 50.76% and the among-landrace genetic diversity accounted for 49.24% of the total observed. Some individual landraces possessed as much as 96.86% of the total genetic diversity occurring among landraces within origins. Differentiation between geographic origins accounted for only 3.14% of the total genetic diversity. Both Yunnan and Guizhou would be the diversity centers and the original centers of waxy maize.

Genetic Diversity and Relationship of Weedy Rice in Taizhou City, Jiangsu Province, China

Microsatellite markers and morphological characteristics were used to explore the genetic diversity and possible origin of weedy rice in Taizhou City, Jiangsu Province, China. Fifty-two weedy rice (Oryza sativa L.) accessions were compared with two wild rice, four hybrid rice and five cultivars using 22 simple sequence repeat (SSR) primer pairs. A total of 107 fragments were amplified, averaging 5.6 alleles per primer pair. The polymorphic index content (PIC) values ranged from 0.3077 to 0.7951, averaging at 0.5870. The average genetic distance of all samples ranged from 0.02 to 0.46 with an average of 0.262. The genetic distance among Taizhou weedy rice ranged from 0.03 to 0.44 with an average of 0.224. Cluster analysis showed that all the weedy rice accessions from Taizhou City were indica, and could be subdivided into different genotypes. The majority (86%) of weedy rice was most closely related to hybrid rice. The Taizhou weedy rice accessions were morphologically similar, but still could be delineated into indica or japonica group by some morphological traits. It is suggested that the levels of genetic and morphological diversities of weedy rice in Taizhou City are low and these weedy rice plants originated from the segregating progenies of hybrid rice that had naturally introgressed with cultivated rice.

Analysis of Genetic Diversity of Processing Apple Varieties

Genetic diversity of 18 processing apple varieties and two fresh varieties were evaluated using 12 simple sequence repeats （SSR） primer pairs previously identified in Malus domestica Borkh. A total of 87 alleles in 10 loci were detected using 10 polymorphic SSR markers selected within the range of 5-14 alleles per locus. All the 20 varieties could be distinguished using two primer pairs and they were divided into four groups using cluster analysis. The genetic similarity （GS） of groups analyzed using cluster analysis varied from 0.14 to 0.83. High acid variety Avrolles separated from other varieties with GS less than 0.42. The second group contained Longfeng and Dolgo from Northeast of China, the inherited genes of Chinese crab apple. The five cider varieties with high tannin contents, namely, Dabinette, Frequin rouge, Kermerrien, M.Menard, and D.Coetligne were clustered into the third group. The fourth group was mainly composed of 12 juice and fresh varieties. Principal coordinate analysis （PCO） also divided all the varieties into four groups. Juice and fresh apple varieties, Longfeng and Dolgo were clustered together, respectively, using both the analyses. Both the analyses showed there was much difference between cider and juice varieties, cider and fresh varieties, as well as Chinese crab apple and western European crab apple, whereas juice varieties and fresh varieties had a similar genetic background. The genetic diversity and differentiation could be sufficiently reflected by combining the two analytical methods.

The complete mitogenome of the Chinese bush cricket,Gampsocleis gratiosa(Orthoptera:Tettigonioidea)

The complete mitochondrial genome （mitogenome） of Gampsocleis gratiosa was determined. The 15,929 bp in the size of G. gratiosa mitogenome contains a typical gene content, base composition, and codon usage found in metazoan. All 13 protein coding genes （PCGs） of the G gratiosa mitogenome start with a typical ATN codon. The usual termination codons （TAA and TAG） were found from 10 PCGs. However, the atp6, had4, and had5 had incomplete termination codon （T）. The anticodons of all tRNAs are identical to those observed in Drosophila yakuba and Locusta migratoria, and can be folded in the form of a typical clover leaf structure except for trnS （AGN）. The secondary structure of trnS （AGN） was drawn according with the Steinberg-Cedergren tertiary structure. The A＋T content （67.4%） of the A＋T-rich region is relatively lower among the mitogenome regions, in contrast, it usually contains the highest A＋T content for most insects. Two isolated sequence repeat regions （202 bp） were found in the A＋T-rich region with mapping and secondary structure information.

Relationship Between Heterosis and Parental Genetic Distance Based on Molecular Markers for Functional Genes Related to Yield Traits in Rice

The genetic distances among 18 cytoplasmic male sterile lines and 11 restorer lines were analyzed with molecular markers derived from yield-related functional genes. The correlation between parental genetic distance and heterosis was investigated by analyzing the performance of 47 combinations. The results showed that the genetic distance was significantly correlated with yield heterosis （r=0.29^＊）, but not significantly correlated with heterosis for other traits, such as number of effective panicles per plant, seed setting rate, 1000-grain weight, number of grains per panicle and theoretical yield. However, the correlation coefficient was so small that the parental genetic distance could not to be used to predict heterosis.

Major QTL Conferring Resistance to Rice Bacterial Leaf Streak

Bacterial leaf streak （BLS） is one of the important limiting factors to rice production in southern China and other tropical and sub-tropical areas in Asia. Resistance to BLS was found to be a quantitative trait and no major resistant gene was located in rice until date. In the present study, a new major quantitative trait locus （QTL） conferring resistance to BLS was identified from a highly resistant variety Dular by the employment of Dular/Balilla （DB） and Dular/IR24 （DI） segregation populations and was designated qBLSR-11-1. This QTL was located between the simple sequence repeat （SSR） markers RM120 and RM441 on chromosome 11 and could account for 18.1-21.7% and 36.3% of the variance in DB and DI populations, respectively. The genetic pattern of rice resistance to BLS was discussed.

ISSR Analysis on Genetic Diversity of the 34 Populations of Oryza meyeriana Distributing in Yunnan Province,China

The genetic diversity of the 34 populations of wild rice Oryza meyeriana Baill. distributed in Yunnan Province, China was analyzed using 13 inter-simple sequence repeat （ISSR） markers. A total of 168 bands were amplified, of which 135 polymorphic bands were discovered and the percentage of polymorphic bands （PPB） was 80.36%. A genetic diversity was revealed as Nei＇s gene diversity （H） = 0.2666 and Shannon information index （I） = 0.4028 at population level. The 34 populations were divided into different groups based on administrative regions, latitude and longitudes, river areas, altitudes of their origins, and their indexes such as Na （number of alleles）, Ne （effective number of alleles）, H, I and PPB were calculated. Richer genetic diversity was found in the wild rice populations distributed in Simao Prefecture than that in Lingcang Prefecture or Xishuangbanna Prefecture whereas the least genetic diversity was in Baoshan Prefecture or Dehong Prefecture. Rich genetic diversity was also discovered in the wild rice populations originated from higher than 710 m altitude around the middle and lower reaches of the Lancang River belonging to the Pacific Ocean drainage system. The 34 populations could be classified into two groups, one group covered the wild rice distributing in Simao Prefecture only while the other group covered ones in Lingcang, Xishuangbanna and Dehong Prefectures. The issue on how to effectively conserve the wild rice germplasm was discussed.

A SSR Linkage Map of Maizex Teosinte F_2 Population and Analysis of Segregation Distortion

In this study,a linkage genetic map was constructed using a F2 population derived from a cross between a elite maize inbred,B73,and its progenitor,Teosinte（Z.mays ssp.mexicana）,through 205 simple sequence repeat（SSR） markers and one morphological marker.By Mapmaker 3.0,polymorphic markers were clustered into 10 groups,covering 10 chromosomes of maizexteosinte,with a total length of 2 002.4 cM and an average interval of 9.7 cM.Genotyping errors were detected using R/QTL（LOD=2.0） in 109 markers referring to 176 individuals,distributed across all 10 chromosomes with a ratio 1.2%.Projected error loci were re-run and 304 out of the 460 were confirmed as errors and replaced.A new linkage map was constructed,in which markers maintained the same order but the total map length decreased to 1 947.8 cM,with an average interval of 9.4 cM between markers.In total,25.2%（P0.05） markers were identified to have segregation distortion,in which 34.6% deviated towards the pollination parent（B73）,30.8% deviated towards Teosinte,32.7% deviated towards heterozygote and 1.9% deviated towards both parents.This map was also compared with published maizexteosinte and maize IBM map.

Genetic Diversity of Tropical Hybrid Rice Germplasm Measured by Molecular Markers

Investigation of genetic diversity and relationships among breeding lines is of great importance to facilitate parent selection in hybrid rice breeding programs.In this study,we characterized 168 hybrid rice parents from International Rice Research Institute with 207 simple sequence repeat （SSR） and 353 single nucleotide polymorphism （SNP） markers.A total of 1 267 SSR and 706 SNP alleles were detected with the averages of 6.1 （SSR） and 2.0 （SNP） alleles per locus respectively across all lines.Based on the genetic distances estimated from the SSR and SNP markers separately and combined,the unrooted neighbor-joining cluster and STRUCTURE analyses consistently separated the 168 hybrid rice parents into two major groups：B-line and R-line,which is consistent with known parent pedigree information.The genetic distance matrices derived from the SSR and SNP genotyping were highly correlated （r=0.81,P 0.001）,indicating that both of the SSR and SNP markers have distinguishable power to detect polymorphism and are appropriate for genetic diversity analysis among tropical hybrid rice parents.A subset of 60 SSR markers were also chosen by the Core Hunter with 368 alleles,and the cluster analysis based on the total and subset of SSR markers highly corresponded at r=0.91 （P 0.001）,suggesting that fewer SSR markers can be used to classify and evaluate genetic diversity among parental lines.