R-ISSR as a new tool for genomic fingerprinting, mapping, and gene tagging

In the present study we propose and test the concept of R-ISSR, a new tool for genomic fingerprinting, mapping, and gene tagging. The concept is based on the fact that primers for inter-simple sequence repeat (ISSR) and random-amplified polymorphic DNA (RAPD) analysis elicit different genomic information, and the combined use of these 2 kinds of primers in the same polymerase chain reaction (PCR) reactions might reveal new genomic loci that could not be detected with either technique alone. The feasibility of this tool was first electronically simulated with sequence analysis software andArabidopsis chromosome sequence. Next, different combinations of ISSR and RAPD primers were applied in real PCR reactions to detect new genomic loci in 2 maize lines (Q319 and 1145). Sequencing gels were used to separate PCR products and showed good resolving ability in comparison with agarose gels. RAPD primers could be successfully used with ISSR primers for the detection of new genomic loci and applied in a new way for genomic mapping, fingerprinting, and gene tagging.     

Development and characterization of microsatellite markers from the yellow passion fruit (Passiflora edulis f. flavicarpa)

Here we described the development of the first set of Passiflora microsatellite loci isolated from an enriched genomic library. A sample of 43 individuals from 12 accessions of the yellow passion fruit was used to characterize those loci, which revealed up to 20 alleles per locus. Two loci were monomorphic. The observed (H_O) and expected (H_E) heterozygosities were very similar, as expected for a self‐incompatible species. Allelic diversity (H_T) was 0.444. This set of markers will permit genetic structure analyses of cultivated and wild populations of Passiflora, and contribute for integrating genetic maps based on dominant markers, as they can provide bridge alleles.     

利用SSR分子标记进行海岛棉遗传多样性研究

利用SSR分子标记,对20世纪50年代我国引入海岛棉以来培育的45个国内品种(系)及8个国外品种的遗传多样性进行研究.通过256对SSR引物的筛选,选择24对扩增效果好的引物对53个海岛棉种质资源进行遗传多样性的检测分析,共检测出106个等位位点,每对引物等位位点数在2～8之间,平均为4.4.其中多态性等位基因变异97个,占91.5%.位点多态性信息含量平均为0.688,最高为0.848,最低为0.245.利用NTSYSpc2.1软件,分别计算农艺经济性状的欧氏距离(Euclid)和分子标记数据的Jaccard系数矩阵,采用UPGMA法对所选材料进行聚类分析.结果表明,两个树状聚类图基本吻合,53个品种被分为两大类,与系谱来源一致.实验证明SSR分子标记在鉴别品种和品种遗传多样性研究方面具有重要作用.     

水稻早世代稳定相关的SSR标记

以具有早世代稳定特性的9个水稻品系和7个栽培稻进行杂交得到130个杂交组合。发现在同一个组合的F2群体中除出现孟德尔式分离株系外还出现性状整齐一致的稳定株系,在F2群体中出现了32个不同农艺性状的稳定株系。SSR标记结果表明：稳定株系F2与其F1单株的标记一致,都是纯合的,且双亲的标记在后代都有出现,双亲的标记位点在后代的染色体上同时出现,说明稳定株系为真杂种;分离株系F2与其F1单株SSR标记均为杂合;大部分稳定株系在RM276、RM258、RM248、RM1这4个标记位点都较高频率地出现非父母带型。推测出现变异的配子激活杂种合子体细胞减数分裂,合子经过减数分裂后,4个单倍体细胞经过胚胎发育选择,其中一个细胞染色体加倍而发育为纯合的二倍体植株。     

矮泰引-3中半矮秆基因的分子定位

矮泰引-3的矮生性状受两对独立遗传的半矮秆基因控制,利用SSR标记将这两个矮秆基因分别定位到第1和第4染色体上。等位性测交的结果表明,位于第1染色体上的矮秆基因与sd1是等位的,所以仍然称其为sd1;而位于第4染色体上的矮秆基因是一个新基因,暂命名为sdt2。利用SSR标记将sd1定位于RM297、RM302和RM212的同一侧,而与OSR3共分离,它们之间的位置关系可能是RM297-RM302-RM212-OSR3-sd1,遗传距离分别为4．7cM、0cM、0．8cM和0cM,这与sd1在第1染色体长臂上的确切位置是基本一致的。利用已有的SSR标记和拓展的SSR标记将sdt2定位于SSR332、RM1305和RM5633、RM307、RM401之间,它们的排列位置可能是SSR332-RM1305-sdt2-RM5633-RM307-RM401,它们之间的遗传距离分别为11．6cM、3．8cM、0．4cM、0cM和0．4cM。     

Structure of Pinus sylvestris L. populations in Bulgaria revealed by chloroplast microsatellites and terpenes analysis: Provenance tests

In the present study we investigated the genetic structure and genetic diversity of Pinus sylvestris populations in Bulgaria using chloroplast microsatellite markers and terpene analysis. We were interested in addressing the following questions: (1) can population structure in Scots pine be detected via chloroplast microsatellites markers and terpenes; (2) are there differences in population differentiation between the two analyses; and (3) how are the patterns related to geographic distances. Twelve provenances were chosen throughout the species' range in Bulgaria. Following DNA extraction, chloroplast microsatellite (cpSSR) loci were surveyed using six primer pairs. Between 4 to 8 size variants were identified at each locus. A total of 35 size variants at the six loci were identified, 11 occurring at low frequencies (<1%). They were combined in 134 different haplotypes, of which seven represent 1/3 of the genetic structure. AMOVA analysis revealed that 10.99% of the variation was found among populations, while 89.01% was expressed within populations. The cpSSR analysis divided Scots pine populations into two groups, the first represented by populations located in the south-western part of the Rhodopes and Pirin mountains, while the second group is located in the northeast of Rhodopes and Rila mountains. Terpene analysis revealed that on average, 53% of the monoterpene pool in P. sylvestris was accounted for by -pinene (range 47–59%) followed by β-pinene (range 6–12%). The presence of two distinct groups is weekly consistent with physical distances between populations, similar significant correlation between genetic distance determined by chloroplast microsatellites analysis and chemotype distance (determined by terpenes) was observed. Our results suggest that the structural pattern of genetic diversity of cpDNA in Scots pine populations is the consequence of historical biogeographic processes.     

Patterns of morphological and genetic diversity of Valeriana jatamansi Jones in different habitats and altitudinal range of West Himalaya,India

Importance to know and understand diversity of Himalayan plants is increasingly recognized considering the fact that various natural and anthropogenic pressures might bring about serious influences to morphological and genetic diversity of the vegetation in the region. In this context, Valeriana jatamansi was investigated in detail, taking into account its importance in various Ayurvedic and modern medicines. Randomly selected mature plants from twenty five different populations (located between 1215 m to 2775 m asl) of V. jatamansi were analysed for their morphological attributes. Further, ISSR markers were used to detect genetic variation among 151 plants of selected 25 populations. Use of 20 primers yielded 125 reproducible polymorphic loci which were used to estimate different parameters of genetic diversity. These parameters were in turn applied to develop relationships with habitat types and altitude range. Significant variation (p < 0.05) in above ground dry weight (AGDW) and below ground dry weight (BGDW) across the populations was observed. Nei's genetic diversity index (He) ranged from 0.25 to 0.37 across the populations, with a mean of 0.31. Genetic diversity exhibited a decreasing trend with increasing altitude, and maximum diversity (He = 0.325) was observed in the range of 1201–1500 m asl. Among the different habitat conditions, highest genetic diversity (He = 0.334; Pp = 84.38) was observed in grassland habitats while minimum in mixed forest habitats (He = 0.285; Pp = 72.433). The genetic diversity (He) had significant negative relationships with AGDW, BGDW and rhizome diameter (Pearson r = −0.359, −0.424 and −0.317, respectively; p < 0.05). The genetic characterization of V. jatamansi from the western Himalaya by this study suggests influences of habitat types and the altitudinal range upon genetic diversity, and based on these proposals for conservation strategies in favour of the species are made.     

Morphological and molecular identification to secure cultivar maintenance and management of self-sterile Rubus arcticus

Background and Aims

Preservation of cultivar purity creates a particular challenge for plants that are self-incompatible, require insects for cross-pollination, and have easily germinating seeds and vigorously spreading rhizomes. As the fields must be planted with mixed populations, and a balance must be maintained between the cultivars to achieve effective pollination, methods for field monitoring of the relative density of different cultivars must be practical. Furthermore, a DNA-based method is needed for cultivar verification in the collections and outside of the growing season. The aim of this study was to develop both types of methods for Rubus arcticus (arctic bramble).

Methods

Morphological parameters were measured from six cultivars grown on three farms. Observations from the flowers and fruits included: petal and sepal number, flower diameter, arrangement of petals, size of calyx in relation to corolla, fruit weight, yield and soluble sugars. Observations from the leaves included: width and height of middle leaflet, shape of the base of terminal leaflet, shape of terminal leaflet, leaf margin serration and fingertip touch. The applicability of simple sequence repeat (SSR) or microsatellite DNA markers developed for red raspberry was tested on eight arctic bramble cultivars.

Key Results and Conclusions

Morphological and molecular identification methods were developed for R. arcticus. The best morphological characteristics were the length-to-width ratio of the middle leaflet and leaf margin serration. A particular characteristic, fingertip touch, was shown by electron microscopy to be related to the density and quality of the leaf hairs. Red raspberry SSR marker no. 126 proved to be applicable for differentiation of the eight arctic bramble cultivars tested. These identification methods are critical to secure the maintenance and management of R. arcticus. However, the challenges faced and approaches taken are equally applicable to other species with similar biology.     

Recombinase-mediated cassette exchange (RMCE) — A rapidly-expanding toolbox for targeted genomic modifications

Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After “gene swapping” the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided (“serial RMCE”). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites (“heterospecific FRT-doublets”), into the LTRs of lentiviral vectors. These “twin sites” enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single “FRT-twin”. Such a strategy combines the established expression potential of the common retro- and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic “safe harbors” (GOIs) for re-use. This is enabled by the capacity of “FRT-twins” to accommodate any incoming RMCE-donor cassette with a compatible design.     

Signatures of diversifying selection at EST‐SSR loci and association with climate in natural Eucalyptus populations

Understanding the environmental parameters that drive adaptation among populations is important in predicting how species may respond to global climatic changes and how gene pools might be managed to conserve adaptive genetic diversity. Here, we used Bayesian F_ST outlier tests and allele–climate association analyses to reveal two Eucalyptus EST‐SSR loci as strong candidates for diversifying selection in natural populations of a southwestern Australian forest tree, Eucalyptus gomphocephala (Myrtaceae). The Eucalyptus homolog of a CONSTANS‐like gene was an F_ST outlier, and allelic variation showed significant latitudinal clinal associations with annual and winter solar radiation, potential evaporation, summer precipitation and aridity. A second F_ST outlier locus, homologous to quinone oxidoreductase, was significantly associated with measures of temperature range, high summer temperature and summer solar radiation, with important implications for predicting the effect of temperature on natural populations in the context of climate change. We complemented these data with investigations into neutral population genetic structure and diversity throughout the species range. This study provides an investigation into selection signatures at gene‐homologous EST‐SSRs in natural Eucalyptus populations, and contributes to our understanding of the relationship between climate and adaptive genetic variation, informing the conservation of both putatively neutral and adaptive components of genetic diversity.