Genome-wide association studies reveal the genetic basis of amino acid content variation in tea plants

Tea is one of the most popular non-alcoholic beverages in the world,and free amino acids,especially theanine,make a major contribution to the umami taste of tea.However,the genetic basis of the variation in amino acid content in tea plants remains largely unknown.Here,we measured the free amino acid content in fresh leaves of 174 tea accessions over two years using a targeted metabolomics approach and obtained genotype data via RNA sequencing.Genome-wide association studies were conducted to investigate loci affecting the content of free amino acids.A total of 69 quantitative trait loci(–log10(P-value)>5)were identified.Functional annotation revealed that branched-chain amino acid aminotransferase,glutamine synthetase,nitrate transporter,and glutamate decarboxylase might be important for amino acid metabolism.Two significant loci,glutamine synthetase(Glu1,P=3.71×10^(−4);Arg1,P=4.61×10^(−5))and branched-chain amino acid aminotransferase(Val1,P=4.67×10^(−5);I_Leu1,P=3.56×10^(−6)),were identified,respectively.Based on the genotyping result,two alleles of CsGS(CsGS-L and CsGS-H)and CsBCAT(CsBCAT-L and CsBCAT-H)were selected to perform function verification.Overexpression of CsGS-L and CsGS-H enhanced the contents of glutamate and arginine in transgenic plants,and overexpression of CsBCAT-L and CsBCAT-H promoted the accumulation of valine,isoleucine and leucine.Enzyme activity assay uncovered that SNP1054 is important for CsGS catalyzing glutamate into glutamine.Furthermore,CsGS-L and CsGS-H differentially regulated the accumulation of glutamine,and CsBCAT-L and CsBCAT-H differentially regulated the accumulation of branched-chain amino acids.In summary,the findings in our study would provide new insights into the genetic basis of amino acids contents variation in tea plants and facilitate the identification of elite genes to enhance amino acids content.

Genetic variation of Laminaria japonica (Phaeophyta) populations in China as revealed by RAPD markers

For the population genetics analysis of the naturally grown brown seaweed Laminaria japonica （Laminariales,Phaeophyta） sampled from Dalian,Yantai,Weihai,Rongcheng and Qingdao in China,ten primers were employed to produce 88 bands as revealed by randomly amplified polymorphic DNA （RAPD） markers,and all these bands were polymorphic.According to these band patterns,there were 94 distinct phenotypes occurred in 100 samples indicating the high heterozygosity of this kelp.Dalian population samples showed the highest percentage of polymorphism （71.67%）,and also the higher diversity estimated on the basis of the Shannon’s index （8.498）,suggesting that this population could be chosen as the best resource for genetic breeding.The highest diversity of Yantai population possibly resulted from the introduction of L.longissima used for interspecific cross breeding with L.japonica cultivated in China.From Dalian southwards to Qingdao,the genetic variation of the five populations became less with a decrease in latitude,possibly due to the natural selection especially of high temperature.The genetic distance （Φ ST values） of the five populations was a little significantly correlated with the geographical distance （r=0.496） at P =0.05 by Mantel’s test.Weihai,Rongcheng and Yantai populations were closely grouped genetically together by Neighbor-joining cluster analysis probably in that the dispersal of the kelp by propagules more easily occurring in the range of relatively short distance.The analysis of molecular variance （AMOVA） also demonstrated that the relatively higher variation occurred among populations （71.49%） at an extremely significant level （P 0.000 1）.All these evidence showed that there was a relatively distinct genetic differentiation among the sampled kelp populations,and L.japonica grown in China was also rather heterozygous in heredity.

Genetic Variation in Triticum turgidum L. ssp. turgidum Landraces from China Assessed by EST-SSR Markers

It was helpful for the wheat improvement to evaluate the genetic resources of Triticum turgidum L. ssp. turgidum landraces. In this study, 68 turgidum landraces accessions, belonging to four geographic populations in China, were investigated by using EST-SSR markers. A total of 63 alleles were detected on 22 EST-SSR loci, and the number of alleles on each locus ranged from 1 to 5, with an average of 2.9. The results of the analysis of molecular variance （AMOVA） indicated that 92.5% of the total variations was attributed to the genetic variations within population, whereas only 7.5% variations among populations. Although the four populations had similar genetic diversity parameters, Sichuan population was yet distinguished from other populations when comparing the population samples in pairs. Significant correlations were detected by the statistic analysis among six genetic diversity parameters among each other. The selection difference between heterozygosty and homozygosty was also observed among different EST-SSR locus. The genetic similarity （GS） ranged from 0.18 to 0.98, with the mean of 0.72, and all accessions could be clustered into 7 groups. The dendrogram suggested that the genetic relationships among turgidum accessions evaluated by EST-SSR markers were unrelated to their geographic distributions. These results implied that turgidum landraces from China had the unique characters of genetic diversity.

Genetic variation and division of Pinus sylvestris provenances by ISSR markers

Inter Simple Sequence Repeat （ISSR） was used to detect genetic variation among nine provenances, including 135 individuals. A total of 108 loci were amplified using 10 random primers. The differentiation of the percentage of polymorphic bands （PPB） among different provenances was evident, ranging from 27% to 54%. of which Honghuaerji provenance had the highest PPB and Kalunshan provenance had the lowest PPB. Shannon＇s Information index （1） at species level was 0.1581 and Nei＇s gene diversity （h） was 0.2393. Coefficient of gene differentiation （Gsr） calculated by Popgene was 0.3965, these results indicated that majority of genetic variation （60.35%） was found within provenances. According to dendrogram among pinus sylvestriv provenances, nine provenances were divided into two provenance areas. namely Daxing＇an and Xiaoxing＇an Mountains provenance area and Hulunbeier provenance area.

Genetic Variation Analysis on the Whole Genomic Sequence of a H9N2 Subtype Avian Influenza Virus Isolate

A Objective3 This study was to understand the genetic variation characters of the H9N2 subtype avian influenza virus isolate （A/Chicken/ Hebei/WD/98, abbreviated as WD98） by comparing with other reference strains. I-Method3 Eight complete genes were amplified by RT-PCR and sequenced. The homology and genetic evolution relationship were analyzed between these sequences and that of the seven reference strains. [Result] The whole genomic sequence of WD98 strain was 91.1% -95.8% homologous to that of seven reference strains tested. This isolate shared the highest homology （95.8%） to D/HK/Y280/97 and the lowest homology （91.1% ） to C/Pak/2/99. The HA cleavage site of the WD98 strain was R-S-S-R G, and the 226th amino acid at receptor-binding site was Gin. [ Condmion] WD98 strain belongs to mildly pathogenic avian in- fluenza virus and may not infect human. The genetic relationship is the closest between A/Chicken/Hebei/wD/98 and A/duck/HongKong/Y280/ 97, both of which belong to the sub-line of A/Chicken/Beijing/1/94 in Eurasian line. And A/Chicken/Hebei/WD/98 and A/Chicken/Beijing/1/94 are genetically distant within the same sub-line.

Analysis of drought tolerance and genetic and epigenetic variations in a somatic hybrid between Ipomoea batatas (L.) Lam. and I. triloba L.

The somatic hybrid KT1 was previously obtained from protoplast fusion between sweetpotato （Ipomoea batatas （L.） Lam.） cv. Kokei No. 14 and its wild relative I. triloba L. However, its genetic and epigenetic variations have not been investigated. This study showed that KT1 exhibited significantly higher drought tolerance compared to the cultivated parent Kokei No. 14. The content of proline and activities of superoxide dismutase （SOD） and photosynthesis were significantly increased, while malonaldehyde （MDA） content was significantly decreased compared to Kokei No. 14 under drought stress. KT1 also showed higher expression level of well-known drought stress-responsive genes compared to Kokei No. 14 under drought stress. Amplified fragment length polymorphism （AFLP） and methylation-sensitive amplified polymorphism （MSAP） analyses indicated that KT1 had AFLP and MSAP band patterns consisting of both parent specific bands and changed bands. Fur- ther analysis demonstrated that in KT1. the proportions of Kokei No. 14 specific genome components and methylation sites were much greater than those of I. triloba. KT1 had the same chloroplast and mitochondrial genomes as Kokei No. 14. These results will aid in developing the useful genes ofI. triloba and understanding the evolution and phylogeny of the cultivated sweetpotato.

Genetic variations in the SMAD4 gene and gastric cancer susceptibility

AIM:To explore the association between mothers against decapentaplegic homolog 4 (SMAD4) gene polymorphisms and gastric cancer risk.METHODS:Five tagging single nucleotide polymor-phisms (tSNPs) in the SMAD4 gene were selected and genotyped in 322 gastric cancer cases and 351 cancerfree controls in a Chinese population by using the polymerase chain reactionrestriction fragment length polymorphism method.Immunohistochemistry was used to examine SMAD4 protein expression in 10 normal gastric tissues adjacent to tumors.RESULTS:In the single-locus analysis,two significantly decreased risk polymorphisms for gastric cancer were observed:the SNP3 rs17663887 TC genotype (adjusted odds ratio=0.38,95% confidence interval:0.21-0.71),compared with the wild-type TT genotype and the SNP5 rs12456284 GG genotype (0.31,0.16-0.60),and with the wild-type AA genotype.In the combined analyses of these two tSNPs,the combined genotypes with 2-3 protective alleles (SNP3 C and SNP5 G allele) had a significantly decreased risk of gastric cancer (0.28,0.16-0.49) than those with 0-1 protective allele.Furthermore,individuals with 0-1 protective allele had significantly decreased SMAD4 protein expression levels in the norma tissues adjacent to tumors than those with 2-3 protective alleles (P=0.025).CONCLUSION:These results suggest that genetic variants in the SMAD4 gene play a protective role in gastric cancer in a Chinese population.

Genome-wide identification,evolutionary selection,and genetic variation of DNA methylation-related genes in Brassica rapa and Brassica oleracea

DNA methylation plays an important role in plant growth and development,and in regulating the activity of transposable elements(TEs).Research on DNA methylation-related(DMR)genes has been reported in Arabidopsis,but little research on DMR genes has been reported in Brassica rapa and Brassica oleracea,the genomes of which exhibit significant differences in TE content.In this study,we identified 78 and 77 DMR genes in Brassica rapa and Brassica oleracea,respectively.Detailed analysis revealed that the numbers of DMR genes in different DMR pathways varied in B.rapa and B.oleracea.The evolutionary selection pressure of DMR genes in B.rapa and B.oleracea was compared,and the DMR genes showed differential evolution between these two species.The nucleotide diversity(π)and selective sweep(Tajima’s D)revealed footprints of selection in the B.rapa and B.oleracea populations.Transcriptome analysis showed that most DMR genes exhibited similar expression characteristics in B.rapa and B.oleracea.This study dissects the evolutionary differences and genetic variations of the DMR genes in B.rapa and B.oleracea,and will provide valuable resources for future research on the divergent evolution of DNA methylation between B.rapa and B.oleracea.

Genetic variation of wood tracheid traits and their relationships with growth and wood density in clones of Pinus tabuliformis

To improve wood quality for pulpwood industries, it is important to examine not only wood density but also its components, especially tracheid characteristics. We studied genetic variations in the following tracheid traits by earlywood （EW） and latewood （LW）： tracheid length （TL）, double wall thickness （WT）, radial lumen diameter （R_D1）, tangential lumen diameter （T_D1）, radial central diameter （R_D2）, and tangential central diameter （T_D2）. We also studied the relationship with the following growth traits： diameter at breast height （DBH）, height （H）, crown breadth south-north axis （NSC）, crown breadth east-west axis （EWC）, ring width （RW）, latewood percentage （LWP）, and wood density （WD）. All sample materials were collected from a 33-year old clonal seed orchard of Pinus tabuliformis Carr. Genetic variation among clones was moderate for all tracheid traits, 9.49-26.03%. Clones significantly affected WT, R_D1, R_D2, T_D1, T_D2, and the two ratios WT/R_D1 and TL/T_D2 in EW but had no effects in LW. Clones significantly affected TL in LW but had no effects in EW. H2/C was higher in LW （0.50） than in EW （0.20） for TL, while H 2/C was higher in EW （0.27-0.46） for other tracheid traits and the two ratios （TL/T_D2 and WT/R_D1） than in EW （0.06-0.22）. WD and TL were significantly positively correlated, but WT and TL were negatively correlated both at individual and clone levels; all tracheid diameters and the four ratio values （EW_WT/ R_D1, LW WT/R_D1, EW_TL/T_D2 and LW_TL/ T_D2）, were strongly positively correlated with DBH, H, NSC, WEC and RW, and strongly negatively correlated with WD both at individual and clone levels. The most important variables for predicting WD were LW_TL, EW_WT and R_D1 in both EW and LW （r2= 0.22）. Selecting the top 10% of the clones by DBH would improve DBH growth by 12.19% （wood density was reduced by 0.14%） and produced similar responses between EW and LW for all tracheid traits： a reduction of 0.94 and 3.69% in tracheid length and increases in tracheid diameters （from 0.36 to 5.24%） and double wall thickness （0.07 and 0.87%）. The two ratios WT/R_D1 and TL/T_D2 across tissues （EW and LW） declined 0.59 and 4.56%, respectively. The decreased tracheid length and the ratio between tracheid length and diameter is disadvantageous for pulp production. The unfavorable relationship of tracheid traits with wood density indicate that multiple trait selection using optimal economic weights and optimal breeding strategies are recommended for the current longterm breeding program for P. tabuliformis.

Analysis of Genetic Variation of Seed Proteins in the Genus Vigna and among Its Relatives Cultivated in China

The genetic variation of seed proteins was assayed by SDSPAGE for 24 cultivars belonging to 5 species in Vigna and 7 species in its 7 relative genera cultivated in China. There were 48 polymorphic subunit bands discriminated from electrophoretic profiles. Two dendrograms were constructed by UPGMA cluster analyses using PHYLIP3.6 respectively. Variation among genera or species was larger than that among lower taxonomic categories level. Little variation among cuhivars of yardlong bean （Vigna sesquipedalis ） and small variation of lablab （ Lablab purpureus）, pea （Pisum sativum）, or sword bean （Canavalia gladiata）, but large variation of soybean or rice bean in their origin of China were all revealed. The seed proteins profiles of traditionally regarded as typical species in Vigna such as yardlong bean, rice bean and small bean were more similar than mungbean （Vigna radiata） and black gram （Vigna mungo） were. Mungbean and black gram had distinct seed proteins pattern, they should be of two species.

Investigation of Genetic Variation of B-G Gene with PCR-SSCP and RFLP in Chinese Indigenous Chickens

To reveal genetic variation of MHC B-G gene at Chinese native chickens, two PCR primer pairs were designed to hybridize specifically with conserved sequences surrounding hypervariable regions within the B-G gene and used to amplify two DNA fragments in ten Chinese indigenous chicken breeds and one introduced breed. The fragments were cloned and sequenced to assure that the expected sequences of chicken B-G gene were isolated. Of which the 189 bp fragment encompassing the most variable region within exon 2 of B-G gene was employed for PCR-SSCP assay, this method provided evidence for the presence of at least 56 B-G genotypes in the Chinese chickens sampled. It revealed a high degree of diversity in B-G genes of Chinese local breeds; particularly, high variation of B-G gene was confirmed with the presence of 48 B-G genotypes within Tibetan chicken population. Not only can the B-G genotypes be used to preliminarily screen new B-G alleles, but also they would be utilized to investigate MHC haplotypes and matched unrelated donors for bone marrow transplantation in immune researches. Another fragment of 401 bp size spanning over partial intron 1 and exon 2 of B-G gene was employed for PCR-RFLP analysis with two restriction enzymes of Msp Ⅰ and Tas Ⅰ in the breeds sampled. In this part of the gene, three novel SNPs were detected at the two restriction sites. It was more generally found the transition of two nucleotides of A294G and T295C occurred at Tas Ⅰ restriction site, and consequently led to a non-synonymous substitution of asparagine into serine at position 54 within the deduced amino acid sequence of immunoglobulin variable-region-like domain encoded by the exon 2 of B-G gene. It was observed at rare frequency that an alone mutation of A294G occurring at the site, which also caused the substitution of amino acid, asparagine 54-to-serine; and we haven＇t found only single mutation occurred at position 295 of the restriction site. At the Msp Ⅰ site, the transversion of G318C led to a non-synonymous substitution, glutamine 62-to-histidine. The variations at expression level caused by the genetic variability of B-G gene may bring about the changes in immune specificity of B-G antigen finally. Furthermore, two alleles, A and B, were identified at Msp Ⅰ and Tas Ⅰ loci of B-G gene, respectively. The allele frequencies were estimated, which gave a nonsymmetrical distribution either in the eight Chinese local breeds or in the introduced breed. By comparison, allele A at Msp Ⅰ locus was tended to be dominative, while, the allele B at Tas Ⅰ locus was tended to be prevalent in the breeds analyzed. It is concluded that the genetic variability of B-G gene revealed by the PCR-SSCP and RFLP assays in Chinese native chickens provide molecular data for further investigating the varied immune functions of B-G antigen; and the PCR-RFLPs at Msp Ⅰ and Tas Ⅰ loci of B-G gene might be used as genetic markers in selecting for the traits of disease resistance in chicken breeding.

Genetic variation of Chinese and Japanese wild Pacific abalone(Haliotis discus hannai)measured by microsatellite DNA markers

Population differentiation and relationships among three wild populations of the Pacific abalone Haliotis discus hannai collected from coastal seas around China and Japan were estimated using microsatellite DNA analysis. The results obtained with six microsatellite loci showed a high genetic diversity for China and Japan populations. The mean number of alleles per locus ranged from 11.7 to 23.0, and the average of observed and expected heterozygosity ranged from 0. 656 to 0. 721, and from 0. 721 to 0. 793, respectively. The observed genotype frequencies at each locus were mostly in agreement with Hardy - Weinberg expectations with five exceptions. Significant differences were detected between Chinese and Japanese H. discus hannai populations [ Weir and Cocker-ham＇ s fixation index（ Fst ） range ： 0. 020～ 0.023 ; Slatkin＇ s fixation index （Rst） range： 0. 016～ 0. 044 ], and no obvious difference was detected between the samples of Japanese H. discus hannm populations （ Fst = 0. 002 ; Rst = 0. 007）. The level of differentiation among populations is further evidenced by the nNeighbor-joining tree topology on which the Japanese samples were closely clustered, and the Chinese population formed a separate cluster. These results suggest that care should be taken in future management of different populations.

AFLP analysis of genetic variation of Hyphantria cunea(Drury) populations in Beijing and a nearby site

Hyphantria cunea （Drury） is a severe invasive pest in Beijing. It is important to understand the reason for its successful invasion and outbreak. Accordingly, this study was tailored to assess molecular variation and genetic relationships among three H. cunea populations in Beijing and one population from Sanhe in Hebei Province as control （totally 100 individuals）. A silver-labeled amplified fragment length polymorphisms （AFLP） DNA profiling method was used to detect the genetic diversity and relationship among the populations. Five EcoRI and MseI primer combinations produced a total of 171 （91.2% polymorphic） informative fragments. The average Nei＇s genetic diversity in four H. cunea populations analyzed by Popgene3.2 software was 0.2287, and the level of genetic diversity ranked in the order BDR （reared population in Daxing, Beijing） 〉 BDN （natural population in Daxing, Beijing） 〉 HSN （natural population in Sanhe, Hebei Province） 〉 BFN （natural population in Fengtai, Beijing）. The value of genetic differentia- tion among populations was 0.3321, and the gene flow was 1.0057. The genetic distance between these populations ranged from 0.0735 to 0.3309, 0.0531 on average. The result of UPGMA （unweighed pair group method with arithmetic averages） clustering showed that populations BFN and HSN were closely related, while BFN and BDN had the largest genetic distance. These results indicate that H. cunea populations in Beijing might originate from several areas.

Genetic variation and population structure of the mushroom Pleurotus ferulae in China inferred from nuclear DNA analysis

To investigate the genetic diversity of an edible fungus Pleurotus ferulae, a total of 89 wild samples collected from six geographical locations in the Xinjiang Uygur Autonomous Region of China and two geographical locations in Italy, were analyzed using three DNA fragments including the translation elongation factor（EF1α）, the second largest subunit of t he RNA polymerase II（RPB2） and the largest subunit of the RNA polymerase II（RPB1）. The results indicated relatively abundant genetic variability in the wild resources of P. ferulae. The analysis of molecular variance（AMOVA） showed that the vast majority of the genetic variation was found within geographical populations. Both the Chinese populations and the Italian populations of P. ferulae displayed a limited genetic differentiation. The degree of differentiation between the Chinese populations and the Italian populations was obviously higher than that between the populations from the same region, and moreover the genetic differentiation among all the tested populations was correlated to the geographical distance. T he phylogeny analyses confirmed that samples from China and Italy belonged to another genetic group separated from Pleurotus eryngii. They were closely related to each other but were clustered according to their geographical origins, which implied the Chinese populations were highly differentiated from the Italian populations because of distance isolation, and the two populations from different regions might be still in the process of allopatric divergence.

RAD-sequencing improves the genetic characterization of a threatened tree peony(Paeonia ludlowii)endemic to China:Implications for conservation

Compared with traditional genetic markers,genomic approaches have proved valuable to the conservation of endangered species.Paeonia ludlowii having rarely and pure yellow flowers,is one of the world’s most famous tree peonies.However,only several wild populations remain in the Yarlung Zangbo Valley(Nyingchi and Shannan regions,Xizang)in China due to increasing anthropogenic impact on the natural habitats.We used genome-wide single nucleotide polymorphisms to elucidate the spatial pattern of genetic variation,population structure and demographic history of P.ludlowii from the fragmented region comprising the entire range of this species,aiming to provide a basis for conserving the genetic resources of this species.Unlike genetic uniformity among populations revealed in previous studies,we found low but varied levels of intra-population genetic diversity,in which lower genetic diversity was detected in the population in Shannan region compared to those in Nyingzhi region.These spatial patterns may be likely associated with different population sizes caused by micro-environment differences in these two regions.Additionally,low genetic differentiation among populations(Fst=0.0037)were detected at the species level.This line of evidence,combined with the result of significant genetic differentiation between the two closest populations and lack of isolation by distance,suggested that shared ancestry among now remnant populations rather than contemporary genetic connectivity resulted in subtle population structure.Demographic inference suggested that P.ludlowii probably experienced a temporal history of sharp population decline during the period of Last Glacial Maximum,and a subsequent bottleneck event resulting from prehistoric human activities on the Qinghai-Tibet Plateau.All these events,together with current habitat fragment and excavation might contribute to the endangered status of P.ludlowii.Our study improved the genetic characterization of the endangered tree peony(P.ludlowii)in China,and these genetic inferences should be considered when making different in situ and ex situ conservation actions for P.ludlowii in this evolutionary hotspot region.

Genetic variation and superior provenances selection for wood properties of Larix olgensis at four trials

Larix olgensis,one of the most important timber species in northeastern China,is used for paper making and construction.In this study,10 wood properties(wood density,fiber length,fiber width,fiber length-to width ratio,hemicellulose content,cellulose content,holocellulose content,lignin content,ash content,and carbon content)of 10 provenances of L.olgensis planted at sites of CuoHai(CH),JiaGeDaQi(JGDQ),LiangShui(LS),and Mao'erShan(MES)were analyzed.The results of ANOVA showed that almost traits differed significantly among locations and provenances,with a significant interaction effect.Each trait also differed significant among provenances within sites.The phenotypic and genetic coefficient of variation(PCV and GCV)and provenance heritability(H 2)for wood properties ranged from 1.122 to 27.365%,from 0.564%to 21.113%and from 0.332 to 0.996,respectively.A correlation analysis showed that wood density was significantly negatively correlated with cellulose content and holocellulose content at sites CH,JGDQ,and LS,but were significantly positively correlated at site MES.Wood density was significantly negatively correlated with lignin content at CH and JGDQ,but not at LS and MES.Fiber width(FW)was negatively correlated with the ratio of fiber length(FL)to width across sites,and FW and FL/W were all positively correlated with FL.Lignin content was significantly positively correlated with hemicellulose content at site JGDQ and significantly negatively correlated with cellulose content and with holocellulose content at site MES.Interestingly,carbon content was positively correlated with cellulose content and holocellulose content at CH,but negatively correlated with these two traits at site MES.In a correlation analysis of wood properties with geographic,soil and climatic characteristics at the four sites,wood properties were mainly correlated with latitude and altitude of the site and affected by the average annual precipitation and temperature simultaneously.To select superior pulpwood provenances and high carbon storage provenances,we selected the two best provenances with excellent wood properties for each location based on a multi-trait comprehensive evaluation,which can be used as the preferred materials for the establishment of large-scale plantations in specific locations.

Attenuation of Virulent Porcine Reproductive and Respiratory Syndrome Virus Strain CH-1a and Genetic Variation of ORF5 Gene

To develop a modified live vaccine （MLV） against porcine reproductive and respiratory syndrome virus （PRRSV）, virulent CH-Ia strain was attenuated by serial passages up to 130 passage （P130） in Marc-145 cells. The virulence and immune efficacy of the attenuated CH-1 a were evaluated in pigs. The results showed that animals inoculated with P130 did not develop any clinical sign of the disease, but produced rapid and effective humoral immune responses against PRRSV challenge, indicating that attenuated CH-1 a P 130 is the candidate as the effective vaccine against PRRSV. To define the potential mutations in the attenuated CH-la genome, we sequenced and analyzed the ORF5 gene of CH-la strain of different passages （P39, P55, P65, P70, P85, P100, P115, P120, P125, and P130） and found that three mutations （C5Y, H38Q and L146Q） which may be related with the attenuation of CH-la. In addition, we also found a unique restriction enzyme site （TspEI） in the ORF5 gene of attenuated CH-la, which can be used as a genetic marker to distinguish original and attenuated CH- 1 a.

Genetic Variation of the Novel Badnaviruses Infecting Nelumbo Nucifera Based on the RT/RNase H Coding Region Sequences

Lotus(Nelumbo nucifera Gaertn.)is an economically important perennial aquatic herb plant in China.Presently,viral diseases caused by Cucumber mosaic virus(CMV)and Dasheen mosaic virus(DsMV)are detected in lotus.In this study,the putatively novel badnaviruses from lotus plants(LBVs)were identified using next-generation sequencing of siRNAs and conventional Sanger sequencing based on the RT/RNase H coding region sequences.Lotus plants infected by LBVs harbored virus quasispecies.A unique recombination event of LBVs was found in a single lotus plant.Sap inoculation showed that LBVs could actively replicate in the lotus plants,but without a wide host range.The field survey of 43 lotus plants in Jiangsu Province showed a prevalence of 62.8%for LBVs,confirming that it is widely distributed in Jiangsu Province of China.

Genetic Variation of Grey-rumped Pheasant (Torquatus group) Populations in China

The polymerase chain reaction combined with direct sequencing was employed to deduce the variety of the grey-rumped pheasant populations in China. Totally 1079 basepair sequences of mitochondrial DNA （mtDNA） control-region gene were analyzed from 121 grey-rumpod pheasants obtained from 13 Chinese lo- calities, 76 variable sites resulted in 91 haplotypes. These populations possessed a high level of genetic diversity, indicating that during their evolution effective population size and polymorphism were not reduced by climatic fluctuations in the Pleistocene ice ages. Phylogenetie analysis indicates that all populations were clustered into two groups, western and eastern. The degree of population differentiation presumably results from a low gene flow （ N, = 0. 33, F,, = 0. 60, P 〈 0. 001 ） between the two groups, and the divergence occurred in the years （2.1 ×10^5 - 2.2 × 105 ）. The genetic structure of grey-rumped pheasants in China ap- pears to be the result of the uplift of Qinghai-Tibet Plateau, the existence of Qinling Mountains and Liupan Mountains and cyclical climatic oscillations during the late Pleistocene.

Assessment of Genetic Variation in Soybean (<i>Glycine max</i>) Accessions from International Gene Pools Using RAPD Markers: Comparison with the ISSR System

Soybean (Glycine max) is one of the most important crops in the world in terms of total production and usage. It is also among the least diverse species. The main objectives of the present study were to 1) assess the level of genetic variation among soybean (G. max) accessions from different countries using Random Amplified Polymorphic DNA (RAPD) markers and 2) compare Inter Simple Sequence Repeats (ISSR) and RAPD marker systems in detecting polymorphic loci in soybeans (G. max). Genomic DNAs from 108 soybeans (G. max) accessions from 11 different gene pools were analyzed using several ISSR and RAPD primers. The average level of polymorphic loci detected with the RAPD primers was 35%. The soybean accessions from the China, Netherlands, and Canada gene pools were the least genetically variable with 25%, 26%, and 30% of polymorphic loci, respectively. Accessions from Hungary (43%) and France (48%) showed the highest level of polymorphism based on the RAPD analysis. Overall, RAPD data revealed that the accessions from different countries are closely related with 64% genetic distance values below 0.40. The levels of polymorphic loci detected with the RAPD and ISSR marker systems were in general moderate and similar even if they target different regions of the genome. A combination of different marker systems that include RAPD/ISSR, microsatellites (SSR), and SNPs should provide the most accurate information on genetic variation of soybean (G. max) accessions.