Simple sequence repeat marker diversity in cassava landraces: genetic diversity and differentiation in an asexually propagated crop

Cassava (Manihot esculenta) is an allogamous, vegetatively propagated, Neotropical crop that is also widely grown in tropical Africa and Southeast Asia. To elucidate genetic diversity and differentiation in the crop's primary and secondary centers of diversity, and the forces shaping them, SSR marker variation was assessed at 67 loci in 283 accessions of cassava landraces from Africa (Tanzania and Nigeria) and the Neotropics (Brazil, Colombia, Peru, Venezuela, Guatemala, Mexico and Argentina). Average gene diversity (i.e., genetic diversity) was high in all countries, with an average heterozygosity of 0.5358 ± 0.1184. Although the highest was found in Brazilian and Colombian accessions, genetic diversity in Neotropical and African materials is comparable. Despite the low level of differentiation [F_st(theta) = 0.091 ± 0.005] found among country samples, sufficient genetic distance (1-proportion of shared alleles) existed between individual genotypes to separate African from Neotropical accessions and to reveal a more pronounced substructure in the African landraces. Forces shaping differences in allele frequency at SSR loci and possibly counterbalancing successive founder effects involve probably spontaneous recombination, as assessed by parent-offspring relationships, and farmer-selection for adaptation.Communicated by H.C. Becker     

Genetic diversity of SSR markers in wild populations of Tapiscia sinensis,an endangered tree species

Tapiscia sinensis is a Tertiary relict and endangered tree species with unique scientific research value and great economic value. In this study, we assessed the genetic diversity of five wild T. sinensis populations from different geographical regions using 10 polymorphic simple sequence repeat (SSR) markers. Our results reveal that the natural populations of T. sinensis have rich genetic diversity (PPL = 100%, He = 0.6904, I = 1.4368), with Shannon's index indicating that the T. sinensis populations are at a relatively stable stage. Of the genetic relationships among populations, the distance between the Hunan Yanling (YL) and Guizhou Xifeng (XF) populations is the smallest (0.4829); the genetic distance between the Shaanxi Ningshan (NS) and the Guizhou Xifeng (XF) populations is the largest (0.9821). A Mantel test shows that there is no correlation among the populations between geographic distance and genetic distance. AMOVA suggest that 33.3% of the genetic variation arose among the populations, while 66.7% of the variation arose within them. The moderate gene flow among populations (Nm = 0.7274) is not sufficient to counteract genetic drift within the populations and result in significant differentiation (Fst = 0.2987). Our results will benefit the conservation and exploitation of T. sinensis and provide a theoretical basis for further study of the evolution and phylogeography of the species.     

羽叶金合欢的DNA提取和SSR引物筛选

利用SSR分子标记对羽叶金合欢( Acacia pennata )的野生种和栽培种进行了分析,利用改良的CTAB方法优化了其总DNA的提取方法,并优化了PCR扩增条件.从已有的金合欢属植物的82对SSR引物中筛选出多态性高,稳定性好的12对引物作为羽叶金合欢的SSR分析引物,为进一步对其进行遗传多样性研究提供了依据.     

Cotton genome mapping with new microsatellites from Acala ‘Maxxa’ BAC-ends

Fine mapping and positional cloning will eventually improve with the anchoring of additional markers derived from genomic clones such as BACs. From 2,603 new BAC-end genomic sequences from Gossypium hirsutum Acala ‘Maxxa’, 1,316 PCR primer pairs (designated as MUSB) were designed to flank microsatellite or simple sequence repeat motif sequences. Most (1164 or 88%) MUSB primer pairs successfully amplified DNA from three species of cotton with an average of three amplicons per marker and 365 markers (21%) were polymorphic between G. hirsutum and G. barbadense. An interspecific RIL population developed from the above two entries was used to map 433 marker loci and 46 linkage groups with a genetic distance of 2,126.3 cM covering approximately 45% of the cotton genome and an average distance between two loci of 4.9 cM. Based on genome-specific chromosomes identified in G. hirsutum tetraploid (A and D), 56.9% of the coverage was located on the A subgenome while 39.7% was assigned to the D subgenome in the genetic map, suggesting that the A subgenome may be more polymorphic and recombinationally active than originally thought. The linkage groups were assigned to 23 of the 26 chromosomes. This is the first genetic map in which the linkage groups A01 and A02/D03 have been assigned to specific chromosomes. In addition the MUSB-derived markers from BAC-end sequences markers allows fine genetic and QTL mapping of important traits and for the first time provides reconciliation of the genetic and physical maps. Limited QTL analyses suggested that loci on chromosomes 2, 3, 12, 15 and 18 may affect variation in fiber quality traits. The original BAC clones containing the newly mapped MUSB that tag the QTLs provide critical DNA regions for the discovery of gene sequences involved in biological processes such as fiber development and pest resistance in cotton. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

基于免疫磁分离的三重荧光定量PCR检测食品中沙门氏菌、志贺氏菌和金黄色葡萄球菌

【目的】开发一种同时对食品中沙门氏菌、志贺氏菌和金黄色葡萄球菌快速、灵敏、准确的检测方法。【方法】利用特异性免疫磁球,在37°C条件下从250 m L猪肉增菌液体系中边富集边循环捕获目标菌。快速提取DNA后,利用特异性的引物与探针,对3种食源性致病菌进行三重荧光定量PCR检测。【结果】针对沙门氏菌、志贺氏菌和金黄色葡萄球菌的检测限分别达到2.0、6.8和9.6 CFU/g。方法总体灵敏度、特异性和准确度达到99.2%、100%及99.5%。对151份实际样品进行检测,与国标(GB/T 4789.4-2010、GB 4789.5-2012和GB/T4789.10-2010)方法的检测结果相比,金黄色葡萄球菌有一例阴性偏差。【结论】开发的基于免疫磁分离的三重荧光定量PCR方法,能够在8 h内完成对食品中3种致病菌检测,并且灵敏度高、特异性好、检测准确,可以作为快速应对此类食品安全突发事件的检测手段。     

检测PCV2、PPV、PRV疫苗株与野毒株的多重PCR方法

本文建立了一种同时检测猪圆环病毒2型(PCV2)、细小病毒(PPV)、及伪狂犬病毒(PRV)疫苗株与野毒株的多重PCR方法.根据GenBank上发表的PCV2、PPV和PRV gB、gE基因序列,针对各自保守区各设计一对特异性引物,用这四对引物对同一样品中的PCV2、PPV和PRV gB、gE进行检测,结果可同时扩增出269bp(PCV2)、581bp(PPV)、372bP(PRV gB)及147bp(PRV gE)四条特异性片段.对JEV、PRRSRV、大肠杆菌和双蒸水的PCR扩增结果均为阴性;敏感性测定结果表明,该多重PCR能检出10pg PCV2、PPV和PRV gB、gE检测敏感度分别为10-6.2、10-3.8、10-5.8TCID50的模板.该方法的建立对临床上进行这三种疾病的鉴别诊断和混合感染的检测具有重要意义.     

全细胞多重PCR检测蓝藻、微囊藻及产毒微囊藻方法初探

选取三对分别针对微囊藻、蓝藻16S rDNA及微囊藻毒素合成酶基因mcyB的保守序列的特异性引物209F/409R、27F1/409R、MTR/MTF,其中409R为一条共用引物。设计并优化了一种可以同时检测蓝藻和微囊藻的两重全细胞PCR方法和一种可以同时检测蓝藻、微囊藻和可产毒微囊藻的三重全细胞PCR方法,并且测试了这两种PCR反应的灵敏度区间,分别为10⁵～10³cell·mL^-1、10⁵～10²cell·mL^-1。对采集水库水样检测结果表明双重全细胞PCR方法可以直接应用于对天然水样的检测,三重全细胞PCR方法可用于实验室培养藻细胞的筛查。全细胞多重PCR方法具有快速、简便、准确等特点,在水体微囊藻毒素检测预警方面具有应用价值。     

珍稀濒危树种毛红椿微卫星DNA分离及SSR反应体系优化

本研究以江西宜丰种源毛红椿为材料,成功提取其基因组DNA。利用改良的链亲和素磁珠法亲和捕捉出毛红椿基因组微卫星DNA片断,并构建了富含微卫星的基因组文库。从构建的基因组文库中随机挑选了63个单克隆进行测序,其中50个单克隆成功测序,含有微卫星的单克隆有18个,并根据测序结果设计并合成SSR引物18对。利用所合成的引物优化SSR反应体系,对影响SSR反应的各个因子进行了探讨。确定了模板DNA浓度最适浓度为30ng;dNTP的最适浓度为0.3mmol·L-1;0.3μmol·L-1是引物在反应体系中的最合适浓度。建立了重复性好、稳定性好的SSR反应体系,为下一步进行毛红椿群体遗传结构和遗传变异研究提供了技术支持。     

A cherry map from the inter-specific cross Prunus avium ‘Napoleon’ × P. nipponica based on microsatellite, gene-specific and isoenzyme markers

One hundred and sixty microsatellite (simple sequence repeat (SSR)) and six gene-specific markers revealing 174 loci were scored in 94 seedlings from the inter-specific cross of Prunus avium ‘Napoleon’ × Prunus nipponica accession F1292. The co-segregation data from these markers were used to construct a linkage map for cherry which spanned 680 cM over eight linkage groups with an average marker spacing of 3.9 cM per marker and just six gaps longer than 15 cM. Markers previously mapped in Prunus dulcis ‘Texas’ × Prunus persica ‘Earlygold’ allowed the cherry map to be anchored to the peach × almond map and showed the high level of synteny between the species. Eighty-four loci segregated in P. avium ‘Napoleon’ versus 159 in P. nipponica. The segregations of 32 isoenzyme loci in a subset of 47 seedlings from the progeny were scored, using polyacrylamide gel electrophoresis and/or isoelectric focusing separation followed by activity staining, and the co-segregation data were analysed along with those for 39 isoenzymes reported previously and for the 174 sequence-tagged site loci plus an additional two SSR loci. The second map incorporates 233 loci and spans 736 cM over eight linkage groups with an average marker spacing of 3.2 cM per marker and just two gaps greater than 15 cM. The microsatellite map will provide a useful tool for cherry breeding and marker-assisted selection and for synteny studies within Prunus; the gene-specific markers and isoenzymes will be useful for comparisons with maps of other rosaceous fruit crops. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.