Development of retrotransposon primers and their utilization for germplasm identification in Diospyros spp. (Ebenaceae)

Retrotransposons play an important role in plant genetic instability and genome evolution. Retrotransposon-based molecular markers are valuable tools to reveal the behavior of retrotransposons in their host genome. In this study, suppression polymerase chain reaction was used, for the first time, to develop retrotransposon long terminal repeat (LTR) and polypurine tract (PPT) primers in Japanese persimmon (Diospyros kaki Thunb.), which were then employed for germplasm identification by means of interretrotransposon-amplified polymorphism (IRAP), sequence-specific amplified polymorphism (SSAP) and retrotransposon-microsatellite-amplified polymorphism (REMAP) molecular markers. The results showed that 16 out of 26 primers produced expected amplifications and abundant polymorphisms by IRAP in 28 genotypes of Diospyros. Moreover, some of these primers were further successfully used in REMAP and SSAP analysis. Each type of molecular markers produced unique fingerprint in 28 genotypes analyzed. Among the primers/primer combinations, two IRAP primers and four SSAP primer combinations could discriminate all of the germplasm solely. Further comparative analysis indicated that IRAP was the most sensitive marker system for detecting variability. High level of retrotransposon insertion polymorphisms between bud sports were detected by IRAP and SSAP, and the primers/primer combinations with powerful discrimination capacity for two pairs of bud sports lines were further obtained. Additionally, possible genetic relationships between several Japanese persimmon were discussed. To our knowledge, this is the first report on the development of retrotransposon LTR and PPT primers in Diospyros, and the retrotransposon primers developed herein might open new avenue for research in the future.     

Development of mapped simple sequence repeat markers from common bean (Phaseolus vulgaris L.) based on genome sequences of a Chinese landrace and diversity evaluation

Microsatellite or single sequence repeat (SSR) markers have been commonly used in genetic research in many crop species, including common bean (Phaseolus vulgaris L.). A limited number of existing SSR markers have been designed from high-throughput sequencing of the genome, warranting the exploitation of new SSR markers from genomic regions. In this paper, we sequenced total DNA from the genotype Hong Yundou with a 454-FLX pyrosequencer and found numerous SSR loci. Based on these, a large number of SSR markers were developed and 90 genomic-SSR markers with clear bands were tested for mapping and diversity detection. The new SSR markers proved to be highly polymorphic for molecular polymorphism, with an average polymorphism information content value of 0.44 in 131 Chinese genotypes and breeding lines, effective for distinguishing Andean and Mesoamerican genotypes. In addition, we integrated 85 primers of the 90 polymorphism markers into the bean map using an F2 segregating population derived from Hong Yundou crossed with Jingdou. The distribution of SSR markers among 11 chromosomes was not random and tended to cluster on the linkage map, with 14 new markers mapped on chromosome Pv01, whereas only four loci were located on chromosome Pv04. Overall, these new markers have potential for genetic mapping, genetic diversity studies and map-based cloning in common bean.     

柿树炭疽菌侵染不同柿树种、品种和部位的细胞学特征

用柿树炭疽病菌Colletotrichumgloeosporioides的分生孢子制备孢子悬浮液,接种无核柿、野柿、冬柿和浙江柿的新梢、叶柄和叶片,并观察致病性、附着胞形成和侵染特性。柿树炭疽菌可以侵染无核柿枝条和叶柄以及野柿枝条,但不侵染无核柿叶片、野柿叶柄和叶片,也不侵染冬柿和浙江柿枝条、叶柄和叶片。室内接种试验与田间自然发病结果一致。柿树炭疽菌在不同柿树表面均能形成附着胞,附着胞产生在寄主表皮背斜细胞壁间结合处(JACWs)或近结合处的百分率达81%~93%。接种12h后,不同柿树表面都有附着胞形成;36h后,无核柿枝条、叶柄中有侵染菌丝存在;48h后,无核柿枝条、叶柄中观察到膨大初生菌丝和较细次生菌丝,初生菌丝可扩展到相邻细胞中,而野柿枝条中仅观察到侵染菌丝;60h后,野柿枝条中也观察到膨大的初生菌丝和较细的次生菌丝,但初生菌丝仅局限在最初侵染的细胞中,无核柿枝条和叶柄以及野柿枝条中都有分枝的次生菌丝在细胞内、细胞间或相邻的细胞中扩展;直到接种90h后,在冬柿和浙江柿上都未观察到侵染菌丝的形成。结果表明,柿树炭疽菌在不同柿树种和品种上侵染菌丝的形成和扩展方式可能是其寄主专化性(或致病性)差异的重要机制之一。     

基于叶绿体DNA ndhA和nrDNA ITS序列变异分析栽培柿及其近缘种亲缘关系

以柿属植物（Diospyros spp.）中与柿近缘的8种共30个基因型为试材,进行核糖体DNA(nrDNA)内转录间隔区（ITS）和叶绿体DNA ndhA序列变异分析,并通过软件计算两个序列及合并后的进化模型,依据进化模型采用ML法(maximum likelihood method)分析进化关系。为进一步弄清柿属植物种间亲缘关系和供试柿（Diospyros kaki Thunb.）种内分子差异提供了理论依据。结果表明：（1）ndhA序列长度变异范围在1 492~1 511,14个信息位点;ITS序列长度变异范围在660~761,56个信息位点。ITS、ndhA和ndhA+ITS（ndhA和ITS合并）最适碱基进化模型分别为（TrN+I+G）、（F81+I）和（GTR+I+G）。综合ITS和ndhA序列分析表明：柿与油柿和云南野毛柿亲缘关系最近,与美洲柿和乌柿最远。（2）21份柿品种材料的ITS长度均为730,包括4个变异位点,据此4个变异位点对供试柿种内21个品种进行聚类分析。研究认为,ndhA和ITS能较清楚解释了柿与其近缘种间的亲缘关系,并通过柿品种ITS的差异位点分析鉴别出栽培柿种内的差异。     

Regulation of the peripheral body temperature by foods: a temperature decrease induced by the Japanese persimmon (kaki,Diospyros kaki)

We investigated whether the ingestion of the Japanese persimmon (kaki, Diospyros kaki) could lower the human peripheral body temperature. It was found that the temperatures recorded at the foot and wrist were depressed after kaki consumption compared to after the same amount of water consumption. The effects of ingesting freeze-dried kaki and eating a cookie (as its nutritional counterpart) containing the same amount of carbohydrate, protein, fat, and water were compared. A similar temperature-reducing effect of kaki was observed. The recovery of finger temperature after soaking the finger in ice-cooled water was also studied. The temperature recovery was delayed after kaki consumption. It was thus quantitatively demonstrated that ingesting kaki indeed had the effect of lowering (or repressing the rise) of the peripheral human body temperature, as has been traditionally believed in China for many hundreds of years.     

Genetic relationships of Japanese and Indian mulberry (Morus spp.) genotypes revealed by DNA fingerprinting

Mulberry (Morus spp.), a deciduous tree, originated at the foothills of the Himalayas and is used in sericulture for its leaf to feed the silkworm Bombyx mori L. Species differentiation among the genotypes of the genus Morus has never been out of debate as inter-specific hybridization events are often fertile. In the present study attempts were made to elucidate the genetic relationships among 18 mulberry genotypes collected from India and Japan using 15 Inter Simple Sequence Repeat (ISSR) and 15 Random Amplified Polymorphic DNA (RAPD) primers. The ISSR primers generated 81.13% polymorphism while the RAPDs generated 71.78% polymorphism. The polymorphic index of the primers identified UBC-812, UBC-826, UBC827, UBC-881, OPA-01, OPA-02, OPA-04 and OPH-17 as informative primers in mulberry. The genetic similarity coefficients and the dendrograms showed considerable genetic similarity among the genotypes. However, using the DNA markers, these genotypes were discriminated into two major groups in accordance with their geographic origin and species status. Distribution of the genotypes on a two-dimensional figure on the basis of the ALSCAL algorithm using Euclidean distance further confirmed the genetic divergence between these two groups. From the study it can be concluded that though morphologically Japanese and Indian mulberry genotypes show little divergence, genetic analysis using DNA markers could unravel significant genetic variation between these two groups. Similarly, while the species status of Japanese mulberry genotypes agrees with the genetic analysis, the same does not apply to Indian genotypes, in agreement with many earlier reports. The information generated in this study is of much use for taxonomical grouping and also for utilization in breeding and conservation programs.     

Isolation and expression of gene encoding leucoanthocyanidin reductase from <Emphasis Type="Italic">Diospyros kaki</Emphasis> during fruit development

Leucoanthocyanidin reductase (LAR) converts leucoanthocyanidin to (+)-catechin, a precursor of proanthocyanidins abundant in Japanese persimmon (Diospyros kaki Thunb.) fruits. A putative LAR gene (DkLAR) was isolated by rapid amplification of cDNA ends from young fruits. The full-length cDNA of DkLAR gene was 1 356 bp long and encoded an open reading frame of 349 residues. The deduced DkLAR protein was closely related to the homolog in other plant species. The expression of the DkLAR gene in Chinese pollination-constant non-astringent (PCNA) genotype was coincident with the tannin cell development, but was not in Japanese PCNA and Chinese pollination-variant astringent (PCA) genotypes.     

Analysis of Genetic Diversity in Cicer arietinum L Using Random Amplified Polymorphic DNA Markers

PCR-based random amplified polymorphic DNA (RAPD) markers were employed to assess genetic diversity in 23 chickpea genotypes. Forty of the 100 random primers screened revealed polymorphism among the genotypes. Most of the primers revealed single polymorphic band, and only 14.1 2% of the products were polymorphic. Estimates of genetic similarity based on Jaccard’s coefficient ranged from 0.92 to 0.99, indicating narrow genetic variability among the genotypes based on RAPD markers.The 23 chickpea genotypes formed two major clusters in the dendrogram.The low RAPD polymorphism among chickpea genotypes suggests that more number of polymorphic primers need to be analysed to determine genetic relationships. It was observed that RAPD analysis employing 30 polymorphic primers could provide better estimates of genetic relationships in chickpea.     

Development and characterization of EST-derived simple sequence repeat (SSR) markers for pasture grass endophytes.

Fungal endophytes of the genus Neotyphodium are common in temperate pasture grass species and confer both beneficial and deleterious agronomic characteristics to their hosts. The aim of this study was to develop molecular markers based on simple sequence repeat (SSR) loci for the identification and assessment of genetic diversity among Neotyphodium endophytes in grasses. Expressed sequence tags (ESTs) from both Neptyphodium coenophialum and Neotyphodium lolii were examined, and unique SSR loci were identified in 9.7% of the N. coenophialum sequences and 6.3% of the N. lolii sequences. A variety of SSRs were present, although perfect trinucleotide repeat arrays were the most common. Primers were designed to 50 SSR loci from N. coenophialum and 57 SSR loci from N. lolii and were evaluated using 20 Neotyphodium and Epichlo? isolates. A high proportion of the N. coenophialum and N. lolii primers produced amplification products from the majority of isolates and most of these primers detected genetic variation. SSR markers from both N. coenophialum and N. lolii detected high levels of polymorphism between Neotyphodium and Epichlo? species, and low levels of polymorphism within N. coenophialum and N. lolii. SSR markers may be used in appropriate combinations to discriminate between species. Comparison with amplified fragment length polymorphism (AFLP) data demonstrated that the SSR markers were informative for the assessment of genetic variation within and between endophyte species. These markers may be used to identify endophyte taxa and to evaluate intraspecific population diversity, which may be correlated with variation for endophyte-derived agronomic traits.     

Genetic diversity and taxonomic studies of date plum (Diospyros lotus L.) using morphological traits and SCoT markers

Date plum persimmon (Diospyros lotus L.) is an important fruit crop in China due to its better compatibility as a major root stock for Diospyros kaki Thunb. The objective of this study was to reveal the diversity and taxonomic status based on morphological and molecular markers for 26 date plum persimmon accessions from different ecogeographical areas. Significant variability was observed in the 26 date plum persimmon accessions using the analysis of 5 quantitative and 16 qualitative morphological characters of branches, fruits and seeds. Molecular diversity was analyzed using start codon targeted (SCoT) markers. A total of 118 bands were produced with 11 SCoT primers; genetic similarity was ranged from 0.42 to 0.83, which indicating higher genetic variation among accessions. Based on the cluster results of morphological and molecular data, the results showed that date plum persimmon may be divided into five taxonomic types which including male date plum persimmon, rounded seedless date plum persimmon, rounded seedy date plum persimmon, obround seedless date plum persimmon, and oblate seedy date plum persimmon.     

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.     

Comparative evaluation of genetic diversity using RAPD,SSR and cytochrome P450 gene based markers with respect to calcium content in finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> L. Gaertn.)

Genetic relationships among 52 Eleusine coracana (finger millet) genotypes collected from different districts of Uttarakhand were investigated by using randomly amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and cytochrome P450 gene based markers. A total of 18 RAPD primers, 10 SSR primers, and 10 pairs of cytochrome P450 gene based markers, respectively, revealed 49.4%, 50.2% and 58.7% polymorphism in 52 genotypes of E. coracana. Mean polymorphic information content (PIC) for each of these marker systems (0.351 for RAPD, 0.505 for SSR and 0.406 for cyt P450 gene based markers) suggested that all the marker systems were effective in determining polymorphisms. Pair-wise similarity index values ranged from 0.011 to 0.999 (RAPD), 0.010 to 0.999 (SSR) and 0.001 to 0.998 (cyt P450 gene based markers) and mean similarity index value of 0.505, 0.504 and 0.499, respectively. The dendrogram developed by RAPD, SSR and cytochrome P450 gene based primers analyses revealed that the genotypes are grouped in different clusters according to high calcium (300–450 mg/100 g), medium calcium (200–300 mg/100 g) and low calcium (100–200 mg/100 g). Mantel test employed for detection of goodness of fit established cophenetic correlation values above 0.95 for all the three marker systems. The dendrograms and principal coordinate analysis (PCA) plots derived from the binary data matrices of the three marker systems are highly concordant. High bootstrap values were obtained at major nodes of phenograms through WINBOOT software. Comparison of RAPD, SSR and cytochrome P450 gene based markers, in terms of the quality of data output, indicated that SSRs and cyt P450 gene based markers are particularly promising for the analysis of plant genome diversity. The genotypes of finger millet collected from different districts of Uttarakhand constitute a wide genetic base and clustered according to calcium contents. The identified genotypes could be used in breeding programmes and amajor input into conservation biology of cereal crops.     

Genetic diversity and relationships between and within persimmon (Diospyros L.) wild species and cultivated varieties by SRAP markers

Persimmon (Diospyros L.) is one of the most important fruits worldwide and it has been recognized as a health nutrient supply for human consumption. In this study, sequence-related amplified polymorphism (SRAP) markers were used to characterize the genetic diversity of seven species of Persimmon, aiming at exploiting valuable wild resources for breeding new cultivars. Our results show that 303 out of a total of 303 bands amplified with 11 SRAP primer combinations reveal highly polymorphism. The 48 genotypes of persimmons were divided into five groups using the unweighted pair-group method of arithmetic average cluster analysis and principal coordinated analysis. The clusters results show that not only interspecies among seven persimmon genus, but also inner-species of each genus could be clearly differentiated. The phylogenetics positions of Deyang yeshi, Yemaoshi and Jinzaoshi have been elucidated. The results also indicated that wild Chinese persimmon species bear diverse genetic makeup.     

开发SSR引物方法之研究动态

SSR标记是一种基于DNA长度多态性的分子标记技术,是进行群体遗传结构分析、构建遗传连锁图谱非常有效的工具。由于SSR标记是特异引物标记,必须在知道某个物种DNA序列的前提下,才能设计引物进行PCR扩增,故而存在一个引物开发的问题。从SSR标记的发展历程来看,开发SSR引物的方法有经典的构建与筛选基因组文库的方法、微卫星富集法、省略筛库法和数据库搜索法等四种。本文综述了这四种方法的操作流程及其在实际应用中的优缺点,并对近年来SSR引物在相近的物种间转移使用的情况作了介绍. Abstract: SSRs is one of molecular markers technology based on DNA length polymorphism and an efficient tool for population genetic studies and primary genetic linkage maps construction. Because of a special primer marker, It’s necessary to know a species DNA sequence in order to design primers for PCR testing. That is to say, there is a problem of SSR primer development. For the progress of SSR marker technology, the methods of developing SSR primer could be divided into four kinds: traditional constructing and screening genome library procedure, the SSR richment procedure, avoiding screening genome library procedure and database search procedure. This paper reviewed these four methods’operation processes and their advantages and disadvantages. In addition, transferability of SSR primers in closely related species were introduced in recent years.     

Simple sequence repeat (SSR) analysis in relation to calcium transport and signaling genes reveals transferability among grasses and a conserved behavior within finger millet genotypes

Being an excellent source of calcium, finger millet crop has nutraceutical importance. Mineral accumulation, being a polygenic trait, becomes essential to target potential candidate genes directly or indirectly involved in the regulation of calcium transport and signaling in cereals and might have influence on grain calcium accumulation. In view of this, genic microsatellite markers were developed from the coding and non-coding sequences of calcium signaling and transport genes viz. calcium transporters (channels; ATPases and antiporters), calcium-binding proteins and calcium-regulated protein kinases available in rice and sorghum. In total, 146 genic "simple sequence repeat" (SSR) primers were designed and evaluated for cross-transferability across a panel of nine grass species including finger millet. The average transferability of genic SSR markers from sorghum to other grasses was highest (73.2 %) followed by rice (63.4 %) with an overall average of 68.3 % which establishes the importance of these major crops as a useful resource of genomic information for minor crops. The transfer rate of SSR markers was also correlated with the phylogenetic relationship (or genetic relatedness) of the species. Primers with successful amplification in finger millet were further used to screen for polymorphism across a set of high and low calcium containing genotypes. The results reveal a conserved behavior across the finger millet genotypes indicating that the mineral transport and the storage machinery largely remain conserved in plants and even SSR variations in them remain suppressed during the course of evolution. Single nucleotide polymorphism and differential expression patterns of candidate genes, therefore, might be a plausible reason to explain variations in grain calcium contents among finger millet genotypes.     

32个柿主栽品种SSR图谱构建及遗传变异分析

以32份柿主栽品种为试验材料,利用SSR标记技术构建其指纹图谱并进行遗传多样性分析。从78对候选引物中筛选出20对多态性高、稳定性好的引物作为核心引物,构建柿主栽品种SSR指纹图谱。结果显示:(1)20对引物在32份材料中共扩增出183条多态性条带,每对引物扩增出3～20条不等,平均每对引物扩增出9.15条,多态性比率为81.3%。各个位点的杂合度在0.410 3～0.914 3之间,平均为0.702 7。(2)8对引物在12个品种上具有特征带型,采用5对引物进行组合鉴定即可将32个柿品种完全区分开。(3)依据SSR带型特征,对每对引物生成的不同带型直接编号,简化柿SSR带型记录方法,并利用每个品种的带型编号,建立其DNA指纹图谱,结果表明,核心引物组合法比特征谱带法更适用于构建中国柿主栽品种DNA指纹图谱。(4)根据系统聚类分析将32个柿主栽品种分为两大类,品种间的亲缘关系与地理来源具有一定的相关性。     

Genomic distribution of three repetitive DNAs in cultivated hexaploid Diospyros spp. (D. kaki and D. virginiana) and their wild relatives

To understand the genomic organization of Diospyros species with different ploidy levels, we cloned three different repetitive DNAs and compared their genomic distributions in ten Diospyros species, including hexaploid D. kaki and D. virginiana. Genomic Southern hybridization demonstrated that the EcoRV-repetitive DNA was present in tandem in the genomes of D. glandulosa (2n=2x=30), D. oleifera (2n=2x=30), D. lotus (2n=2x=30), D. virginiana (2n=6x=90) and D. kaki (2n=6x=90). All of these species except D. virginiana also contained the HincII-repetitive DNA in tandem. Fluorescent in situ hybridization showed that the EcoRV- and HincII-repetitive DNAs were predominantly located at the proximal or centromeric regions of chromosomes. The DraI-repetitive sequence cloned from D. ehretioides (2n=2x=30) was not found in the other Diospyros species tested. This suggests that D. ehretioides has a genomic organization different from that of the other Diospyros species. Speciation of hexaploid Diospyros species is also discussed with respect to the genomic distribution of the three repetitive DNAs cloned.