Genetic diversity of switchgrass and its relative species in Panicum genus using molecular markers

Switchgrass (Panicum virgatum), a warm season C4 grass, is a promising crop for bioenergy-dedicated biomass production. Understanding of genetic diversity within Panicum genus will facilitate switchgrass breeding. Genetic relationships of 22 Panicum species from six continents including ninety-one USDA germplasm accessions were investigated by Sequence-Related Amplified Polymorphism (SRAP) and Expressed Sequence Tags-Simple Sequence Repeat (EST-SSR) markers. Eight hundred and twenty-six markers from 28 pairs of SRAP and 25 pairs of EST-SSR Primers were used to differentiate between accessions of a bulk of 25 genotypes. The results showed that there was high genetic diversity found in Panicum species. Most genetic variation was present among the different species and cluster analysis indicated that all the Panicum accessions could be distinguished by SRAP or EST-SSR. Dendrogram results reflected the phylogenetic relationships between Panicum species and Panicum amarum was found to be the closest species to switchgrass. Comparison between molecular markers revealed that SRAP methods were considered more efficient than EST-SSR for screening Panicum accessions.     

Development of EST-SSR markers and investigation of genetic relatedness in tung tree

The tung tree is an important non-edible oilseed source and consists of two species Vernicia fordii and Vernicia montana native to southern China and southeast Asia. In this study, the frequency and distribution of simple sequence repeats (SSRs) in expressed sequence tags (ESTs) of V. fordii were characterized based on 2,407 available EST sequences from the National Center for Biotechnology Information database. Twenty-two EST-SSR markers were developed by screening the genomic DNAs of six individuals of V. fordii from three accessions and 120 individuals of V. montana from 30 indigenous V. montana accessions collected from different geographic areas in southwestern China and northern Laos. The 22 EST-SSR markers exhibited a moderate level of polymorphism in V. montana with an average of 3.36 alleles per locus and PIC of 0.401. Genetic relatedness investigation showed that there was not only distinct genetic differentiation among tung trees but also a distinct geographic pattern among V. montana accessions. The current study is the first report of the development of EST-SSRs and genetic relatedness investigation in tung trees. These EST-SSR markers reported here will be valuable resources for future genetic studies, like construction of linkage maps, diversity analysis, quantitative trait locus/association mapping, and molecular breeding of the tung tree. The genetic relatedness identified in V. montana would provide potential clues in choosing germplasms in interest as progenitors for cross breeding and variety improvement of V. montana in practice.     

Genetic diversity of Crotalaria germplasm assessed through phylogenetic analysis of EST-SSR markers.

The genetic diversity of the genus Crotalaria is unknown even though many species in this genus are economically valuable. We report the first study in which polymorphic expressed sequence tag-simple sequence repeat (EST-SSR) markers derived from Medicago and soybean were used to assess the genetic diversity of the Crotalaria germplasm collection. This collection consisted of 26 accessions representing 4 morphologically characterized species. Phylogenetic analysis partitioned accessions into 4 main groups generally along species lines and revealed that 2 accessions were incorrectly identified as Crotalaria juncea and Crotalaria spectabilis instead of Crotalaria retusa. Morphological re-examination confirmed that these 2 accessions were misclassified during curation or conservation and were indeed C. retusa. Some amplicons from Crotalaria were sequenced and their sequences showed a high similarity (89% sequence identity) to Medicago truncatula from which the EST-SSR primers were designed; however, the SSRs were completely deleted in Crotalaria. Highly distinguishing markers or more sequences are required to further classify accessions within C. juncea.     

基于SRAP和TRAP标记的河北野生狗牙根种质遗传多样性分析

以SRAP和TRAP 2种标记技术对36份狗牙根材料的遗传多样性及亲缘关系进行了分析,其中包含34份河北省野生狗牙根种质资源。分别由238对SRAP和85对TRAP引物组合中筛选获得具有多态性的SRAP和TRAP引物组合各10对,PCR扩增总条带分别为186和161条,多态性条带156和132条,平均每对引物扩增出多态性条带各15.6和13.2条,多态性位点比率分别为83.4%和81.0%。2种标记合并进行聚类分析,所有供试的36份狗牙根材料遗传相似系数GS=0.519~0.983,平均为0.7。当GS=0.68时,可将36份供试材料分为4个类群。本研究结果表明河北野生狗牙根种质资源存在较丰富的遗传多样性,可为种质资源保护和选育优良狗牙根新品种提供科学依据。     

Comparative analysis of genetic diversity in sacred lotus (<Emphasis Type="Italic">Nelumbo nucifera</Emphasis> Gaertn.) using AFLP and SSR markers

The sacred lotus (Nelumbo nucifera Gaertn.) is an aquatic plant of economic and ornamental importance in China. In this study, we developed twenty novel sacred lotus SSR markers, and used AFLP and SSR markers to investigate the genetic diversity and genetic relationships among 58 accessions of N. nucifera including 15 seed lotus, 12 rhizome lotus, 24 flower lotus and 7 wild lotus. Our results showed that sacred lotus exhibited a low level of genetic diversity, which may attribute to asexual reproduction and long-term artificial selection. A dendrogram based on both AFLP and SSR clustering data showed that: (1) the seed lotus accessions and rhizome lotus accessions were distinctly clustered into different groups, which indicated the significant genetic differentiation between them. This may be attributed to the two modes of reproduction and lack of genetic exchange; (2) the accessions of Thailand wild lotus were separated from other wild lotus accessions. This implied that the Thailand lotus might be genetically differentiated from other wild lotuses. In addition, Mantel test conducted gave highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with the values of r = 0.941 and r = 0.879, respectively, indicating the higher efficiency of the combination of these techniques (AFLP and SSR) in estimation and validation of the genetic diversity among the accession of sacred lotus. This knowledge of the genetic diversity and genetic relatedness of N. nucifera is potentially useful to improve the current strategies in breeding and germplasm conservation to enhance the ornamental and economic value of sacred lotus.     

Genetic diversity assessment of a germplasm collection of Salvia miltiorrhiza Bunge. based on morphology,ISSR and SRAP markers

Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants for its therapeutic effects. In the present study, morphological traits, ISSR (inter-simple sequence related) and SRAP (sequence-related amplified polymorphism) markers were used to analyze the genetic diversity of 59 S. miltiorrhiza phenotypes. Out of the 100 ISSR primers and 100 SRAP primer combinations screened, 13 ISSRs and 7 SRAPs were exploited to evaluate the level of polymorphism and discriminating capacity. The results showed that the 13 ISSRs generated 190 repeatable amplified bands, of which 177 (93.2%) were polymorphic, with an average of 13.6 polymorphic fragments per primer. The 7 SRAPs produced 286 repeatable amplified bands, of which 266 (93.4%) were polymorphic, with an average of 38.1 polymorphic fragments per primer. Cluster analysis readily separated different morphological accessions, wild and cultivated controls based on morphological traits, ISSR and SRAP markers. The study indicated that morphological traits, ISSR and SRAP markers were reliable and effective for assessing the genetic diversity of phenotypic S. miltiorrhiza accessions. The overall results suggested that the introduction of genetic variation from morphology-based germplasms enlarged the genetic base for the collection, conservation and further breeding program of S. miltiorrhiza germplasm.     

Genetic diversity and differentiation of lotus (Nelumbo nucifera) accessions assessed by simple sequence repeats

Nelumbo nucifera (lotus) is a perennial aquatic crop of substantial economical and ecological importance. Currently, the evaluation of the genetic variation of lotus germplasm accessions using codominant simple sequence repeat (SSR) markers is significant, and it is essential for understanding the population structure of N. nucifera. Here we report the genetic diversity and differentiation of 92 N. nucifera accessions (82 cultivated varieties and 10 wild lotus) using 50 polymorphic SSR markers. A total of 195 alleles were detected, with an average of 3.9 alleles/locus. The mean polymorphic information content (PIC) and the mean expected heterozygosity were 0.43 and 0.50, respectively. The genetic relationships among accessions were estimated using an unweighted pair‐group method with arithmetic average (UPGMA) cluster and principal coordinate analysis (PCA). Both methods revealed that the lotus accessions from China and those from its adjacent Asian countries formed a single cluster, respectively. The cultivated varieties were correlated with their major characteristics in cultivation (the seed, rhizome and flower type) rather than their geographic distribution. On the basis of the Bayesian model‐based analyses, two genetically distinct groups (the seed lotus group and the rhizome lotus group) were generated, with a strong differentiation between them (F_ST = 0.57). The seed lotus group exhibited higher genetic diversity than did the rhizome lotus group. The results herein indicated that the current levels of genetic diversity and differentiation between the lotuses have been greatly influenced by artificial selection.     

Evaluation of bamboo genetic diversity using morphological and SRAP analyses

Bamboo is an important member of the giant grass subfamily Bambusoideae of Poaceae. In this study, 13 bamboo accessions belonging to 5 different genera were subjected to morphological evaluation and sequence-related amplified polymorphism (SRAP) analysis. Unweighted pair-group method of arithmetic averages (UPGMA) cluster analysis was used to construct a dendrogram and to estimate the genetic distances among accessions. On the basis of morphological characteristics, the 13 accessions were distinctly classified into 2 major clusters; 3 varieties, PPYX, PGNK, and PLYY were grouped as cluster A, and 10 accessions were categorized under cluster B. Similarity coefficients ranging from 0.23 to 0.96 indicated abundant genetic variation among bamboo varieties. Approximately 38 SRAP primer combinations generated 186 bands, with 150 bands (80.65%) showing polymorphisms among the 13 accessions. Based on SRAP analysis, 13 bamboo accessions were grouped into 3 major clusters. Five species comprised Cluster I (PASL, PLYY, PTSC, SCNK, and BMAK), which belongs to genus Phyllostachys. Cluster II consisted of 5 varieties, PASL, PLYY, PTSC, SCNK, and BMAK; Cluster III included 3 varieties, PGNK, PLSY, and BMRS. Comparison of the results generated by morphological and SRAP analyses showed that the classification based on SRAP markers was more concordant to the taxonomic results of Gamble than that performed using morphological characters, thus suggesting that SRAP analysis is more efficient in evaluating genetic diversity in bamboos compared to morphological analysis. The SRAP technique serves as an alternative method in assessing genetic diversity within bamboo collections.     

利用SRAP标记分析四川省芋种质资源遗传多样性

在分子水平研究四川省芋资源的遗传多样性和亲缘关系,为芋种质资源的分类、保护和有效利用遗传资源以及新品种选育提供依据。本研究利用SRAP分子标记技术,使用28对SRAP引物组合对65份四川省不同地区芋种质资源材料进行遗传多样性分析,采用NT-SYS 2.1统计软件对数据进行分析,建立树状聚类图。扩增出并检测到341条条带,平均每个引物组合扩增检测出12.18条带,多态性带251条,多态率73.6%。UPGMA树形图表明,所用的SRAP引物组合可以将65份材料分成5类,分别与这些材料在园艺分类学上按母芋和子芋的生长习性分类基本相符,与以芋叶心色斑颜色、叶柄中下部颜色、母芋芽色及母芋肉色4种形态性状组合描述具有相关性。研究表明,从四川省不同地区、不同生态环境下收集的不同类型芋种质资源间存在着较丰富的遗传多样性,SRAP分析聚类结果与主要形态学性状分类基本一致,可以解释芋栽培种的进化关系。     

茄子耐盐种质资源遗传多样性的SRAP和ISSR分析

利用SRAP和ISSR分子标记,研究了14份耐盐茄子种质资源的遗传多样性,结果表明,2种标记均能揭示材料间较高的遗传多样性,其中ISSR标记多态性略高于SRAP标记。在SRAP分析中,每对引物组合可扩增出8-15条DNA片段,平均为12．12条：26对SRAP引物组合共扩增出315条DNA片段,其中263条具有多态性,多态性比率为83．49％;材料间遗传相似系数变化范围为0．212～0．923,平均值为0．755。在ISSR分析中,每个引物可获得5～16条DNA片段,平均为10．87条;15个ISSR引物共扩增出163条DNA片段,其中141条具有多态性,多态性比率为86．50％;材料间遗传相似系数变幅为0．333-0．957,平均值为0．736。聚类分析表明,2种标记都能将供试材料完全区分开来,聚类结果具有一定的相似性,但也存在明显差异。Mantel相关分析表明,SRAP分析与ISSR分析的相关性达到极显著性水平（r=0．904,P〈0．01）。     

应用SRAP标记对莲藕资源的聚类分析

利用一种新的分子标记SRAP技术对17个莲藕品种进行了DNA多态性分析。选取7对引物扩增基因组DNA,共获得168条带,其中159条为多态性条带,每对引物平均提供24个标记信息。由UPMGA方法得到的聚类分析结果表明了17个品种间的遗传关系:(1)亚洲莲与美洲莲之间有明显的差异;(2)在亚洲莲内部,藕莲和花莲有明显的遗传分化,在花莲内部亚洲莲与美洲莲的杂交后代和其它花莲品种之间存在明显的差异;(3)SRAP标记是作分子图谱的好标记,但很难区分遗传关系较近的品种。     

植物功能基因组研究中出现的新型分子标记

随着功能基因组学的发展,表达序列标签(Expressed Sequence Tags, ESTs)已经成为开发以PCR为基础的新型分子标记的重要资源。本文综述了植物功能基因组研究中出现的EST-SSR、CAPS、SNP、SRAP和TRAP等新型分子标记的基本原理和特点。这些标记具有其显著的优势,如开发简便、信息量高和通用性好等,尤其是由于它们来源于基因编码区（ORFs),因此具有很高的物种间通用性,并且其多态性与基因功能变异相关联。目前,这些功能基因分子标记已广泛应用于遗传图谱构建、重要性状基因定位、比较作图、遗传多样性和品种鉴别、分子标记辅助选择育种等研究中。在文章最后简要介绍了作者所在实验室近年来所开展的功能基因分子标记工作,并说明了在使用这些功能基因分子标记时可能会出现的问题。     

Genetic relationship among wild,landraces and cultivars of hazelnut (Corylus avellana) from Portugal revealed through ISSR and AFLP markers

The genus Corylus, a member of the birch family Betulaceae, includes several species that are widely distributed throughout temperate regions of the Northern Hemisphere. This study assesses the genetic diversity in 26 international cultivars and 32 accessions of Corylus avellana L. from Portugal: 13 wild genotypes and 19 landraces. The genetic relationships among the 58 hazelnuts (Corylus avellana L.) were analyzed using inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) markers. Eighteen ISSR primers and seven AFLP primer pairs generated a total of 570 unambiguous and repeatable bands, respectively, from which 541 (95.03 %) were polymorphic for both markers. Genetic similarity index values ranged from 0.239 for wild types and cultivars to 0.143 for landraces and wild types. The genetic relationships were presented as a Neighbor-Joining method dendrogram and a two-dimensional principal coordinate analysis (PCoA) plot. The Neighbor-Joining dendrogram showed three main clusters, and the PCoA analysis has shown to be congruent with the hierarchical analysis. Bayesian analysis clustered all individuals into three groups showing a good separation among wild genotypes, landraces and cultivars. The genetic diversity found on wild genotypes and Portuguese landraces may provide relevant information for the diversity conservation and it will be useful in breeding programs and to identify local selections for preservation.     

SSR markers development and their application in genetic diversity evaluation of garlic (Allium sativum) germplasm

Garlic (Allium sativum), an asexually propagated vegetable and medicinal crop, has abundant genetic variation. Genetic diversity evaluation based on molecular markers has apparent advantages since their genomic abundance, environment insensitivity, and non-tissue specific features. However, the limited number of available DNA markers, especially SSR markers, are insufficient to conduct related genetic diversity assessment studies in garlic. In this study, 4372 EST-SSR markers were newly developed, and 12 polymorphic markers together with other 17 garlic SSR markers were used to assess the genetic diversity and population structure of 127 garlic accessions. The averaged polymorphism information content (PIC) of these 29 SSR markers was 0.36, ranging from 0.22 to 0.49. Seventy-nine polymorphic loci were detected among these accessions, with an average of 3.48 polymorphic loci per SSR. Both the clustering analyses based on either the genotype data of SSR markers or the phenotypic data of morphological traits obtained genetic distance divided the 127 garlic accessions into three clusters. Moreover, the Mantel test showed that genetic distance had no significant correlations with geographic distance, and weak correlations were found between genetic distance and the phenotypic traits. AMOVA analysis showed that the main genetic variation of this garlic germplasm collection existed in the within-population or cluster. Results of this study will be of great value for the genetic/breeding studies in garlic and enhance the utilization of these garlic germplasms.     

普通小麦Genomic-SSR和EST-SSR分子标记遗传差异及其与系谱遗传距离的比较研究

采用Genomic-SSR和EST—SSR标记技术,对来自我国北方冬麦区的18份普通小麦品种(系)的遗传多样性进行了探讨,并与系谱遗传距离进行了比较分析。研究发现,平均每个Genomic—SSR检测到的等位基因数为3．34个,明显高于EST-SSR的2．31个。遗传距离(GD)计算结果显示,18个小麦基因型之间的EST—SSR平均遗传距离较小,仅为0．3996,低于Genomic—SSR的GD平均值0．5458。尽管EST-SSR揭示出的多态性明显低于Genomic-SSR,但系谱分析和聚类结果均表明,与Genomic—SSR相比,EST—SSR标记能更准确地反映出不同小麦基因型之间的遗传和亲缘关系。据此可以认为,EST—SSR是评价小麦遗传多样性的一种理想标记形式。研究还证实,一个骨干亲本与由其衍生出来的品种(系)之间的遗传差异一般较小,并对拓宽普通小麦遗传基础的策略和方法进行了讨论。     

Genetic characterization of an elite coffee germplasm assessed by gSSR and EST-SSR markers

Coffee is one of the main agrifood commodities traded worldwide. In 2009, coffee accounted for 6.1% of the value of Brazilian agricultural production, generating a revenue of US$6 billion. Despite the importance of coffee production in Brazil, it is supported by a narrow genetic base, with few accessions. Molecular differentiation and diversity of a coffee breeding program were assessed with gSSR and EST-SSR markers. The study comprised 24 coffee accessions according to their genetic origin: arabica accessions (six traditional genotypes of C. arabica), resistant arabica (six leaf rust-resistant C. arabica genotypes with introgression of Híbrido de Timor), robusta (five C. canephora genotypes), Híbrido de Timor (three C. arabica x C. canephora), triploids (three C. arabica x C. racemosa), and racemosa (one C. racemosa). Allele and polymorphism analysis, AMOVA, the Student t-test, Jaccard's dissimilarity coefficient, cluster analysis, correlation of genetic distances, and discriminant analysis, were performed. EST-SSR markers gave 25 exclusive alleles per genetic group, while gSSR showed 47, which will be useful for differentiating accessions and for fingerprinting varieties. The gSSR markers detected a higher percentage of polymorphism among (35% higher on average) and within (42.9% higher on average) the genetic groups, compared to EST-SSR markers. The highest percentage of polymorphism within the genetic groups was found with gSSR markers for robusta (89.2%) and for resistant arabica (39.5%). It was possible to differentiate all genotypes including the arabica-related accessions. Nevertheless, combined use of gSSR and EST-SSR markers is recommended for coffee molecular characterization, because EST-SSRs can provide complementary information.     

Mining EST-Derived SSR Markers to Assess Genetic Diversity in Cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Crantz) is a very important staple and industrial crop in tropical and subtropical regions of the world. The paucity of markers is a serious limitation in marker-assisted breeding. A total of 35,992 expressed sequence tags (ESTs) from cassava, which were clustered in 13,173 unigenes, were used in this study. A total of 1,889 microsatellites were identified, with an average density of one simple sequence repeats (SSRs) every 4.40 kb. Of the 1,058 designed EST-SSRs from cultivars SC06, TMS60444, and W14, 431 were polymorphic. Then, 31 randomly selected EST-SSRs from the 431 polymorphic EST-SSRs were used to evaluate the genetic diversity of 76 cassava accessions. A total of 93 alleles were identified, and the number detected for each EST-SSR ranged from one to four. Based on the 93 alleles, the 76 cassava accessions could be classified into six groups, and the genetic similarity coefficient ranged from 0.55 to 0.94. This study demonstrated the potential of EST-derived SSRs in cassava. The resources developed in this study enriched the available molecular markers for cassava.     

Genetic diversity and phylogenetic relationship of kenaf (Hibiscus cannabinus L.) accessions evaluated by SRAP and ISSR

Understanding genetic diversity and phylogenetic relationships is useful for plant breeding. In this study, we assessed the genetic diversity in a panel of 84 accessions of kenaf from 26 countries using SRAP and ISSR markers. The kenaf accessions could be divided into L1 (60 cultivated varieties) and L2 (24 wild accessions) at the level of 0.145 genetic dissimilarity coefficient by UPGMA. The L₂ group was further divided into two subgroups (16 relative-wide and 9 origin wide accessions) at the level of 0.207 genetic dissimilarity. Out of the 9 wild accessions in the L₂ group, 6 were from Tanzania and the remaining 3 lines were from Kenya. These results suggest that the center of origin for kenaf might be Tanzania and Kenya.