Assessment of genetic diversity in Azadirachta indica using AFLP markers

 Genetic diversity was estimated in 37 neem accessions from different eco-geographic regions of India and four exotic lines from Thailand using AFLP markers. Seven AFLP selective primer combinations generated a total of 422 amplification products. The average number of scorable fragments was 60 per experiment, and a high degree (69.8%) of polymorphism was obtained per assay with values ranging from 58% to 83.8%. Several rare and accession-specific bands were identified which could be effectively used to distinguish the different genotypes. Genetic relationships within the accessions were evaluated by generating a similarity matrix based on the Jaccard index. The phenetic dendrogram generated by UPGMA as well as principal correspondence analysis separated the 37 Indian genotypes from the four Thai lines. The cluster analysis indicated that neem germplasm within India constitutes a broad genetic base with the values of genetic similarity coefficient ranging from 0.74 to 0.93. Also, the Indian genotypes were more dispersed on the principal correspondence plot, indicating a wide genetic base. The four lines from Thailand, on the other hand, formed a narrow genetic base with similarity coefficients ranging from 0.88 to 0.92. The lowest genetic similarity coefficient value (0.47) was observed between an Indian and an exotic genotype. The level of genetic variation detected within the neem accessions with AFLP analysis suggests that it is an efficient marker technology for delineating genetic relationships amongst genotypes and estimating genetic diversity, thereby enabling the formulation of appropriate strategies for conservation and tree improvement programs. Received: 20 October 1998 / Accepted: 28 November 1998     

SSR and AFLP based genetic diversity of soybean germplasm differing in photoperiod sensitivity

Forty-four soybean genotypes with different photoperiod response were selected after screening of 1000 soybean accessions under artificial condition and were profiled using 40 SSR and 5 AFLP primer pairs. The average polymorphism information content (PIC) for SSR and AFLP marker systems was 0.507 and 0.120, respectively. Clustering of genotypes was done using UPGMA method for SSR and AFLP and correlation was 0.337 and 0.504, respectively. Mantel's correlation coefficients between Jaccard's similarity coefficient and the cophenetic values were fairly high in both the marker systems (SSR = 0.924; AFLP = 0.958) indicating very good fit for the clustering pattern. UPGMA based cluster analysis classified soybean genotypes into four major groups with fairly moderate bootstrap support. These major clusters corresponded with the photoperiod response and place of origin. The results indicate that the photoperiod insensitive genotypes, 11/2/1939 (EC 325097) and MACS 330 would be better choice for broadening the genetic base of soybean for this trait.     

Assessment of genetic variation and differentiation of hop genotypes by microsatellite and AFLP markers.

Microsatellites have many desirable marker properties and have been increasingly used in crop plants in genetic diversity studies. Here we report on the characterisation of microsatellite markers and on their use for the determination of genetic identities and the assessment of genetic variability among accessions from a germplasm collection of hop. Thirty-two polymorphic alleles were found in the 55 diploid genotypes, with an average number of eight alleles (3.4 effective alleles) for four microsatellite loci. Calculated polymorphic information content values classified three loci as informative markers and two loci as suitable for mapping. The average observed heterozygosity was 0.7 and the common probability of identical genotypes was 3.271 x 10(-4). An additional locus, amplified by one primer pair, was confirmed by segregation analysis of two crosses. The locus discovered was heterozygous, with a null allele in the segregating population. The same range of alleles was detected in nine triploid and five tetraploid hop genotypes. Cultivar heterozygosity varied among all 69 accessions, with only one cultivar being homozygous at four loci. Microsatellite allele polymorphisms distinguished 81% of all genotypes; the same allelic profile was found mainly in clonally selected cultivars. Cultivar-specific alleles were found in some genotypes, as well as a specific distribution of alleles in geographically distinct hop germplasms. The genetic relationship among 41 hop accessions was compared on the basis of microsatellite and AFLP polymorphisms. Genetic similarity dendrograms showed low correlation between the two marker systems. The microsatellite dendrogram grouped genetically related accessions reasonably well, while the AFLP dendrogram showed good clustering of closely related accessions and, additionally, separated two geographically distinct hop germplasms. The results of microsatellite and AFLP analysis are discussed from the point of view of the applicability of the two marker systems for different aspects of germplasm evaluation.     

Genetic characterization of Paspalum notatum accessions by AFLP markers

Paspalum notatum Flügge is a warm-season forage grass with sexual diploid and apomictic tetraploid races. Genetic improvement was achieved in out-breeding diploids. The acquisition of artificial sexual tetraploids has raised the possibility of performing crosses and plant improvement at the tetraploid level. The objective of our study was to obtain a genetic and cytoembryological characterization of a germplasm collection of P. notatum, including 31 accessions from seven countries of America and 11 experimentally obtained genotypes. Morphology of mature gametophytes was observed to assess the mode of reproduction of the accessions. A total of 1342 AFLP fragments were generated across the 42 genotypes and from two reference taxa: P. urvillei and P. procurrens. AFLP data were converted into a binary matrix and similarity relationships were established. The genetic distance among all the accessions showed a maximum value of 0.36. In addition, eleven AFLP fragments were observed exclusively in apomictic plants, which could be linked to genomic regions implicated in the control of apospory.     

中国食用向日葵种质资源遗传变异的RAPD及AFLP分析

本研究采用RAPD和AFLP方法对23个中国不同地区的食用向日葵（Helianthus annuus L.)骨干品种进行了遗传变异分析,同时对两种标记系统进行了比较。26个RAPD引物产生了总计192条DNA条带,大小分布 于0.26kb-1.98kb之间,其中165条（86．12％）具有多态性,每条引物产生DNA条带的平均数为7．38。8对AFLP引物组合共产生了576条带,分布于100bp-500bp之间,其中的341条具有多态性,多态百分率为76．00％,每对引物组合产生DNA条带的平均数为72。RAPD方法检测的每位点有效等位基因数（1．76）大于AFLP（1．65）,AFLP标记位点的平均多态性信息量（PIC）（0．38）低于RAPD标记位点PIC（0．41）,但AFLP标记具有很高的多态性检测效率（Ai=38．52）。用RAPD标记分析23个食用向日葵材料的亲缘关系,Nei氏相似性系数分布在47．84％－82．06％,平均相似性系数为0．6495,而采用AFLP的Nei氏相似性系数分布在54．15％－83．52％,平均相似性系数为0．6884。RAPD数据的标准差为0．13,而AFLP数据的标准差为0．08。因此,采用RAPD和AFLP方法分析食用向日葵遗传变异,RAPD标记具有较低相似性系数和较高方差而AFLP则相反。源于两种不同标记的遗传相似矩阵的相关系数为0．51,说明采用RAPD和AFLP系统分析食用向日葵遗传变异得到的结果有一定的相关性,无论采用RAPD还是AFLP标记进行聚类分析,都将23个不同基因型的食用向日葵材料分成了三个类群。     

Comparison of four molecular markers for genetic analysis in Diospyros L. (Ebenaceae)

Four molecular markers, including inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP), sequence-specific amplified polymorphism (SSAP), and amplified fragment length polymorphism (AFLP), were compared in terms of their informativeness and efficiency for analysis of genetic relationships among 28 genotypes in the genus Diospyros. The results were as follows: (1) the highest level of detected polymorphism were observed for IRAP; (2) AFLP was the most efficient marker system due to the simultaneous detection of abundant polymorphism markers per single reaction; (3) the marker index (MI) value was lower for SSAP than for AFLP, but SSAP had a higher level of detected polymorphism than AFLP; (4) the correlation coefficients of similarity were statistically significant for all four marker systems; (5) the four molecular markers yielded similar phylogenetic trees. To our knowledge, this was the first detailed report of a comparison of performance among three retrotransposon-based molecular markers (IRAP, REMAP, SSAP) and the AFLP technique (DNA-based molecular marker) on a set of samples of Diospyros. The results provide guidance for future efficient use of these molecular methods in the genetic analysis of Diospyros.     

Comparison of PCR-based molecular marker analyses of Musa breeding populations

Progress in the breeding of plantain and banana has been restricted by the complex genetic structure and behaviour of cultivated polyploid Musa. Genetic improvement has been hindered due to the large amount of space required for growth and maintenance of plant populations, in addition to the long growth cycle and the low levels of fertility and seed viability characteristic of cultivated genotypes. Molecular marker assisted breeding has the potential to dramatically enhance the pace and efficiency of genetic improvement in Musa. This study was conducted to compare different PCR-based marker systems (RAPD, VNTR and AFLP) for the analysis of breeding populations generated from two diverse Musa breeding schemes. All three assays detected a high level of polymorphism between parental genotypes and within progeny populations. As expected, AFLP assays had by far the highest multiplex ratio while VNTR analysis detected the highest levels of polymorphism. AFLP analysis of a full-sib tetraploid hybrid population confirmed previous reports based on VNTR analysis, of a high frequency of recombination during 2n (3x) gamete formation by a triploid plantain landrace. In addition, both VNTR and RAPD analyses of a full-sib triploid hybrid population suggested a high frequency of homoeologous recombination during n (2x) gamete formation by tetraploid hybrids. In general, there was a poor correlation between estimates of genetic similarity based on different types of marker. The implications of these findings for the molecular breeding of Musa crops are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic variability of cultivated Chaenomeles speciosa (Sweet) Nakai based on AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis was performed to evaluate genetic relationships among 52 Chaenomeles speciosa accessions grown in China. A total of 208 polymorphic markers were generated from eight selective primer pair combinations. Genetic variations were remarkably observed in a wide range of dissimilarities, percent polymorphisms, and average polymorphism information. Genetic similarity values were calculated using Jaccard's coefficient between individuals of different flowering quince accessions; these values ranged from 0.296 to 0.931 with a mean of 0.597. Cluster analysis and principal coordinate analysis were then performed on the basis of AFLP profiles. In our generated dendrogram, accessions were clustered into seven major groups. Mantel's test was performed to determine cophenetic correlation (r = 0.9); this result indicated a very good fit of the dendrogram. We conducted analysis of molecular variance and found greater variations within cultivars than among cultivars. AFLP analysis results further revealed relevant information regarding the genetic background of flowering quince accessions essential for future breeding programs related to plant improvement.     

AFLP marking of the genotypes of leek (Allium porrum) varieties

The results of the AFLP analysis of 16 leek (Allium porrum) accessions and related species of the sections of the genus Allium are presented. Restriction enzymes and primer combinations for the identification of the genotypes of the A. porrum accessions were chosen. As a result, 265 polymorphic AFLP fragments were amplified for 25 analyzed genotypes, and specific spectra of DNA fragments were obtained for each accession. A total of 24 fragments specific for the A. porrum genome was detected, of which only two characterized the genotypes of individual accessions. A wide range of genetic diversity (0.11-0.32) was revealed for the A. porrum varieties and lines used in the analysis. The highest level of similarity in the analyzed set of accessions was found between A. porrum and sand leek (A. scorodoprasum).     

Genetic differentiation among Sri Lankan traditional rice (Oryza sativa) varieties and wild rice species by AFLP markers

Traditional rice varieties are one important component of the biodiversity of Sri Lanka. However, no proper studies have been performed on genetic diversity of the Sri Lankan traditional rice varieties used in our breeding programs. In the present study, the genetic diversity of 46 traditional rice (Oryza sativa) varieties and 5 wild rice species is assesed using Amplified Fragment Length Polymorphism (AFLP) markers. Ten primer combinations generated a total of 784 fragments. Of these, 772 fragments were polymorphic (98.4%). UPGMA analysis based on Jaccard's similarity separated the accessions into four major clusters. A cophenetic correlation with r=0.786 strongly supported the clustering pattern of UPGMA dendrogram. A principal coordinate analysis (PCoA) also confirmed the UPGMA clusters. Accessions referred to the same cluster showed similar morphological characteristics (e.g. height, grain colour etc.) while accessions which are known to be morphologically distinct appeared genetically separated. In addition, the clustering pattern distinctly separated lowland and upland rice varieties. This genetic diversity assessment at the molecular level provides reliable information for selection of germplasm in the development of new rice varieties and in conservation of traditional rice genetic resources.     

AFLP analysis of the diversity and phylogeny of Lens and its comparison with RAPD analysis

AFLP and RAPD marker techniques have been used to evaluate and study the diversity and phylogeny of 54 lentil accessions representing six populations of cultivated lentil and its wild relatives. Four AFLP primer combinations revealed 23, 25, 52 and 48 AFLPs respectively, which were used to partition variation within and among Lens taxa. The results of AFLP analysis is compared to previous RAPD analysis of the same material. The two methods provide similar conclusions as far as the phylogeny of Lens is concerned. The AFLP technique detected a much higher level of polymorphyism than the RAPD analysis. The use of 148 AFLPs arising from four primer combinations was able to discriminate between genotypes which could not be distinguished using 88 RAPDs. The level of variation detected within the cultivated lentil with AFLP analysis indicates that it may be a more efficient marker technology than RAPD analysis for the construction of genetic linkage maps between carefully chosen cultivated lentil accessions.     

Genetic diversity in cultivars and landraces of Oryza sativa subsp. indica as revealed by AFLP markers.

Genetic diversity among 49 Indian accessions of rice (Oryza sativa subsp. indica), including 29 landraces from Jeypore, 12 modern cultivars, and 8 traditional cultivars from Tamil Nadu, was investigated using AFLP markers. In total, nine primer combinations revealed 664 AFLPs, 408 of which were found to be polymorphic. The percentage of polymorphic AFLPs was approximately the same within the cultivars and landraces. Similar results were obtained when genetic diversity values were estimated using the Shannon-Weiner index of diversity. Genetic diversity was slightly higher in the modern cultivars than in the traditional cultivars from Tamil Nadu. Among the landraces from Jeypore, the lowland landraces showed the highest diversity. The present study showed that the process of breeding modern cultivars did not appear to cause significant genetic erosion in rice. Cluster analysis and the first component of principle component analysis (PCA) both showed a clear demarcation between the cultivars and landraces as separate groups, although the genetic distance between them was narrow. The modern cultivars were positioned between the landraces from Jeypore and the traditional cultivars from Tamil Nadu. The second component of PCA further separated medium and upland landraces from lowland landraces, with the lowland landraces found closest to the traditional and modern cultivars.     

Assessment of genetic diversity in a Morus germplasm collection using fluorescence-based AFLP markers

To meet various breeding objectives and to conserve the existing genetic resources of mulberry for future use, the present study was undertaken to investigate the amount of genetic diversity and to establish the relationships between mulberry genotypes using fluorescence-based AFLP markers. Genetic diversity was estimated in 45 mulberry accessions from different eco-geographic regions of Japan and other parts of the world. Five primer combinations amplified an average of 110 AFLP markers per primer combination, ranging in size from 35 to 500 bp. A high degree of polymorphism was revealed by these combinations that ranged from 69.7 to 82.3% across all the genotypes studied. Several rare genotype-specific bands were also identified which could be effectively utilized to distinguish different genotypes. The wide range in genetic similarity coefficients (0.58–0.99) indicated that the mulberry germplasm collection represents a genetically diverse popu-lation. The phenetic dendrogram generated by the UPGMA method grouped 45 accessions into four major clusters, which was in agreement with the results from conventional methods. Clustering of some genotypes into strictly separate groups was not readily apparent and no clear interrelationships could be depicted, in spite of their different geographic origin. In addition, AFLP analysis provided sufficient polymorphism for DNA typing and contributed additional insights into the genetic structure of the mulberry germplasm. These results will help in the formulation of appropriate strategies for conservation and variety improvement in mulberry, for which little or no knowledge of genetic diversity is currently available. Received: 30 December 1999 / Accepted: 14 March 2000     

Genetic diversity analysis of common beans based on molecular markers

A core collection of the common bean (Phaseolus vulgaris L.), representing genetic diversity in the entire Mexican holding, is kept at the INIFAP (Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Mexico) Germplasm Bank. After evaluation, the genetic structure of this collection (200 accessions) was compared with that of landraces from the states of Oaxaca, Chiapas and Veracruz (10 genotypes from each), as well as a further 10 cultivars, by means of four amplified fragment length polymorphisms (AFLP) +3/+3 primer combinations and seven simple sequence repeats (SSR) loci, in order to define genetic diversity, variability and mutual relationships. Data underwent cluster (UPGMA) and molecular variance (AMOVA) analyses. AFLP analysis produced 530 bands (88.5% polymorphic) while SSR primers amplified 174 alleles, all polymorphic (8.2 alleles per locus). AFLP indicated that the highest genetic diversity was to be found in ten commercial-seed classes from two major groups of accessions from Central Mexico and Chiapas, which seems to be an important center of diversity in the south. A third group included genotypes from Nueva Granada, Mesoamerica, Jalisco and Durango races. Here, SSR analysis indicated a reduced number of shared haplotypes among accessions, whereas the highest genetic components of AMOVA variation were found within accessions. Genetic diversity observed in the common-bean core collection represents an important sample of the total Phaseolus genetic variability at the main Germplasm Bank of INIFAP. Molecular marker strategies could contribute to a better understanding of the genetic structure of the core collection as well as to its improvement and validation.     

Morphological and molecular genetic variations of oat genotypes grown in Kermanshah,Iran

Morphological traits and molecular markers are two common methods for genetic variation studies. Molecular markers, morphological traits methods and relationship between the two were used to study genetic variation among 43 oat genotypes and varieties. For this purpose, an augmented design was conducted in three replicates at 2008–2009 cropping season in the experimental field of Campus of Agriculture and Natural Resources of Razi University, Kermanshah, Iran. Four wild oat accessions (Avena sterilis) were added to evaluated genotypes in molecular experiment. Results showed a significant variation among genotypes for all morphological traits and they were classified based on this variation in four groups by WARD cluster analysis. In molecular experiment, 28 inter simple sequence repeat (ISSR) primers amplified 206 polymorph bands. Based on Jaccard similarity matrix, similarity among genotypes was varied from 0.23 to 0.66 and cluster analysis classified genotypes in seven groups by complete linkage method. The correlation between ISSR marker and morphological traits classifications was not significant. ISSR showed to be a helpful marker for genotype identity and separation as it put wild accessions in a group.     

Genetic diversity analysis in sorghum germplasm as estimated by AFLP, SSR and morpho-agronomical markers

A comparison of the different methods of the estimation of genetic diversity is important to evaluate their utility as a tool in germplasm conservation and plant breeding. Amplified fragment length polymorphism (AFLP), microsatellites or SSR and morphological traits markers were used to evaluate 45 sorghum germplasm for genetic diversity assessment and discrimination power. The mean polymorphism information content (PIC) values were 0.65 (AFLPs) and 0.46 (SSRs). The average pairwise genetic distance estimates were 0.57 (morphological traits), 0.62 (AFLPs) and 0.60 (SSRs) markers data sets. The Shannon diversity index was higher for morphological traits (0.678) than AFLP (0.487) and SSR (0.539). The correlation coefficients obtained by the Mantel matrix correspondence test, which was used to compare the cophenetic matrices for the different markers, showed that estimated values of genetic relationship given for AFLP and SSR markers, as well as for morphological and SSR markers were significantly related (p <0.001). However, morphological and AFLP data showed non-significant correlation (p >0.05). Both data sets from AFLP and SSR allowed all accessions to be uniquely identified; two accessions could not be distinguished by the morphological data. In summary, AFLP and SSR markers proved to be efficient tools in assessing the genetic variability among sorghum genotypes. The patterns of variation appeared to be consistent for the three marker systems, and they can be used for designing breeding programmes, conservation of germplasm and management of sorghum genetic resources.     

陆地棉主要产量相关性状的SSR标记关联分析

高产优质育种是我国棉花育种的主要目标。寻找与目标性状关联的分子标记,可克服常规育种的盲目性,提高分子标记辅助选择育种的准确性。本研究对118份陆地棉种质资源的衣分、单铃重、单株铃数及子指等4个产量相关性状进行2年2点的表型鉴定,并利用覆盖全基因组的、有多态性的214对SSR标记进行标记与性状的关联分析。结果表明:118份材料的4个产量相关性状表型变异丰富,平均变异系数的变幅在6.1%~19.1%之间,且在各环境中表现较为稳定;基因型分析表明,214对标记共检测到460个等位变异,基因多样性指数平均为0.5151,PIC值平均为0.4587,表明该批标记具有较多的等位变异数和较高的基因多样性;群体结构分析表明该批材料可分为4个亚群,且各类群中材料与地理来源无对应关系;关联分析结果显示,在显著条件下(-log10P1.3,P0.05),共有39个标记位点能够在2个及2个以上的环境中同时检测到,其中有4个标记位点同时与2个以上性状相关联,进一步比较发现,有7个位点与前人研究结果一致,其余32个位点为新发现的位点。研究结果可为陆地棉产量性状遗传改良的分子标记辅助选择提供理论依据。     

山东省无花果种质资源多样性的RAPD分析

利用随机扩增多态性DNA(RAPD)技术,对58份山东省无花果(Ficus caricaL.)资源的多样性进行了评价,经15个随机引物的初步分析表明,其中至少有23个不同的基因型。继而从36个随机引物中筛选出28个多态性好的引物,进一步对这23个不同基因型材料进行RAPD扩增分析,共得到309个清晰稳定的扩增位点,其中多态性位点236个,占76.4%。利用UPGMA法分析发现,这些基因型间的遗传相似系数在0.592~0.960之间,在此基础上,构建了各基因型间的遗传关系树状图。结果表明,山东省内的无花果资源具有较为丰富的遗传多样性。     

Large-scale development of expressed sequence tag-derived simple sequence repeat markers and diversity analysis in Arachis spp.

Large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed in peanut (Arachis hypogaea L.) to obtain more informative genetic markers. A total of 10,102 potential non-redundant EST sequences, including 3,445 contigs and 6,657 singletons, were generated from cDNA libraries of the gynophore, roots, leaves and seedlings. A total of 3,187 primer pairs were designed on flanking regions of SSRs, some of which allowed one and two base mismatches. Among the 3,187 markers generated, 2,540 (80%) were trinucleotide repeats, 302 (9%) were dinucleotide repeats, and 345 (11%) were tetranucleotide repeats. Pre-polymorphic analyses of 24 Arachis accessions were performed using 10% polyacrylamide gels. A total of 1,571 EST-SSR markers showing clear polymorphisms were selected for further polymorphic analysis with a Fluoro-fragment Analyzer. The 16 Arachis accessions examined included cultivated peanut varieties as well as diploid species with the A or B genome. Altogether 1,281 (81.5%) of the 1,571 markers were polymorphic among the 16 accessions, and 366 (23.3%) were polymorphic among the 12 cultivated varieties. Diversity analysis was performed and the genotypes of all 16 Arachis accessions showed similarity coefficients ranging from 0.37 to 0.97. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9604-8) contains supplementary material, which is available to authorized users.     

Genetic diversity of Carica papaya as revealed by AFLP markers.

Genetic relationships among Carica papaya cultivars, breeding lines, unimproved germplasm, and related species were established using amplified fragment length polymorphism (AFLP) markers. Seventy-one papaya accessions and related species were analyzed with nine EcoRI-MseI primer combinations. A total of 186 informative AFLP markers was generated and analyzed. Cluster analysis suggested limited genetic variation in papaya, with an average genetic similarity among 63 papaya accessions of 0.880. Genetic diversity among cultivars derived from the same or similar gene pools was smaller, such as Hawaiian Solo hermaphrodite cultivars and Australian dioecious cultivars with genetic similarity at 0.921 and 0.912, respectively. The results indicated that self-pollinated hermaphrodite cultivars were as variable as open-pollinated dioecious cultivars. Genetic diversity between C. papaya and six other Carica species was also evaluated. Carica papaya shared the least genetic similarity with these species, with an average genetic similarity of 0.432; the average genetic similarity among the six other species was 0.729. The results from AFLP markers provided detailed estimates of the genetic variation within and among papaya cultivars, and supported the notion that C. papaya diverged from the rest of Carica species early in the evolution of this genus.