芍药属牡丹组（Paeonia sect. Moutan）种间关系的分子证据：GPAT基因的PCR-RFLP和序列分析

为了探讨芍药属牡丹组Paeonia sect. Moutan的种间关系,对采自15个野生居群,代表牡丹组全部8个野生种的15份材料的GPAT 基因片段（外显子5和6之间2 kb的内含子）进行了PCR-RFLP分析,并对代表牡丹组全部8个野生种的9份材料进行了测序。 根据12个限制性内切酶的PCR-RFLP数据,使用Network 3.0计算机程序的RM (reduced-median)法建立了牡丹组种间亲缘关系网络树。同时根据8个种9份材料的GPAT基因片段序列,利用PAUP*4.0计算机程序建立了牡丹组GPAT基因的最大简约（MP）树和邻接(NJ)树。结果获得了具有很高自展值支持、分辨良好的牡丹组种间关系（GPAT基因）树。最重要的是,该基因树所显示的牡丹组种间关系与根据形态学证据提出的牡丹组的种间关系基本吻合,并得到其他研究证据的支持。根据这一结果,对牡丹组的种间关系进行了详细的讨论。     

Phylogenetic analysis of Paeonia sect. Moutan (Paeoniaceae) based on multiple DNA fragments and morphological data

Tree peony, being crowned the title “King of Flowers” in China, is of great medicinal, ornamental, and economic values. In the present study, the phylogeny of the wild tree peony species (section Moutan, Paeonia, Paeoniaceae), represented by twelve accessions collected from all eight species in the section, was investigated based on the DNA sequence in five DNA fragments from both nuclear (Adh1A, Adh2 and GPAT) and chloroplast (trnS-trnG and rps16-trnQ) genomes, as well as morphological characters. Both maximum parsimony (MP) and Bayesian inference of phylogeny (BI) trees were reconstructed based on the combined data of the DNA sequences and morphological data, respectively. The MP and BI trees have the similar topology, and the sect. Moutan clearly branched into two clades. One clade consists of two species, P. delavayi and P. ludlowii, corresponding to the subsect. Delavayanae, and another clade is composed of other six species. Within the second clade, the six species can be divided into three subclades consisting of P. rockii and P. decomposita, P. jishanensis and P. qiui, P. suffruticosa and P. ostii, respectively. Among the three subclades, P. jishanensis/P. qiui is most closely related to P. suffruticosa/P. ostii. These results provide up to date the clearest picture of the phylogeny of wild tree peony species in the sect. Moutan.     

基于多基因序列和形态性状的牡丹组种间关系

牡丹被认为是中国的国花,具有很高的医学、观赏和经济价值.野生牡丹被认为是栽培牡丹的野生祖先,因此弄清牡丹组的种间亲缘关系具有重要的理论和实践意义.由于受到信息量的限制,根据单基凼数据或形态数据往往无法对牡丹组的种间关系得到明确的结果.本研究用12份样品代表野生牡丹组(Paeonia section Moutan DC.,Paeoniaceae)8个种,利用包括核基因(Adh1A、Adh2和GPAT)和叶绿体基因(trnS-trnG和rps16-trnQ)的DNA序列以及形态性状的多套数据来探讨野牛牡丹的种间关系.合并分析得到具高支持率的牡丹组物种间的系统发育关系.结果表明,芍药属牡丹组8个野生种分为两个亚组,即肉质花盘亚组subseet.Delavayanae和革质花盘亚组subsect.Vaginatae.肉质花盘亚组包括滇牡丹P delavayi和大花黄牡丹P.ludlowii;革质花盘亚组包括其余6个种.革质花盘亚组中,四川牡丹P.decomposita ssp.decomposita和紫斑牡丹P. rockii ssp. rockii关系密切;卵叶牡丹P.qiui和矮牡丹P. jishanenMs关系密切;银屏牡丹P. suffruticosa ssp.yinpingmudan与风丹P. ostii关系 密切,并且后两个分支为姊妹群.     

芍药属牡丹组基于形态学证据的系统发育关系分析

对芍药属牡丹组Paeonia L.sect.Moutan DC.（全部野生种）40个居群进行了基于形态学证据的系统学分析,试图建立组内种间的系统发育关系。利用PAUP （4.0）计算机程序分别构建了建立在25个形态学性状基础上的所有研究类群的距离树（UPGMA、NJ）和最大简约树（MP）。所得树的拓扑结构基本一致,差异只发生在距离树和简约树之间,在由形态和细胞学关系都很近的5个种（牡丹P.suffruticosa、矮牡丹P.jishanensis、卵叶牡丹P.qiui、紫斑牡丹P.rockii和凤丹P.o     

Systematic studies on Paeonia sect. Moutan DC. based on RAPD analysis

Plants in Paeonia sect. Moutan DC., whose wild types are endemic to China, are deciduous subshrubs. Taxonomic treatments of most species in this section have long been in dispute. To address this question, both intraspecific and interspecific relationships of the species in this section were analyzed using RAPD markers. The dendrogram constructed by UPGMA showed that the accessions of the same species were always grouped together earlier than those of different species. The intraspecific similarity coefficients ranged from 0.60 to 0.90, grouping precisely those species of the same subsection together. Hence, the seven species under question can be well distinguished from each other. The similarity coefficient between P. delavayi and P. ludlowii was 0.60, and they were clustered in a clade. The similarity coefficients between P. jishanensis and the three species P. rockii, P. ostii, P. qiui, and between P. jishanensis and P. decomposita were both 0.48. These five species were clustered in another clade. These two clades corresponded well to Subsect. Delavayanae and Subsect. Vaginatae. Our results support the taxonomic treatment of Sect. Moutan re- cently proposed by Hong (1998,1997).     

芍药属牡丹组的系统学研究——基于RAPD分析

芍药属牡丹组(Paeonia sect．Moutan DC)是落叶亚灌木,其野生类群为我国特有。长期以来不同 学者根据形态性状对这个组中种的分类处理不断修正,不断有新种描述。我们采用RAPD标记分析了 牡丹组种内与种间遗传关系。从10个RAPD引物获得121个多态位点。用UPGMA方法构建的树系 图表明每个种的所有个体都各自聚为一支,种内的相似性系数为0.60～0.90,因此现有的7个种能很好 地区分开来。P．delavayi与P．ludlowii相似性系数为0.60,聚为一支;P．jishanensis与P．rockii、P. ostii、P．qiui以及P．decomposita之间的相似性系数为0.48,聚为一大支。这两支与肉质花盘亚组和革 质花盘亚组相对应。这些结果与洪德元根据形态性状对该组所做的分类处理基本相符。我们认为RAPD技术用于牡丹基因组分析是灵敏而行之有效的工具。     

红麻及其近缘种的RAPD分析

利用随机扩增多态性DNA（RAPD）技术分析了木槿属（Hibiscus)Furcaria组中纤维作物红麻（H.cannabinus)及其6个近缘种植物的25份材料,用筛选出的16个引物扩增出192个RAPD条带,它们表现出丰富的多态性。根据得到的RAPD指纹图谱,计算其Nei氏相似系数和遗传距离。并构建了它们的系统树。结果表明,25份材料可划分为7个组。H.penduriformis和H.calyphyllus两个种为一组;红麻种分为两个组,一组为栽培品种,另1组为野生型材料;其余4个种各成为1组。玫瑰麻（H.sabdariffa)和金线吊芙蓉（H.radiatus)的关系较近,而且两者与红麻的亲缘关系也较近,而其它四个种与红麻的关系较远。H.trionum与其它六个种的关系较远。     

Phylogenetic analyses of Paeonia section Moutan（treepeonies，Paeoniaceae）based on morphological data

Cladistic analysis of the morphological relationship of40populations of Paeonia sect.Moutan DC.（including all wild species）was carried out with an attempt to have a better understanding of the phylogeny of tree peonies.Twenty-five morphological characters     

Application of High-Throughput Sequencing to Evaluate the Genetic Diversity Among Wild Apple Species Indigenous to Shandong,China, and Introduced Cultivars

The Taiyi mountainous region of Shandong province in eastern China has an abundance of wild Malus species. We evaluated the genetic diversity of 88 Malus accessions (45 Asian apple cultivars, 10 American apple cultivars, 12 European apple cultivars, 19 Chinese wild apples, and two apple cultivars with unknown origins) based on single-nucleotide polymorphism (SNP) markers. A total of 38,364 SNPs were obtained with an average of 2256 SNPs per chromosome. The average of the polymorphism information content (PIC), gene diversity, and allele frequency for SNPs was 0.268, 0.306, and 0.364, respectively. A circular phylogenetic tree constructed based on SNP data revealed that the 88 Malus accessions could be divided into three groups. However, a population structure analysis suggested the 88 Malus accessions could be divided into four groups. A principal component analysis (PCA) revealed some population stratification. The first three PCs accounted for 41.62% of the population-wide SNP variation, with PC1 accounting for 33.9%. Moreover, the kinship values of the 88 Malus accessions ranged from 0 to 2.36, with 96.42% of the kinship values between 0 and 0.2. A phylogenetic tree and a PCA indicated the Chinese wild apples widely distributed among the cultivated apples had a diverse genetic background. Characterizing the genetic relationships between cultivated apples and Chinese wild apples is essential for increasing the genetic diversity of the germplasms used by apple breeders.

     

Genetic structure and diversity analysis of the primary gene pool of chickpea using SSR markers

Members of the primary gene pool of the chickpea, including 38 accessions of Cicer arietinum, six of C. reticulatum and four of C. echinospermum grown in India were investigated using 100 SSR markers to analyze their genetic structure, diversity and relationships. We found considerable diversity, with a mean of 4.8 alleles per locus (ranging from 2 to 11); polymorphic information content ranged from 0.040 to 0.803, with a mean of 0.536. Most of the diversity was confined to the wild species, which had higher values of polymorphic information content, gene diversity and heterozygosity than the cultivated species, suggesting a narrow genetic base for cultivated chickpea. An unrooted neighbor-joining tree, principal coordinate analysis and population structure analysis revealed differentiation between the cultivated accessions and the wild species; three cultivated accessions were in an intermediate position, demonstrating introgression within the cultivated group. Better understanding of the structure, diversity and relationships within and among the members of this primary gene pool will contribute to more efficient identification, conservation and utilization of chickpea germplasm for allele mining, association genetics, mapping and cloning gene(s) and applied breeding to widen the genetic base of this cultivated species, for the development of elite lines with superior yield and improved adaptation to diverse environments.     

Extensive ribosomal DNA amplification during Andean common bean (Phaseolus vulgaris L.) evolution

The extent of 5S and 45S ribosomal DNA (rDNA) variation was investigated in wild and domesticated common beans (Phaseolus vulgaris) chosen to represent the known genetic diversity of the species. 5S and 45S rDNA probes were localized on mitotic chromosomes of 37 accessions by fluorescent in situ hybridization (FISH). The two 5S rDNA loci were largely conserved within the species, whereas a high variation in the number of 45S rDNA loci and changes in position of loci and number of repeats per locus were observed. Domesticated accessions from the Mesoamerican gene pool frequently had three 45S rDNA loci per haploid genome, and rarely four. Domesticated accessions from Andean gene pool, particularly from the race Peru, showed six, seven, eight or nine loci, but seven loci were found in all three races of this gene pool. Between three and eight loci were observed in accessions resulting from crosses between Andean and Mesoamerican genotypes. The presence of two to eight 45S rDNA loci in wild common beans from different geographic locations indicates that the 45S rDNA amplification observed in the Andean lineage took place before domestication. Our data suggest that ectopic recombination between terminal chromosomal regions might be the mechanism responsible for this variation.     

Chloroplast diversity in the genus Malus: new insights into the relationship between the European wild apple (Malus sylvestris (L.) Mill.) and the domesticated apple (Malus domestica Borkh.)

To unravel the relationship between the European wild apple, Malus sylvestris (L.) Mill., and its domesticated relative M. domestica Borkh., we studied chloroplast DNA variation in 634 wild and 422 domesticated accessions originating from different regions. Hybridization between M. sylvestris and M. domestica was checked using 10 nuclear microsatellites and a Bayesian assignment approach. This allowed us to identify hybrids and feral plants escaped from cultivation. Sixty-eight genotypes belonging to 12 other wild Malus species, including 20 M. sieversii (Ledeb.) Roem. accessions were also included in the analysis of chloroplast diversity. Marker techniques were developed to type a formerly described duplication and a newly detected transversion in the matK gene. Chloroplast DNA variation was further investigated using PCR-RFLP (Polymerase Chain Reaction-Random Fragment Length Polymorphism), and haplotypes were constructed based on all mutational combinations. A closer relationship than presently accepted between M. sylvestris and M. domestica was established at the cytoplasmic level, with the detection of eight chloroplast haplotypes shared by both species. Hybridization between M. sylvestris and M. domestica was also apparent at the local level with sharing of rare haplotypes among local cultivars and sympatric wild trees. Indications of the use of wild Malus genotypes in the (local) cultivation process of M. domestica and cytoplasmic introgression of chloroplast haplotypes into M. sylvestris from the domesticated apple were found. Only one of the M. sieversii trees studied displayed one of the three main chloroplast haplotypes shared by M. sylvestris and M. domestica. This is surprising as M. sieversii has formerly been described as the main maternal progenitor of the domesticated apple. This study hereby reopens the exciting discussion on the origin of M. domestica.     

Molecular and Cytogenetic Characterization of Wild Musa Species

The production of bananas is threatened by rapid spreading of various diseases and adverse environmental conditions. The preservation and characterization of banana diversity is essential for the purposes of crop improvement. The world''s largest banana germplasm collection maintained at the Bioversity International Transit Centre (ITC) in Belgium is continuously expanded by new accessions of edible cultivars and wild species. Detailed morphological and molecular characterization of the accessions is necessary for efficient management of the collection and utilization of banana diversity. In this work, nuclear DNA content and genomic distribution of 45S and 5S rDNA were examined in 21 diploid accessions recently added to ITC collection, representing both sections of the genus Musa. 2C DNA content in the section Musa ranged from 1.217 to 1.315 pg. Species belonging to section Callimusa had 2C DNA contents ranging from 1.390 to 1.772 pg. While the number of 45S rDNA loci was conserved in the section Musa, it was highly variable in Callimusa species. 5S rRNA gene clusters were found on two to eight chromosomes per diploid cell. The accessions were genotyped using a set of 19 microsatellite markers to establish their relationships with the remaining accessions held at ITC. Genetic diversity done by SSR genotyping platform was extended by phylogenetic analysis of ITS region. ITS sequence data supported the clustering obtained by SSR analysis for most of the accessions. High level of nucleotide diversity and presence of more than two types of ITS sequences in eight wild diploids pointed to their origin by hybridization of different genotypes. This study significantly expands the number of wild Musa species where nuclear genome size and genomic distribution of rDNA loci is known. SSR genotyping identified Musa species that are closely related to the previously characterized accessions and provided data to aid in their classification. Sequence analysis of ITS region provided further information about evolutionary relationships between individual accessions and suggested that some of analyzed accessions were interspecific hybrids and/or backcross progeny.     

Use of microsatellites to evaluate genetic diversity and species relationships in the genus Lycopersicon

In order to determine how informative a set of microsatellites from tomato is across the genus Lycopersicon, 17 microsatellite loci, derived from regions in and around genes, were tested on 31 accessions comprising the nine species of the genus. The microsatellite polymorphisms were used to estimate the distribution of diversity throughout the genus and to evaluate the efficacy of microsatellites for establishing species relationships in comparison with existing phylogeny reconstructions. Gene diversity and genetic distances were calculated. A high level of polymorphism was found, as well as a large number of alleles unique for species. The level of polymorphism detected with the microsatellite loci within and among species was highly correlated with the respective mating systems, cross-pollinating species having a significantly higher gene diversity compared to self-pollinating species. In general, microsatellite-based trees were consistent with a published RFLP-based dendrogram as well as with a published classification based on morphology and the mating system. A tree constructed with low-polymorphic loci (gene diversity <0.245) was shown to represent a more-reliable topology than a tree constructed with more-highly polymorphic loci. Received: 19 February 2001 / Accepted: 26 March 2001     

Phylogenetic relationships among Lactuca (Asteraceae) species and related genera based on ITS-1 DNA sequences

Internal transcribed spacer (ITS-1) sequences from 97 accessions representing 23 species of Lactuca and related genera were determined and used to evaluate species relationships of Lactuca sensu lato (s.l.). The ITS-1 phylogenies, calculated using PAUP and PHYLIP, correspond better to the classification of Feráková than to other classifications evaluated, although the inclusion of sect. Lactuca subsect. Cyanicae is not supported. Therefore, exclusion of subsect. Cyanicae from Lactuca sensu Feráková is proposed. The amended genus contains the entire gene pool (sensu Harlan and De Wet) of cultivated lettuce (Lactuca sativa). The position of the species in the amended classification corresponds to their position in the lettuce gene pool. In the ITS-1 phylogenies, a clade with L. sativa, L. serriola, L. dregeana, L. altaica, and L. aculeata represents the primary gene pool. L. virosa and L. saligna, branching off closest to this clade, encompass the secondary gene pool. L. virosa is possibly of hybrid origin. The primary and secondary gene pool species are classified in sect. Lactuca subsect. Lactuca. The species L. quercina, L. viminea, L. sibirica, and L. tatarica, branching off next, represent the tertiary gene pool. They are classified in Lactuca sect. Lactucopsis, sect. Phaenixopus, and sect. Mulgedium, respectively. L. perennis and L. tenerrima, classified in sect. Lactuca subsect. Cyanicae, form clades with species from related genera and are not part of the lettuce gene pool.     

Diversity and Species Relationship in Pearl Millet (Pennisetum typhoides) and Related Species

Thirteen accessions of pearl millet (Pennisetum typhoides (L) Leeke) collected from different states of India and eight wild species of the genus Pennisetum across the world were analyzed for genetic diversity using AFLP markers. A combined analysis of eight primer combinations showed 35% polymorphism among P. typhoides accessions while analysis with five primer combinations showed 99% polymorphism among the wild species. The dendrogram constructed for the P. typhoides accessions based on the UPGMA method revealed two major clusters with samples from Gujarat forming a separate cluster from the rest of the samples. Principal component analysis of the same data set revealed similar results with the first principal component accounting for 65% of the total variation. The percentage of rare and common alleles contributing to the diversity in the sample was analyzed using the Shannon Weiner diversity index. The SW index revealed that the samples from Gujarat contributed significantly to the overall diversity among the accessions. Among accessions of each geographical region, considerable variation was revealed by SW index with samples from Tamil Nadu being most polymorphic. The genetic diversity in the accessions could be utilized for future breeding work. The dendrogram constructed for the wild species revealed the extent of genetic diversity among them. Analysis with one primer combination showed P. typhoides being closer to P. mollissimum than to the other analyzed species.     

Taxonomic relationships among Arachis sect. Arachis species as revealed by AFLP markers.

Cultivated peanut, Arachis hypogaea L., is a tetraploid (2n = 4x = 40) species thought to be of allopolyploid origin. Its closest relatives are the diploid (2n = 2x = 20) annual and perennial species included with it in Arachis sect. Arachis. Species in section Arachis represent an important source of novel alleles for improvement of cultivated peanut. A better understanding of the level of speciation and taxonomic relationships between taxa within section Arachis is a prerequisite to the effective use of this secondary gene pool in peanut breeding programs. The AFLP technique was used to determine intra- and interspecific relationships among and within 108 accessions of 26 species of this section. A total of 1328 fragments were generated with 8 primer combinations. From those, 239 bands ranging in size from 65 to 760 bp were scored as binary data. Genetic distances among accessions ranged from 0 to 0.50. Average distances among diploid species (0.30) were much higher than that detected between tetraploid species (0.05). Cluster analysis using different methods and principal component analysis were performed. The resulting grouping of accessions and species supports previous taxonomic classifications and genome designations. Based on genetic distances and cluster analysis, A-genome accessions KG 30029 (Arachis helodes) and KSSc 36009 (Arachis simpsonii) and B-genome accession KGBSPSc 30076 (A. ipaensis) were the most closely related to both Arachis hypogaea and Arachis monticola. This finding suggests their involvement in the evolution of the tetraploid peanut species.     

Chloroplast DNA study in wild populations and some cultivars of Prunus avium L.

The PCR-RFLP technique was used to detect chloroplast DNA diversity in wild populations of Prunus avium from five European deciduous forests and some cultivars. A study of 10.8% of the total chloroplast genome detected eight insertion-deletion (indel) mutations, distributed over 12 haplotypes. Six haplotypes (H1, H2, H3, H4, H5 and H6) were found in wild populations and eight (H2, H6, H7, H8, H9, H10, H11 and H12) in the cultivars. Only two haplotypes (H2 and H6) are shared by the wild populations and the cultivars. The most-abundant and frequent haplotype in wild populations is H2 (frequency=78%). The wider geographical distribution along with the high frequency reflects its ancient origin. Of the five populations, three are polymorphic. Populations GA (Scotland) and KE (Germany) have unique haplotypes. The total cpDNA diversity in wild populations is h_T=0.40, and a major portion of it is within populations (h_S=0.37). The genetic differentiation among populations was low (G_STC=0.08) and no genetic structure among wild populations was observed. A minimum-length spanning tree, demonstrating relationships among the haplotypes in wild populations, indicated two possible chloroplast lineages. The ten identified cultivars were represented by seven haplotypes; this result proposes the possible utilisation of the PCR-RFLP technique for the characterisation of sweet cherry cultivars. The cpDNA diversity in P. avium should be considered carefully for phylogenetic studies involving this species. Received: 10 July 2000 / Accepted: 19 October 2000     

Species identification in seven small millet species using polymerase chain reaction-restriction fragment length polymorphism of trnS-psbC gene region.

The chloroplast trnS-psbC gene regions from total genomic DNA of 119 accessions from seven small millet species were amplified by polymerase chain reaction (PCR) and digested with eight restriction enzymes individually as well as in combinations of two enzymes to generate restriction fragment length polymorphism (PCR-RFLP). PCR-RFLP with individual enzymes revealed polymorphism between only some species. However, all the species could be distinguished by using a combination of two enzymes, specifically HaeIII and MspI. PCR-RFLP of 11 to 20 accessions with the same enzyme combination showed no intraspecific variation, which established that the differential banding patterns were species specific. In contrast, the same enzyme combination was not useful for differentiating different species of the genera Cajanus, Rhyncosia, Abies, Rhizophora, Ceriops, and Bruguiera, and it also revealed intraspecies variation in three species of Abies. The present study indicated that digestion of trnS-psbC with two four-base recognizing enzymes reveals more variation than with either enzyme alone and that it may be a method of choice for species identification in some genera.     

Restriction fragment variation in the nuclear and chloroplast genomes of cultivated and wild Sorghum bicolor

Summary Fifty-six accessions of cultivated and wild sorghum were surveyed for genetic diversity using 50 low-copy-number nuclear DNA sequence probes to detect restriction fragment length polymorphisms (RFLPs). These probes revealed greater genetic diversity in wild sorghum than in cultivated sorghum, including a larger number of alleles per locus and a greater portion of polymorphic loci in wild sorghum. In comparison to previously published isozyme analyses of the same accessions, RFLP analysis reveals a greater number of alleles per locus. Furthermore, many RFLP alleles have frequencies between 0.25–0.75, while the vast majority of isozyme alleles are either rare (< 0.25) or near fixation (> 0.75). Correlations between genetic and geographic distances among the accessions were stronger when calculated with RFLP than with isozyme data. Systematic relationships revealed by nuclear and chloroplast restriction site analysis indicate that cultivated sorghum is derived from the wild ssp. arundinaceum. The portion of the wild gene pool most genetically similar to the cultivars is from central-northeastern Africa. Previous published data also suggested that this is most likely the principal area of domestication of sorghum. Introgression between wild and cultivated sorghum was inferred from disconcordant relationships shown by nuclear and chloroplast DNA markers. Introgression apparently occurs infrequently enough that the crop and its wild relatives maintain distinct genetic constitutions.