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.

The Precursory Significance of Cumulative Slip of Repeating Earthquake Sequences Prior to Moderately Strong Earthquakes A Case Study of Four Remarkable Earthquake Sequences of Haicheng- Xiuyan

The Haicheng-Xiuyan region is an earthquake-prone area in Liaoning Province where earthquake sequences frequently occur and is regarded as the regional seismic window. In this area we found many earthquake events with the highest waveform similarity in the records of the same station from some remarkable seismic sequences,namely repeating earthquake sequences. In principle,rupture areas of the repeating events overlap with each other and are most closely located. Therefore these events may reflect the seismic process near the earthquake fault. In this paper, we identified four remarkable earthquake sequences of Haicheng-Xiuyan by waveform cross-correlation. The result shows that the cumulative slip of repeating earthquakes is related to moderately strong earthquakes,among which the Xiuyan M_S5. 4 foreshock sequence has the strongest and most apparent pre-shock accelerating-like slip behavior.

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.

RAPD and ISSR Markers of Fertility Restoring Gene for Aegilops kotschyi Cytoplasmic Male Sterility in Wheat

LK783 was found to be a good fertility restorer for K-type male sterility of wheat (Triticum aestivum L.). RAPD and ISSR (inter-simple sequence repeat polymorphism) markers were employed to map the major restoring gene in LK783. Maintainer and restorer DNA pools were established using the extreme sterile and fertile plants among KJ5418A//911289/LK783 F 1 population, respectively. Four hundred and eighteen RAPD primers and 33 ISSR primers were used for screening polymorphisms between the two pools, and amplification bands using a RAPD primer of OPK18 and an ISSR primer of UBC-845 were found polymorphic between the two pools. Linkage analysis showed that OPK18 450 and UBC-845 800 were linked to the restoring gene in LK783. The distance between the restoring gene and OPK18 450 was (15.07±6.28) cM (centiMorgan), with the distance between the restoring gene and UBC-845 800 being (8.20±4.85) cM. The marker of UBC-845 800 was located on chromosome 1BS by amplifying nulli-tetrasomics and 1B ditelosomics of Chinese Spring with the primer of UBC-845, indicating that the restoring gene in LK783 was located on 1BS. The breeding for new fertility restorer lines of K-type cytoplasmic male sterility of wheat would be facilitated by using the two markers.

cpSSR: a New Tool to Analyze Chloroplast Genome of Citrus Somatic Hybrids

Chloroplast simple sequence repeat (cpSSR) markers in Citrus were developed and successfully used to analyze chloroplast genome inheritance of Citrus somatic hybrids. Twenty-two previously reported cpSSR primer pairs from pine (Pinus thunbergii Parl.), rice (Otyza sativa L.) and tobacco (Nicotiana tabacum L.) were tested in Citrus, nine of which could amplify intensive PCR products by agarose gel electrophoresis. Chloroplast genome inheritance of Citrus somatic hybrids from nine fusions was then analyzed, and five of the nine pre-screened primer pairs showed polymorphisms by polyacrylamide gel electrophoresis. The results revealed the random inheritance nature of chloroplast genome in all analyzed Citrus somatic hybrids, which was in agreement with previous reports based on RFLP or CAPS analyses. It was also shown that cpSSR is a more efficient tool in chloroplast genome analyses of somatic hybrids in higher plants, compared with the conventional RFLP or CAPS analyses.

Genetic relationship of interspecies for eight birch species

Genetic relationships of eight species of genus Betula were evaluated using ISSR marks. A total of 236 loci were generated from 17 ISSR primers. Percentage of polymorphic bands (PPB) varied from 5.93 to 19.92. The highest and the lowest level of genetic differentiation were detected in B. ovalifolia and B. maximowicziana Regel respectively. In these eight species, genetic diversity of birch (HT) was 24.38 %, and the genetic variation (GST ) interspecies was accounting for 79.36% of total genetic variation. According to the cluster results of genetic distance, the eight species were classified into three groups as B. davurica, B. ovalifolia, B. platyphylla and B. pendula for one group; B. schmidtii, B. costata and B. ermanii Cham. var. communis for one group, and B. maximowicziana Regel for another group. The result of cluster is consistent with traditional morphological classification.

Optimization of SSR Reaction System for Identifying the Authenticity of Maize Hybrids

[Objective]The aim was to optimize SSR reaction system applied in the identification of authenticity of maize variety.[Method]The technical parameters of SSR including PCR reaction system,annealing temperature and electrophoresis time were optimized to identify 10 major maize varieties in Liaoning Province.[Result]The optimum PCR reaction system was：14.60 μl sterile ultrapure water,2.00 μl 10 × Buffer（Mg2＋）,1.20 μl dNTPs,0.20 μl Taq enzyme,0.50 μl each of the forward and reverse primers and1.00 μl DNA stock solution.Annealing temperature and electrophoresis time could greatly influence the results of PCR amplification.The optimal annealing temperature and electrophoresis time required for the ideal electrophoresis bands under the same conditions were different when different primers were used.[Conclusion]The system was feasible to be applied in rapid identification of authenticity of hybrid maize varieties.

Optimization of Multiplex PCR and Multiplex Gel Electrophoresis in Sunflower SSR Analysis Using Infrared Fluorescence and Tailed Primers

In an effort to simplify the procedure and to reduce the cost of fluorescence SSR analysis, the conditions of the multiplex PCR and the multiplex gel electrophoresis were optimized in the genetic analysis of sunflower (Helianthus annuus L.) inbred lines. Results indicated that factors for a successful multiplex PCR assay were related to the cycling touchdown annealing temperature, the balance of primer concentration at the various loci, the concentration of PCR buffer and the Taq DNA polymerase. Based on the optimization, a tailed primer strategy was outlined, and the effective ways were proposed to overcome the troubleshootings commonly encountered in the multiplex PCR and the multiplex gel electrophoresis.

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.

Mapping of QTL Associated with Drought Tolerance in a Semi-Automobile Rain Shelter in Maize (Zea mays L.)

Drought is a major constraint in maize production worldwide. We studied quantitative trait loci （QTL） underlying drought tolerance for maize plants grown in two different environments. Traits investigated included ASI, plant height, grain yield, ear height, and ear setting. A genetic linkage map was constructed with 120 simple sequence repeat （SSR） markers based on an F2 population derived from a cross between D5 （resistant parent） and 7924 （susceptible parent）. Correlation and heritability were calculated. QTLs of these traits were identified by composite interval mapping combined with a linkage map covering 1 790.3 cM. The markers were arranged in ten linkage groups. QTL mapping was made of the mean trait performance of the 180 F2：3 population. The results showed five, five, six, four, and five QTLs for ASI, plant height, grain yield, ear height, and ear setting under full irrigation condition, respectively, and four, seven, six, four, and four QTLs for ASI, plant height, grain yield, ear height, and ear setting under severe late stress conditions, respectively. Especially the four QTLs detected for five traits in 2008 and 2009. The universal QTLs information generated in this study will aid in undertaking an integrated breeding strategy for further genetic studies in drought tolerance improvement in maize.

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.