Genetic diversity and relationships among pea cultivars revealed by RAPDs and AFLPs

In order to obtain an overview of the genetic diversity present within the set of pea cultivars released in Germany, 21 cultivars were analysed at the DNA level by random amplified polymorphic DNAs (RAPDs) and amplified fragment length polymorphisms (AFLPs), as well as for agronomic traits. Yield of grain cultivars ranged from 2.95 to 3.87 t/ha. Based on the screening of 60 RAPD primers and 32 Eco RI + 3/Mse I+3 AFLP primer combinations, 20 RAPD primers and 11 Eco RI + 3/MseI+ 3 primer combinations generating polymorphic and distinct fragments were chosen for estimation of genetic diversity. Twenty RAPD primers amplified a total of 314 scorable bands ranging from about 262 bp to 1996 bp. Of these, 175 fragments (55.7%) were polymorphic. Based on these data, genetic similarity (GS) was estimated between 0.80 (‘Lisa’ vs.‘Grapis’) and 0.94 (‘Bohatyr’ vs. ‘Sponsor’; mean GS = 0.88). Eleven AFLP primer combinations led to the amplification of 949 scorable fragments ranging from 43 to 805 bp and of these, 462 (48.7%) were polymorphic. Genetic similarity based on AFLPs was calculated between 0.85 (‘Lisa’ vs.‘Laser’) and 0.94 (‘Bohatyr’ vs. ‘Sponsor’, mean GS = 0.90). Correlation of genetic similarity estimated on RAPDs and AFLPs was estimated at r = 0.79** using Spearman's rank correlation coefficient and at r = 0.84 by the Mantel test, respectively. UPGMA cluster analysis carried out on these data separately for RAPDs and AFLPs and on the combined data reflected, to some extent, pedigree relationships and cophenetic correlations (r = 0.89 for RAPDs, r = 0.88 for AFLPs, and r = 0.93 RAPDs + AFLPs) indicate a good fit of respective clusters to genetic similarity data. The correlation of cluster analyses to pedigree information and the impact on parental genotype selection is discussed.     

中国88个马铃薯审定品种SSR指纹图谱构建与遗传多样性分析

为对马铃薯品种鉴别、优良杂交组合选配提供分子水平上的依据,利用SSR标记构建了中国2000-2007年审定的88个马铃薯品种的指纹图谱并进行了遗传多样性分析。以138对SSR引物对16份遗传差异较大的马铃薯材料的基因组DNA进行了扩增,筛选出10对多态性高、谱带清晰的引物。利用10对SSR引物对全部供试材料进行扩增及电泳检测,共检测到135个等位位点,其中133个为多态性位点,多态性比率达98.52%。每对SSR引物扩增出的等位位点数7~22个,平均13.5个,多态性信息量变化范围为0.7604~0.9375,平均0.8501。通过对电泳检测结果的统计分析,利用S180、S25、S7、S151、S184及S192等6对引物构建了88份供试材料的SSR指纹图谱。聚类分析表明,在相似系数0.620处,所有供试材料被被聚为一类,在相似系数0.652处,81.8%的材料仍然聚在一起,从分子水平上表明供试材料遗传基础非常狭窄。聚类分析结果与供试材料系谱来源有较好一致性,同一栽培区域育成的品种在不同程度上聚在一类。     

Genetic maps of RAPD, AFLP and ISSR markers in Ananas bracteatus and A. comosus using the pseudo-testcross strategy

Genetic maps of random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP) and inter simple sequence repeats (ISSR) markers in pineapple (2n = 2x = 50) are reported for the first time. On the basis of a segregating population of 46 F1 individuals from a cross Ananas comosus x A. bracteatus, genetic maps of these two species were constructed using the two‐way pseudo‐testcross approach. The A. bracteatus map consists of 335 markers (60 RAPDs, 264 AFLPs and 11 ISSRs) assembled into 50 linkage groups, 26 of them with at least four markers. The A. comosus map consists of 157 markers (33 RAPDs, 115 AFLPs, eight ISSRs and the ‘piping’ trait locus) organized into 30 linkage groups, 18 of them with at least four markers. These maps cover, respectively, 57.2% of the A. bracteatus genome estimated as 3693 cM long, and 31.6% of the A. comosus genome calculated as 4146 cM. A rough estimate of 120 and 127 kbp/ cM on average was found for the relationship between physical and genetic distance for A. bracteatus and A. comosus, respectively.     

DNA-RAPDs detect genetic variation and paternity in Malus

Summary DNA amplification fingerprinting using arbitrary primer(s) was applied to the identification of Malus species. Highly variable DNA fragment patterns were clearly detected by polyacrylamide gel electrophoresis of the amplified extension products, although three sports of Delicious exhibited the same fingerprint as the original cultivar. The fingerprinting of two triploid apple cultivars suggested that the parent contributing the 2n gamete was the female. We applied this fingerprinting to paternity analysis of an apple cultivar of which the pollen parent was unknown. By using 5 arbitrary primers and RFLP analysis of the amplified products, one cultivar was singled out for paternity among six putative candidates.     

Genetic variation within and between two cultivars of red clover (Trifolium pratense L.): Comparisons of morphological,isozyme, and RAPD markers

Summary Morphological, isozyme and random amplified polymorphic DNA (RAPD) markers were used to estimate genetic variation within and between cultivars of red clover (Trifolium pratense L.), an important temperate forage legume. Two cultivars of red clover, Essi from Europe and Ottawa from Canada, were evaluated. Six monogenic morphological characters were observed for 80 plants from each of these two cultivars. All six morphological loci were polymorphic in the cultivar Essi whereas only four loci were polymorphic in the cultivar Ottawa. Forty plants from each cultivar were assayed for isozyme markers. A total of 21 enzyme-coding loci with 43 alleles was detected using twelve enzyme systems. Thirteen and nine of these loci were polymorphic in Essi and Ottawa, respectively. The mean number of alleles per locus was 1.81 in Essi and 1.67 in Ottawa. Seventeen random 10-mer primers were screened for RAPD markers. Nine primers which gave clear and consistent amplified products were used to assay 20 individuals from each cultivar. Each primer gave from 7 to 20 amplified bands with an average of 14.8 bands per primer. One hundred and eight of 116 putative loci were polymorphic in Essi and 90 of 98 loci were polymorphic in Ottawa. High within-cultivar variation was observed in both cultivars using both isozyme and RAPD markers. This high polymorphism makes these markers useful for germplasm characterization and genetic studies in red clover.     

我国棉花抗枯、黄萎病骨干品种(系)基于AFLP的遗传多样性

利用AFLP分子标记技术,对我国20世纪50年代开展抗枯、黄萎病育种以来培育的105份骨干品种(系)的遗传多样性进行了研究。结果表明,筛选的20个AFLP引物组合在该品种群体中扩增了1498个AFLP标记,其中多态性标记232个,占总标记数的15.5%。单一引物组合扩增的DNA标记数变化在49111之间,平均每个引物组合扩增的总标记和多态性标记分别为74.9和11.6。在该抗病品种群体中,有46个品种(系)具有特异标记,占品种总数的43.81%,其中引物组合E41/M50可使10个品种产生特异标记。品种(系)之间的成对欧氏距离总平均值为4.353,变幅为1.7326.708,单一品种欧氏距离平均值变幅为3.5315.705,高于总平均值的品种数不足50%,表明该陆地棉抗病品种群体的遗传多样性较低。基于AFLPs多态性数据的聚类分析,105个品种(系)被划分为5个陆地棉品种类群(Uplandcottongroups,UCGs),每个UCG包含的品种数目不同。     

Radiomutants of chrysanthemum (Dendranthema grandiflora Tzvelev) of the Lady group: RAPD analysis of the genetic diversity

Random amplified polymorphic DNA (RAPD) markers were used to study the molecular characterization of 10 new radiomutants of chrysanthemum. The original cultivar ‘Richmond’ differed in genetic distance from its Lady group mutants. The analysis of genetic similarity indices revealed low diversity within the radiomutants. The dendrogram obtained after cluster analysis separated the new cultivars as a group that differed from the original cultivar ‘Richmond’. The Lady group cultivars, derived from one original cultivar by radiomutation, could be distinguished from each other by using RAPD markers of only a single primer or sets of two or three primers. Polymerase chain reaction analysis proved the efficiency of the RAPD method for DNA fingerprinting of the original cultivar ‘Richmond’ and its new radiomutants.     

Male and female genetic linkage map of hops, Humulus lupulus

A male and female linkage map of hop has been constructed using 224 DNA polymorphisms (106 amplified fragment length polymorphisms (AFLPs), three random amplified polymorphic DNAs (RAPDs), one RAPD‐sequence‐tagged‐site (STS), and three microsatellite (STSs) segregating in an F₁ population of the English cultivar ‘Wye Target’‐the German male breeding line ‘85/54/15’. Linkage between these loci was estimated using JOINMAP Version 2.0. The final map for the female parent consisted of 110 loci assigned to eight linkage groups covering a distance of 346.7 cM. For the male map, 57 loci could be mapped on nine linkage groups spanning over 227.4 cM. One of these male linkage groups (Gr09‐M) presumably represents the Y chromosome, since all markers assigned (10 AFLPs, three RAPDs and one STS) were closely linked to the male sex (M). Because of their sex‐specific segregation, 10 doubly heterozygous AFLPs spanning a distance of 18.7 cM could be identified as markers describing the X chromosome, which is part of the male and female map. Three STMSs, which had already proved useful in hop genotyping, could be integrated as codominant locus‐specific markers and thus allowed to produce reliable allelic bridges between the female and male counterparts.     

Genetic instability of bermudagrass (Cynodon) cultivars 'Tifgreen' and 'Tifdwarf' detected by DAF and ASAP analysis of accessions and off-types

DNA amplification fingerprinting (DAF) and arbitrary signatures from amplification profiles (ASAP) were used to evaluate the genetic stability of two important bermudagrass (Cynodon) cultivars, the interspecific cross Tifgreen and its somatic mutant Tifdwarf, and study genetic diversity and origin of derived bermudagrass off-types that exhibit patches of contrasting morphology and performance. Mini-hairpin primers produced complex and reproducible DAF and ASAP profiles with high levels of polymorphic DNA, and established genetic relationships between 11 Tifgreen and 8 Tifdwarf turf plot accessions and 16 off-types. DAF analysis revealed an average 14.1 ± 5.6 (SE) polymorphic bands/primer within cultivar accessions. In contrast, the higher resolving power of ASAP detected 24.5 ± 2.1 polymorphic bands/primer. Similarly, DAF and ASAP produced 13.0 ± 5.5 (SE) and 20 ± 2.8 polymorphic bands/primer within off-types, respectively. Phenetic analysis using cluster (UPGMA) and ordination (PCO) techniques showed that both Tifdwarf and Tifgreen were genetically unstable. The analysis showed that almost all cultivar accessions and one-half of the off-types studied were genetically distinct, but very close to each other. In this case, genetic variation was probably the result of somatic mutations. The other off-types and some Tifgreen accessions represented a genetically distant and diverse bermudagrass group of interspecific hybrid (n=27) origin. Off-types were probably the result of sod contamination. Results complement a previous study that established that the interspecific Tifway bermudagrass was genetically stable whereas derived off-types were contaminants rather than somatic mutants. Tifgreen and Tifdwarf showed genetic instabilities that were readily detected by DNA amplification with mini-hairpin primers. The present study offers a direct molecular alternative capable of evaluating the genetic stability of selected cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

A high-density integrative linkage map for <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

Sequence-related amplified polymorphism (SRAP) combined with SSRs, RAPDs, and RGAPs was used to construct a high density genetic map for a F₂ population derived from the cross DH962 (G. hirsutum accession) × Jimian5 (G. hirsutum cultivar). A total of 4,096 SRAP primer combinations, 6310 SSRs, 600 RAPDs, and 10 RGAPs produced 331, 156, 17 and 2 polymorphic loci, respectively. Among the 506 loci obtained, 471 loci (309 SRAPs, 144 SSRs, 16 RAPDs and 2 RGAPs) were assigned to 51 linkage groups. Of these, 29 linkage groups were assigned to corresponding chromosomes by SSR markers with known chromosome locations. The map covered 3070.2 cM with a mean density of 6.5 cM per locus. The segregation distortion in this population was 9.49%, and these distorted loci tend to cluster at the end of linkage groups or in minor clusters on linkage groups. The majority of SRAPs in this map provided an effective tool for map construction in G. hirsutum despite of its low polymorphism. This high-density linkage map will be useful for further genetic studies in Upland cotton, including mapping of loci controlling quantitative traits, and comparative and integrative analysis with other interspecific and intraspecific linkage maps in cotton.     

Genetic variation among cultivars of red clover (Trifolium pratense L.) detected by RAPD markers amplified from bulk genomic DNA

Summary The use of random amplified polymorphic DNA (RAPD) markers obtained from bulked samples was investigated for cultivar identification in red clover. Pooled samples were examined in order to minimize variation within cultivars. To determine the appropriate number of individuals to include in the bulked samples representing each cultivar, DNA samples from two, three, four, five, ten and twenty individuals were pooled. Twenty was found to be an appropriate number of red clover individuals per bulk in order to amplify only the DNA sequences shared among most individuals in each cultivar. Fourteen 10-mer primers were used to amplify genomic DNA from combined leaf samples of 15 red clover cultivars from European, Japanese and North American origins. A total of 79 amplified products, of which 55 were polymorphic, was obtained. Cultivar-specific bands were observed with 13 primers. The amplification patterns obtained from two primers could distinguish all 15 red clover cultivars. Rogers' genetic distances for all 105 pairwise comparisons were calculated to evaluate relationships among these cultivars. Cluster analysis based on these genetic distances separated these 15 cultivars into three groups, with two of the groups consisting of a single Japanese cultivar each, while the third group included cultivars from European, North American, and Japanese origins.     

Assessment of genotypic variation among cultivated durum wheat based on EST-SSRS and genomic SSRS

Genotypic variation of 64 durum lines, landraces, and varieties wereinvestigated using three sources of microsatellites (SSRs). 245 primer pairswere used to detect polymorphism in this collection. A total of 42polymorphic SSR primer pairs consisting of 22 EST-SSR, 11 XGWM and9 WMC were used for genotyping. The EST-SSRs primers produced highquality markers, but were least polymorphic (25%) compared to 53%for the other two sources. The 42 primers detected 189 polymorphicalleles with an average of 4.5 alleles per locus. The coefficient of similaritybetween accessions ranged from 0.28 to 0.70. The estimate of similaritydiffered when each source of SSRs was analyzed independently. This studyshowed that SSRs are highly polymorphic in durum. The data presentedhere provides a platform to develop a genotypic database for durum wheatthat will facilitate the exploitation of its genetic resources.     

Molecular markers and their applications in wheat breeding

In recent years, considerable emphasis has been placed on the development of molecular markers to be used for a variety of objectives. This review attempts to give an account of different molecular markers—restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), sequence-tagged sites (STS), DNA amplification fingerprinting (DAF), amplified fragment length polymorphisms (AFLPs) and microsatellites (STMS)—currently available for genome mapping and for tagging different traits in wheat. Other markers, including microsatellite-primed polymerase chain reaction (MP-PCR), expressed sequence tags (ESTs) and single nucleotide polymorphisms (SNPs) are also discussed. Recent information on synteny in cereal genomes, marker-assisted selection, marker validation and their relevance to cereal breeding in general and wheat breeding in particular are also examined.     

Stability of cassava plants at the DNA level after retrieval from 10 years of in vitro storage

Summary Cassava (Manihot esculenta Crantz) germplasm collections are conventionally maintained by continuous vegetative propagation in the field. Tissue culture techniques provide a more convenient way to conserve germplasm. The cassava in vitro gene bank held in trust at CIAT comprises nearly 6000 accessions. A study was carried out to determine whether any DNA rearrangements resulting from in vitro storage under slow growth could be detected by molecular analysis in retrieved plants. RFLPs with homologous probes, RAPDs with twenty primers and DNA fingerprinting with M13 probe were tested to detect variation at DNA level in cassava plants after ten-years in vitro storage. The molecular marker data obtained in this study supports the stability of the cassava germplasm under the in vitro storage conditions described in this work.     

3个玉米杂交种和亲本SSR指纹图谱的构建

以3个云南省大面积推广的玉米杂交种及其亲本为试材,从96对SSR引物中筛选出24对引物,这些引物分布在玉米基因组的10条染色体上,每对引物可以检测到1～6个数目不等的多态性片段(allele),共97个多态性片段,平均为4.04个,片段大小介于85～300 bp之间.在比较分析各试材图谱的基础上,探讨了构建品种指纹图谱的统计学方法,建立了适     

利用RAPD和SSR分析36个贵州主要玉米种质的比较研究

利用RAPD和SSR两种标记方法研究了36个玉米自交系的遗传多样性,并对这两种分子标记系统进行了比较.利用筛选出的22条RAPD引物,检测到了148条有多态性的带;利用筛选出的34对SSR引物,检测到158个等位基因.RAPD和SSR分子标记均有很高的多态性,RAPD多态性带比例为95.95%,SSR位点检测出的平均等位基因数位4.65.RAPD分子标记结果将36个玉米自交系划分为6大类,SSR分子标记将其划分为5大类.与系谱分析基本一致,两种分子标记划分的结果也相似.研究认为,RAPD、SSR两种分子标记系统均适合于玉米种质的遗传多样性研究,但SSR更可取.     

Heterogeneity of random amplified polymorphic DNA sequences in individual seedlings and bulked samples of four cultivars of Brassica napus

Using four different random amplified polymorphic DNA (RAPD) primers, a qualitative and quantitative assessment was made of the level of DNA sequence heterogeneity present in the seedlings of four representative Australian rapeseed cultivars. It was found that, depending upon the primer/cultivar combination, the seedlings diverged from total homogeneity to almost complete heterogeneity. The increase or decrease of sample-specific RAPD sequences was evaluated in proportional mixtures of DNA from individual seedlings. These results were then compared with those obtained from bulked DNA samples containing DNA from all the seedlings of a cultivar. From these comparisons, it was found that for a specific RAPD to be detectable in a bulked sample, the particular polymorphism had to be present in at least 15% of the individual seedlings. Even so, the bulked samples produced cultivar-specific RAPD banding patterns with all four primers, showing that any of these primers could be used to identify the different rapeseed cultivars. In contrast to the cultivars ‘Oscar’, ‘Dunkeld’ and ‘Narendra’, the cultivar ‘Rainbow’ was found to be highly heterogeneous—as shown by a diversity of RAPD combinations rather than the presence of differing length RAPDs—and it is suggested that this heterogeneity may be related to the improved tolerance of this cultivar to blackleg infection.     

小麦赤霉病抗性QTL分析

以小麦赤霉病抗源望水白与感病品种Alondra杂交产生的104个重组自交系为材料，采用JoinMap®3.0软件构建了含有2个RAPD、109个SSR和105个AFLP标记共25个连锁群的遗传连锁图，其中24个连锁群可以确定为相应的染色体；采用自然发病和土表接种方法，对该重组自交系群体在建阳和苏州进行了连续两年赤霉病抗性鉴定，结果表明：     

Methodology of generation and characteristics of simple sequence repeat DNA markers in avocado (Persea americana M.)

Summary Generation of Simple Sequence Repeat (SSR) DNA markers was based on the construction of genomic DNA library of avocado (Persea americana M.). The library was screened with the four dinucleotide probes (AG), (AT), (GC) and (CA). Positive clones were sequenced to validate the presence of simple sequence repeats (SSR) and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the simple sequence repeat. Twenty six different pairs of primers which yield a PCR product in the initial screening were synthesized. The SSR A1E11 was found to have eleven alleles while A3F8 has eight alleles. The SSRs in avocado were found to be inherited in a Mendelian fashion.Contribution from the Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel. No. 1335-E, 1994 series.