不同性别类型黄瓜ACC合酶基因的单核苷酸多态性标记和酶切扩增长度多态性标记

黄瓜的性别分化与乙烯密切相关,1-氨基环丙烷-1-羧酸(ACC)合酶是乙烯生物合成过程中的关键酶.根据ACC合酶基因家族的保守序列设计PCR引物,从8个不同性别类型(雌雄同株、强雌性和全雌性)黄瓜品种中克隆了长度为1188bp的ACC合酶基因(CS-ACS2)片段(GenBank登记号为:DQ115884～DQ115886和DQ115875～DQ115879).经测序分析,3个雌雄同株性别类型品种的序列完全相同.与之相比,5个强雌性和全雌性品种中存在8个单核苷酸多态性(SNPs)标记,SNPs标记为4个A←→G和4个T←→C之间的转换.在8个SNPs中,有1个SNP位于内含子区域,其余7个SNPs都位于外显子区域.在7个位于外显子区域的SNPs中,有3个为非编码区的SNPs,4个为cSNPs.而在4个cSNPs中,有3个导致了编码的氨基酸序列改变.研究结果表明,与雌雄同株性别类型相比,雌性系中均存在单核苷酸的变异,这提示ACC合酶基因CS-ACS2的单核苷酸变异可能与黄瓜雌性系的发生形成有关.另一方面,根据SNP多态性还发展了一个酶切扩增长度多态性(CAPS)标记C-MT700.利用CAPS标记C-MT700能将强雌性优良品种MT-705与其他黄瓜品种相区别,该标记在黄瓜育种生产上具有一定的应用价值.此外,研究获得的SNPs标记和CAPS标记丰富了黄瓜的分子标记种类.

珍稀药用金钗石斛组培苗遗传稳定性直接扩增长度多态性检测

目的研究珍稀药用金钗石斛DNA指纹图谱,初步探讨直接扩增长度多态性(DALP)分子标记技术在石斛组织培养过程中遗传稳定性检测上的应用。方法采用直接扩增长度多态性(DALP)技术对金钗石斛野生移栽苗和组培苗进行基因组DNA多态性分析。结果从12对引物组合中筛选出6对能获清晰条带的引物组合,分别利用琼脂糖和聚丙烯酰胺对DALP-PCR产物进行凝胶电泳,发现两组苗扩增的DNA带型基本一致,未发现明显变异,说明金钗石斛茎尖离体继代培养具有较高的遗传稳定性。结论DALP分子标记技术可用于石斛遗传稳定性和遗传多样性研究。

利用AFLP进行榧树雌雄株鉴定的初步研究

为了解决榧树Torreya grandis苗期雌雄株鉴别的问题,利用已建立的扩增片断长度多态(AFLP)体系及T-A克隆测序,就榧树的雌雄株开展了研究。结果从15对引物中用引物对E-AGC/M-CAT得到了一条雌性榧树特异的条带。这条榧树雌性性别特异条带,仅在雌株、雌雄同株的个体中有,雄株中没有,推测榧树雌雄同株由雌性榧树变异而来。GenBank序列比对结果发现,数据库没有与此特异条带相匹配的序列。

提取板栗DNA的CTAB法和SDS法的比较

分别采用十六烷基三乙基溴化铵(CTAB)法和十二烷基磺酸钠(SDS)法提取板栗(Castanea mollis-sima BL.)基因组DNA,取适量DNA进行琼脂糖凝胶电泳,并利用紫外可见分光光度计测定所提取的DNA在260和280 nm的吸光度及其比值,利用Eco RⅠ和MseⅠ双酶切后进行随机扩增长度多态性(AFLP)分析。结果表明,两种方法均能从板栗中提取出一定质量的、比较完整的、纯度比较高的DNA,SDS法提取的板栗DNA的纯度更高、质量更好。

湖南省10个现行栽培桑树品种的AFLP指纹图谱分析

利用扩增长度多态性(AFLP)分子标记技术,构建湖南省现行推广的10个桑树品种的指纹图谱,作为该省区桑树遗传育种辅助选择和品种鉴定的重要依据。从36对AFLP引物中筛选出带型丰富、多态性较高的5对引物对10份桑树品种材料进行扩增,共检测到344条多态性条带,多态性比率达27.88%。品种之间的遗传相似系数以及UPGMA聚类分析结果与基于形态学方面的分类结果一致,在遗传相似系数0.79处,10个桑树品种聚为3类:三倍体品种湘桑6号单独聚为一类,其余9个品种聚为一类,其中广东桑品种苗33又单独聚为一类,8个鲁桑品种聚为一类。

Discussion on AFLP Molecular Markers in Piper methysticum and Pepper

The aim of the research was to discuss the genetic relationships between Piper methysticum, Pepper and other wild species in Pepper genus. DNA was extracted from leaves which belonged to 28 germplasms including 6 materials of P. methysticum, 21 maerials of cultivated and wild Pepper, 1 material of Peperomia pellucida belonged to different genus. Premiers with good band-type and high polymorphism and resolution were selected from 64 pairs of primers for AFLP amplification and the clustering analysis was conducted with MVSP3.13f software. 191 bands were amplified by 4 pairs of premiers, 189 of which had polymorphism, being 98.6%. 28 germplasms were classified into 6 different groups at the genetic similarity coefficient of 0.52 by silver staining AFLP, in which 6 materials of Piper methysticum were clustered into a single group, indicating that P. methysticum belonged to Pepper family of Pepper genus but were distantly related to the others. The research provided the basis for selecting rootstocks for P. methysticum graft, molecular identification of P. methysticum and the fingerprint construction of P. methysticum.

鱼类环境DNA metabarcoding片段的近缘物种识别差异

已知的鱼类环境DNA(eDNA)metabarcoding片段均未被针对性考察其对近缘物种的适用性,实际使用过程中存在“物种丢失”风险。为筛选出物种识别率最高的片段,本研究比较了15个主流片段对106属(共935种)鱼类的识别差异。研究结果如下:(1)蛋白质编码基因(COI,片段15)的物种识别率最高,但其引物通用性最差;片段09、片段11、片段07、片段03、片段12的引物序列总平均遗传距离也较大,均存在eDNA低效扩增的风险;(2)片段长度影响物种识别率,核糖体基因中片段05、片段06、片段01、片段02及片段13的物种识别率较高;(3)非度量多维尺度分析(NMDS)显示,不同基因、同一基因不同片段的识别结果存在较大差异,应考虑多片段、多基因组合应用;片段01与片段02、片段05与片段06等在NMDS图上距离较近,存在相互替代性;(4)物种类群影响识别结果,eDNA研究仍需要进一步开发高识别率片段。综合物种识别率、引物通用性、NMDS分析等多方面因素,本研究推荐2×150 bp测序平台使用片段01(Mi Fish-U)、2×250 bp测序平台使用片段05(Ac12S),辅以片段13(Vert-16S-eDNA)等进行近缘鱼类多样性调查。本研究旨在为提高鱼类eDNA调查结果准确性提供一定技术支撑。

A genetic linkage map of marine shrimp Penaeus (Fenneropenaeus) chinensis based on AFLP, SSR, and RAPD markers

The Chinese shrimp Penaeus (Fenneropaeneus) chinensis is an important species in marine fishery and aquaculture in China. A female Chinese shrimp Penaeus (Fenneropaeneus) chinensis was captured from west coast of the Korean peninsula and mated with a "Yellow Sea No. 1" male to produce the first filial generation (F1) 100 F2 full-sib progeny from brother-sister crosses between F1 families was used for the mapping study. A genetic linkage map of the Chinese shrimp was constructed, based on 354 markers, including 300 amplified fragment length polymorphism (AFLP) markers, 42 microsatellite (SSR) markers, and 12 randomly amplified polymorphism (RAPD) markers. Forty-seven linkage groups (LGs) were identified. The total map length was 4 580.5 cM, with an average spacing of 11.3 cM, covering 75.8% of the estimated genome size. The construction of this genetic linkage map was part of a genetic breeding program. This linkage map will contribute to the discovery of genes and quantitative trait loci (QTLs) in Chinese shrimp.

Genetics and phylogeny of genus Coilia in China based on AFLP markers

The taxonomy of Coilia has been extensively studied in China,and yet phylogenetic relationships among component taxa remain controversial.We used a PCR-based fingerprinting technique,amplified fragment length polymorphism(AFLP) to characterize and identify all four species of Coilia in China.We examined the genetic relationships of the four species of Coilia and a subspecies of Coilia nasus with AFLP.A total of 180 AFLP loci were generated from six primer combinations,of which 76.11% were polymorphic.The mean genetic distance between pairs of taxa ranged from 0.047 to 0.596.The neighbor-joining tree and UPGMA dendrogram resolved the investigated species into three separate lineages:(1) C.mystus,(2) C.grayii and(3) C.brachygnathus,C.nasus,and C.nasus taihuensis.Phylogenetic analysis of the AFLP data is inconsistent with current morphological taxonomic systems.The AFLP data indicated a close relationship among C.brachygnathus,C.nasus taihuensis,and C.nasus.Therefore,the two species described under Coilia(C.brachygnathus and C.nasus taihuensis) are treated as synonyms of C.nasus.

Genetic Variation and Differentiation in Wide Ranging Populations of Razor Clam(Sinonovacula constricta) Inferred from AFLP Markers

The genetic variation and differentiation of the razor clam Sinonovacula constricta distributed along the coast of China were studied through amplified fragment length polymorphism(AFLP) analysis.Six primer combinations generated 193 fragments.The He values varied from 0.322 to 0.463 and the percentage of polymorphic loci ranged from 74.1% to 98.4%,which indicates a high level of genetic diversity.Cluster analysis by Nei's pairwise distance grouped all specimens by geographical origins.AMOVA consistently showed that genetic variation among populations was 8.71%,and most of the variation came from the genetic variation within populations(91.29%).Genetic differentiation among the six populations was moderate;pairwise FST ranged from 0.0282 to 0.1480,which indicated that S.constricta populations along the coast of China are genetically connected.Among all the six populations,the Beihai population is the mostly differentiated from the others,suggesting that Hainan Island and Leizhou Peninsula act as barriers to gene flow.All populations abide isolation by distance model as indicated by Mantel test,except for ZS(Zhoushan) and YQ(Yueqing) populations.Information obtained in this study will provide guidelines for conservation and fishery management of this species in the future.

Application of AFLP markers for population genetic study on half-smooth tongue sole Cynoglossus semilaevis

The genetic diversity of wild and hatchery populations of half-smooth tongue sole Cynoglossus semilaevis, based on observation of amplified fragment length polymorphism (AFLP) was described. Two hundred individuals from four wild populations, Laizhou (LZ), Weihai (WH), Qingdao (QD), Rizhao (RZ), and one hatchery population, Mingbo (MB), were screened using eight different AFLP primer combinations. A total of 384 loci were screened in the five studied populations. 48.4%, 51.3%, 50.7%, 49.3% and 45.8% of these loci were polymorphic among the individuals tested in the LZ, WH, QD, RZ and MB populations, respectively. The number of polymorphic loci detected by single primer combinations ranged from 17 to 35. The average heterozygosity of the LZ, WH, QD, RZ and MB populations was 0.072, 0.093, 0.092, 0.090 and 0.063, respectively. The WH population showed the highest genetic diversity in terms of total number of AFLP bands, total number of polymorphic bands, average heterozygosity and percentage of low frequency (0-0.2) polymorphic loci among all the populations, while the LZ population was the lowest among the wild populations. Compared with the wild populations, the hatchery population showed a low genetic viability.

Preliminary Analysis of Population Genetic Diversity of Cultivated Laminaria japonica Sporophyte via AFLP Technique

The amplified fragment length polymorphic DNA (AFLP) technique was adopted to estimate the population genetic polymorphism among 30 sporophytes of Laminaria japonica collected from a cultivating farm in Rongcheng,China.Three methods were used for genomic DNA extraction from Laminaria japonica sporophyte and only the products obtained using the improved genomic DNA extraction kit method proved qualified for AFLP analysis.The parameters of the method were optimized.Samples of forty milligrams and the cell lysis time of 120 min were suggested to replace the parameters recommended by the manufacturer.Thirty individuals of Laminaria japonica from the same cultivating site were investigated using one pair of selective primers.A total of 21 loci were obtained and 17 of them were polymorphic.The mean percent age of polymorphic loci of this population was 80.95%.The Nei's gene diversity (H) within this population was 0.3028 and the average Shannon's Information index (I) was 0.4498.A genetic distance matrix among different individuals was constructed as well.Through this study,an applicable AFLP genetic analysis working system for Laminaria japonica sporophyte was established.The results of this research also revealed a high level of genetic diversity within the studied population.

Genetic diversity of populations and clones of Rhopilema esculentum in China based on AFLP analysis

Amplified fragment length polymorphisms(AFLP) markers were developed to assess the genetic variation of populations and clones of Rhopilema esculentum Kishinouye(Scyphozoa,Rhizostomatidae).One hundred and seventy-nine loci from 56 individuals of two hatchery populations and two wild populations were genotyped with five primer combinations.The polymorphic ratio,Shannon's diversity index and average heterozygosity were 70.3%,0.346 and 0.228 for the white hatchery population,74.3%,0.313,and 0.201 for the red hatchery population,79.3%,0.349,and 0.224 for the Jiangsu wild population,and 74.9%,0.328 and 0.210 for the Penglai wild population,respectively.Thus,all populations had a relatively high level of genetic diversity.A specific band was identified that could separate the white from the red hatchery population.There was 84.85% genetic differentiation within populations.Individual cluster analysis using unweighted pair-group method with arithmetic mean(UPGMA) suggested that hatchery populations and wild populations could be divided.For the hatchery populations,the white and red populations clustered separately;however,for the wild populations,Penglai and Jiangsu populations clustered together.The genetic diversity at the clone level was also determined.Our data suggest that there are relatively high genetic diversities within populations but low genetic differentiation between populations,which may be related to the long-term use of germplasm resources from Jiangsu Province for artificial seeding and releasing.These findings will benefit the artificial seeding and conservation of the germplasm resources.

Genetic Analysis of Floating Enteromorpha prolifera in the Yellow Sea with AFLP Marker

Extremely large accumulation of green algae Enteromorpha prolifera floated along China'coastal region of the Yellow Sea ever since the summer of 2008.Amplified Fragment Length Polymorphism (AFLP) analysis was applied to assess the genetic diversity and relationships among E. prolifera samples collected from 9 affected areas of the Yellow Sea.Two hundred reproducible fragments were generated with 8 AFLP primer combinations,of which 194 (97%) were polymorphic. The average Nei's genetic diversity, the coefficiency of genetic differentiation (Gst), and the average gene flow estimated from Gst in the 9 populations were 0.4018, 0.6404 and 0.2807 respectively. Cluster analysis based on the unweighed pair group method with arithmetic averages (UPGMA) showed that the genetic relationships within one population or among different populations were all related to their collecting locations and sampling time. Large genetic differentiation was detected among the populations.The E. prolifera originated from different areas and were undergoing a course of mixing.

Discriminating ability of molecular markers and morphological characterization in the establishment of genetic relationships in cultivated genotypes of almond and related wild species

A total 23 morphological traits, 19 AFLP-primer combinations, 80 RAPD primers and 32 SSR primer pair were used to compare the informativeness and efficiency of random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers in establishing genetic relationships among 29 almond cultivars and three related wild species. SSRs presented a high level of polymorphism and greater information content, as assessed by the expected hetrozygosity, compared to AFLPs and RAPDs. The lowest values of expected hetrozygosity were obtained for AFLPs; however AFLPs showed the highest efficiency, owing to their capacity to reveal large numbers of bands per reaction, which led to high values for various types of indices of diversity. All the three techniques discriminated almond genotypes very effectively, except that SSRs failed to discriminate between ‘Monagha’ and ‘Sefied’ almond genotypes. The correlation coefficients of similarity were statistically significant for all the three marker systems, but were lower for the SSR data than for RAPDs and AFLPs. For all the markers, high similarity in dendrogram topologies was obtained, although some differences were observed. All the dendrograms, including that obtained by the combined use of all the marker data, reflect relationships for most of cultivars according to their geographic diffusion. AMOVA detected more variation among cultivated and related wild species of almond within each geographic group. Bootstrap analysis revealed that the number of markers used was sufficient for reliable estimation of genetic similarity and for meaningful comparisons of marker types.

Genetic Diversity Analysis of Cultured Populations of Jade Perch （Scortum bacoo） in China Using AFLP Markers

Genetic diversity of three cultured populations of jade perch （Scortum bacoo） are studied using amplified fragment length polymorphism （AFLP） markers, which is Guangzhou（GZ） population, Foshan （FS） population and Qingdao（QD） population. Nine primer combinations were used and 385 fragments were detected. Among the 385 fragments, 80 bands （20.78%） were polymorphic. And it can be speculated that the genetic diversity of the three cultured populations of jade perch was very poor according to the gene genetic diversity among populations （Ht）, the gene genetic diversity within populations （Hs） and Shannon-Weiner index （I）. From gene differentiation （Gst）, genetic distance （D）, genetic similarity （5）, and UPGMA analysis, it is found that the relationship among the three populations is very closed, and the difference in genetic diversity among the three populations is not significant. Further clustering relationships of the jade perch cultured populations also are correlated to historical-breeding observations and genetic relationships. The GZ population clustered together with the QD population first, then with FS population.

AFLP analysis of genetic variation among three natural populations of horseshoe crab Tachypleus tridentatus along Chinese coast

The AFLP(amplified fragment length polymorphism) technique was used to analyze and compare the genetic diversity of Tachypleus tridentatus from three south-eastern coastal sites of China(Pingtan,Hong Kong and Beihai).Eight pairs of primers generated 361 loci,including 285 polymorphic loci.The ratio of polymorphic loci was 96.97%.Nei's genetic diversity index was 0.420 8 and the Shannon information index was 0.607 5,both of which were higher than that reported for many other arthropods.These results show that the genetic diversity detected was mainly caused by individual differences within a population.Genetic distance showed that the rational division of the three geographic populations of T.tridentatus along the south-eastern coast of China was not significant,in which the genetic distance was not proportional to the geographic distance.All three horseshoe crab populations may belong to a large group,and had a high degree of genetic similarity.The high level of genetic diversity obtained from the present AFLP analysis may be due to the large effective population size of the species in Chinese waters.

Microwave induced mutagenesis of Lactobacillus casei subsp. rhamnosus for enhancing L-lactic acid production

Mutagenesis of Lactobacillus casei subsp, rhamnosus Xl-12 after low power microwave irradiation was investigated. Under a microwave power of 400 W and irradiation length of 3 min, a mutated strain W4-3-9 with high-yield L-lactic acid was obtained by screening. Compared with the starting strain X1-12, the L-lactic acid production of W4-3-9 was increased by 58.0% at a concentration of 115.8 g/L. The strain maintained the capability of producing a high L-lactic acid level after 10 generations. Cell surface morphology and DNA structures of parental and mutated strains were observed by atomic force microscopy （ AFM ）. Amplified fragment length polymorphism （AFLP） analysis suggested the difference in AFLP band pattern between the mutated and non-mutated strains. Sequencing and BLAST analysis revealed that the catalytic site of lactate dehydrogenase （DHL） was changed due to the microwave induced mutation.

Significant population genetic structure detected in the rock bream Oplegnathus fasciatus(Temminck & Schlegel,1844) inferred from fluorescent-AFLP analysis

Oplegnathusfasciatus （rock bream） is a commercial rocky reef fish species in East Asia that has been considered for aquaculture. We estimated the population genetic diversity and population structure of the species along the coastal waters of China using fluorescent-amplifed fragment length polymorphisms technology. Using 53 individuals from three populations and four pairs of selective primers, we amplified 1 264 bands, 98.73% of which were polymorphic. The Zhoushan population showed the highest Nei＇s genetic diversity and Shannon genetic diversity. The results of analysis of molecular variance （AMOVA） showed that 59.55% of genetic variation existed among populations and 40.45% occurred within populations, which indicated that a significant population genetic structure existed in the species. The pairwise fixation index Fst ranged from 0.20 to 0.63 and were significant after sequential Bonferroni correction. The topology of an unweighted pair group method with arithmetic mean tree showed two significant genealogical branches corresponding to the sampling locations of North and South China. The AMOVA and STRUCTURE analyses suggested that the O.fasciatus populations examined should comprise two stocks.

Genomic and proteomic analysis of soybean heritable variations induced by space flight

To analyze the biological effects of space environment,the diversity of genomic DNA between the space flight soybean 194(4126) with phenotype of good yield and good fruit quality induced by space flight and the soybean with ground control was studied by amplified fragment length polymorphism(AFLP) method,and the polymorphism of space flight soybean 194(4126) was 3.56%.The differences of protein expression of seeds and leaves between the two kinds of soybeans were analysed by two-dimensional electrophoresis,PDQuest software and MALDI-TOF mass spectrometry.Results show that the loss and decrease of protein expression in 194(4126) soybean are subjected to the space fight of seeds,and three special proteins including Dehydrin,MAT1 and ceQORH are identified.It is concluded that the space environment changes the phenotype and genotype of soybeans due to the space flight of seeds.