Genetic diversity of Iranian and some European grapes as revealed by nuclear and chloroplast microsatellite and SNP molecular markers

Genetic and chloroplast haplotype variations of 35 Iranian genotypes and 10 European grape cultivars were investigated using 9 nuclear simple sequence repeats (nSSRs), 4 chloroplast simple sequence repeats (cpSSRs) and 46 single nucleotide polymorphism (SNP) markers. In total, 83 alleles were detected at nine nSSRs, giving a mean of 15.66 alleles per locus and polymorphism information content (PIC) values ≥0.75 ranged from 0.75 to 0.90. For SNP markers, PIC values varied from 0.30 to 0.39 with an average of 0.34. Analysis of molecular variance revealed 97 and 93% of partitioned genetic diversity within populations using nSSRs and SNPs markers, respectively. Un-weighted neighbour-joining (NJ) cluster analysis grouped grapes into 10 and 9 major clusters using SSR and SNP markers, respectively. Synonyms and homonyms were identified among the Iranian genotypes. Close genetic relationship among Farkhi and Bidane-Sefid genotypes may probably propose a common ancestor and mutational evolution. Most European cultivars were differentiated from Iranian genotypes, however, clustering of some Iranian genotypes with European cultivars in the same clusters suggests that clonally propagated materials have probably been exchanged from the Middle East to West or vice versa. C and D chloroplast haplotypes were the most frequent within the Iranian genotypes, while A chloroplast haplotype was exclusively observed among European cultivars.     

The origin and dissemination of the cultivated almond as determined by nuclear and chloroplast SSR marker analysis

Sixteen nuclear and 10 chloroplast SSR markers were evaluated for 40 almond genotypes including cultivated almond, 18 related species and 5 interspecific-hybrid populations. Results establish the value of SSR (nuclear and chloroplast) markers for distinguishing different genetic lineages and characterize an extensive gene pool available to almond genetic improvement. Hierarchical analysis using integrated nuclear and chloroplast DNA markers support Prunus fenzliana, a species native to the northeast Iran, as a probable ancestor of the cultivated almond. Results also established the importance of interspecific hybridization and subsequent genetic introgression in the development of cultivated almond and demonstrate continuing value of an interspecific gene pool for modern cultivar improvement. Molecular results implicate a dissemination of the cultivated almond from Asia to the Eastern Mediterranean and subsequently the Western Mediterranean and the New World is supported by the molecular analysis of regional germplasm.     

Development of specific primers for cpSSR analysis in caper,olive and grapevine using consensus chloroplast primer pairs

Chloroplast SSR (cpSSR) markers have demonstrated utility in studying genetic relationships. DNA sequence information of the chloroplast genome is necessary for the development of cpSSR primer pairs. To overcome this limitation, “consensus” primers have been developed to amplify the homologous regions in plants where chloroplast sequences are not available. However, 80% Pinus thunbergi and Nicotiana tabacum developed “consensus” primers tested with grapevine, olive and caper showed multi-locus patterns. The presence of multi-locus patterns requires the use of agarose gel electrophoresis followed from isolation and sequencing of the bands. Herein, a PCR-strategy is proposed to construct specific cpSSR primer pairs without genomic sequence information, giving single-band amplifications that can be directly sequenced. Twelve new specific cpSSR primer pairs were developed for Capparis spinosa L., Olea europea L. and Vitis vinifera L. PCR products were sequenced to confirm the presence of microsatellite sequences, and their transportability was tested on six V. vinifera cultivars. Both single-nucleotide polymorphisms and polymorphic cpSSR were observed in the six grapevine cultivars using the specific cpSSR primers.     

叶绿体DNA分析技术及其在栗属植物中的应用

被子植物叶绿体DNA母性遗传,有独立的进化路线,基于叶绿体DNA的分析技术在分子系统学、资源多样性评价、杂种鉴定等方面具有重要作用。栗属植物资源丰富,分布广泛。概述了叶绿体DNA分析技术在栗属植物中的研究进展,阐述了包括PCR-RFLP、DNA杂交、叶绿体SSR标记技术和叶绿体基因区段测序分析技术在内的分子标记技术的原理、特点及其在栗属植物中的应用。并对今后如何开发叶绿体DNA标记用于栗属植物的研究进行了展望。     

贵州威宁红花油茶的叶绿体基因组特征分析

对威宁红花油茶（Camellia weiningensis）的叶片高通量测序数据进行叶绿体全基因组的组装和注释分析。其叶绿体全基因组长为156 490 bp,包含典型的四分体结构,序列已登录GenBank（MK820035）。叶绿体全基因组比较分析表明,与其他山茶属物种一样,其结构与基因排序均保守,但在基因组的反向重复区边界处表现出明显区别。简单重复序列（SSR）分析表明,全基因组仅包含51个单核苷酸重复的SSR,且仅为A/T重复类型。系统发育分析表明,威宁红花油茶与腾冲红花油茶亲缘关系最近,但其所处分支包含的4个山茶属物种的分组与传统分类不一致。     

Comparison of the use of morphological,protein and DNA markers in the genetic characterization of Iranian wild Prunus species

In this study, the genetic diversity of four Iranian wild Prunus species including Prunus eleagnifolia, Prunus hauskenchtii, Prunus scoparia and Prunus lycioides were investigated using morphological, protein and DNA markers. DNA markers included nuclear and chloroplast SSRs and self-incompatibility (S) allele amplification. At the morphological level, leaf width showed significant differences between the four wild Prunus species. Concerning fruit and kernel characters, their values are relatively similar indicating the high degree of homoplasy described in Prunus. Nuclear SSR markers have been the most abundant markers with a higher polymorphism in comparison with morphological, protein and chloroplast SSR markers. Results also indicated the high variability present in the S locus. On the other hand, the correlation between the clustering based on DNA markers and protein were in general low. Dendogram performed using nuclear and chloroplast SSR indicated a more diffuse clustering between the wild almond species probably due to the natural introgression of genes observed in these wild almond species. Data from the analysis of the total protein seems to be more accurate to establish taxonomy relationships in these very close wild species.     

福建省梨地方品种的遗传多样性研究

利用叶绿体DNA非编码区序列acc D-psa I和17对SSR引物对原产于福建省的49个梨地方品种的遗传多样性进行分析,旨在为育种利用提供参考。获得49份福建地方品种的acc D-psa I序列,并检测到4个多态性位点,包含5个叶绿体单倍型(Hap1~Hap5),其中核苷酸多态性(Pi)为0.00139,单倍型多样性(Hd)为0.660。TCS网络图显示Hap5是最古老的单倍型,其仅在两个样品中检测到。17个SSR位点共检测到211个等位基因位点,有效等位基因数(A)为5~23个,平均值为12.41;观察杂合度(Ho)和期望杂合度(He)的平均值分别为0.5802和0.7549;17个SSR位点的Shannon’s信息指数(I)值为0.5830~2.7683,平均值为1.8661,表现出较高的遗传多样性。基于Nei和Li的遗传距离的邻接法(Neighbor-Joining,NJ)将49份样品聚为9个组,其中大部分聚类结果与单倍型类型表现的亲缘关系较为一致。叶绿体单倍型和SSR多态性位点信息证实福建地方品种具有较为丰富的遗传多样性。     

Assessment of genetic relationships among 29 introduced and 49 local sweet cherry accessions in Turkey using AFLP and SSR markers

Summary

The characterisation of sweet cherry (Prunus avium L.) genetic resources in Turkey may help to increase their use in breeding programmes worldwide, as Turkey is the centre of origin of sweet cherry. Amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers were therefore used to analyse genetic diversity among a total of 78 local and introduced sweet cherry cultivars. Four AFLP primer combinations, and six SSR primer pairs for sweet cherry were used for genetic diversity analysis. A genetic similarity matrix was calculated using the combined data from AFLP and SSR analyses with simple matching coefficient. Genetic similarities among the sweet cherry genotypes studied were higher than 42%. No two accessions had an identical AFLP and SSR marker profile, indicating that all 78 genotypes were unique. An UPGMA dendrogram, based on the similarity matrix, revealed 18 separate Groups at or above the 70% similarity level. While some Groups consisted of both introduced and local genotypes, other Groups had only local genotypes. This result suggests that there was broad genetic diversity among the local Turkish sweet cherry genotypes, which was not present in the introduced sweet cherry accessions. The genetic variation present in local Turkish sweet cherry genotypes may be useful for future breeding programmes. We found that the use of both SSR and AFLP marker systems was effective for distinguishing between genetically-close sweet cherry genotypes. These marker systems can be used to complement pomological and morphological markers during the characterisation and identification of sweet cherry genotypes.     

Phylogenetic analysis in some Diospyros spp. (Ebenaceae) and Japanese persimmon using chloroplast DNA PCR-RFLP markers

Ten universal primer pairs of chloroplast genome were used to amplify non-coding regions of chloroplast DNA (cpDNA) in 7 Diospyros L. species including 29 genotypes and approximately 20.4 kb, 12.6% of the chloroplast genome were analyzed. The amplified products were digested by seven restriction enzymes. The results showed that there were abundant polymorphisms in inter-specific cpDNA within the genus Diospyros. However, it was not observed intra-species variations in 22 tested genotypes of Diospyros kaki (Japanese persimmon), except for ‘Male strain No. 9’. Persimmon had close relationship with Diospyros lotus and Diospyros glaucifolia, but distantly with Diospyros virginiana and Diospyros rhombifolia in diagram based on principal coordinates analysis and Wagner parsimony method. The discrepancy of digesting pattern in cpDNA suggested that the genotype Jinzaoshi was distinct with the remaining Diospyros spp., which revealed that Jinzaoshi may be a new species of the genus Diospyros.     

Functional characterization of watermelon (Citrullus lanatus L.) EST–SSR by gel electrophoresis and high resolution melting analysis

The present work was conducted to characterize the functionality of 257 watermelon EST–SSR primer pairs for their PCR amplification and polymorphisms. EST–SSR markers were tested on DNA sample panels of six watermelon cultigens and two related species of Citrullus lanatus var. citroides and Citrullus colocynthis based on agarose gel electrophoresis and high resolution melting (HRM) analysis. Successful PCRs were shown for 240 primer pairs (79%), and 173 primer pairs (67%) were polymorphic in a watermelon DNA sample panel on agarose gel electrophoresis. In addition, HRM analysis of 24 EST–SSR markers that were monomorphic on agarose gel separation identified an additional 19 polymorphic markers, indicating that HRM is an efficient tool for the rapid screening of sequence variations and allele discrimination. In the assessment of genetic relationships, six watermelon cultivars were closely related together (0.91–0.97) and demonstrated a narrow genetic base in the watermelon genetic pool. A high level of genetic dissimilarity (0.36–0.97) was shown between watermelon species and other related species. Marker transferability to melon species (Cucumis melo L.) was examined by cross-species PCR amplification and genetic diversity assessment in eight melon cultigens. Of the 257 EST–SSR primer pairs, 79 (32.9%) showed successful PCR amplification from melon DNA samples. A dendrogram of the genetic relationship based on 22 EST–SSR markers showed a clear classification of melon genotypes in accordance to fruit characteristics. The EST–SSR markers characterized in this study will contribute to diverse genomic investigations and breeding efforts, including comparative genome mapping, marker-assisted selection, and DNA fingerprinting for genetic diversity and cultivar identification in many cucurbit crops.     

Rose (Rosa hybrida L.) EST-derived microsatellite markers and their transferability to strawberry (Fragaria spp.)

The ‘Genome database for Rosaceae (GDR)’ provides a large collection of expressed sequence tags (ESTs) harboring simple sequence repeats (SSRs) from several Rosaceae genera, including Rosa (rose). Primer pairs flanking SSR were designed for 312 unique Rosa ESTs based on GDR database. Eight rose (Rosa hybrida L.) genotypes were tested for PCR amplification, and 287 (92%) of the primer pairs generated allele-specific PCR bands that were readily scored. From 183 (63.7%) primer pairs that evidenced polymorphic alleles among the eight rose cultivars, 20 pairs evidencing EST sequence homology to known gene functions and high levels of polymorphism were selected and utilized for DNA fingerprinting and genetic diversity assessments of 47 rose hybrids. A total of 202 polymorphic bands were scored and generated unique fingerprints for each rose hybrid. The Nei–Li genetic similarity coefficients among 1081 pair-wise comparisons of 47 cultivars exhibited a broad range of genetic variations from 0.30 (‘Grand King’ and ‘Carnival’) to 0.99 (‘First Red’ and ‘Red Champ’). UPGMA cluster analysis divided 47 hybrids into five major groups and two sub-groups. The cross-species transferability of 273 EST-SSR primer pairs was evaluated using four genotypes of the strawberry, a genus member of the Rosaceae family. PCRs on the DNA samples of strawberry were successful for 165 primer pairs; among these, 123 pairs amplified 243 polymorphic bands. As surrogates of the marker transfer, the phenetic relationship among the four strawberry genotypes was evaluated. Genetic similarity coefficients varied from 0.78 (‘Maehyang’ and ‘Janghyee’) to 0.64 (‘Janghyee’ and ‘Pragana’). The results of cluster analysis showed that the three octaploid strawberry cultivars were quite similar, whereas the diploid ‘Pragana’ was related distantly at the genomic DNA level. The EST-SSR markers developed in the present study can be efficiently utilized for genetic diversity studies in Rosaceae.     

Variety identification and comparative analysis of genetic diversity in yardlong bean (Vigna unguiculata spp. sesquipedalis) using morphological characters,SSR and ISSR analysis

Genetic diversity and relatedness of 23 yardlong bean (Vigna unguiculata spp. sesquipedalis) accessions and 7 accessions of a hybrid between cowpea (V. unguiculata spp. unguiculata) and yardlong bean (dwarf yardlong bean) in Thailand were estimated using morphological characters, simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers. In addition, two mungbean (Vignaradiata (L.) Wilczek) and two blackgram (Vigna mungo (L.) Hepper) accessions were also used as outgroup species for molecular analysis. Five morphological characters were diverse among most accessions. However, five groups of 2–3 accessions could not be distinguished from one another based on these morphological characters alone. Unweighted pair-group arithmetic average (UPGMA) analysis of these characters separated these 30 accessions into 2 major groups; the yardlong bean group and the dwarf yardlong bean group. Eleven of the sixteen SSR primers yielded clear SSRs, ten of which were polymorphic (90.91% polymorphism), detecting a total of 54 alleles with an average of 4.91 alleles per locus. These 10 polymorphic SSR markers successfully distinguished 28 yardlong bean and dwarf yardlong bean accessions. The polymorphic information content (PIC) among genotypes varied from 0.251 to 0.752 with an average of 0.597. Among the 16 ISSR primers used, a total of 312 ISSR fragments were amplified for these three Vigna species, revealing the polymorphism percentage of 91.03%. The average ISSR PIC value (0.197) with the range of 0.137–0.276 was lower than that of SSR. Nevertheless, the average marker index of this multilocus marker was 3.495, which was higher than that of SSR (0.669), owing to the differences in the effective multiplex ratio. In addition, Mantel test cophenetic correlation coefficient was higher for ISSR (0.566) than that of SSR (0.198). These results indicated higher efficiency of ISSR for estimating the levels of genetic diversity and relationships among yardlong beans and dwarf yardlong beans in this study. Pair-wise coefficients of SSR- and ISSR-based genetic similarity among all yardlong bean and dwarf yardlong bean accessions averaged 0.87 and 0.91, respectively, suggesting a narrow genetic base that emphasizes the need to broaden genetic diversity to ensure continued breeding success. Clustering of genotypes within groups was not similar when SSR and ISSR derived dendrograms from UPGMA analysis were compared. It appeared that ISSR was the most effective marker system in determining the genetic variability and relationships among yardlong bean and dwarf yardlong bean accessions and differentiating three Vigna species. In addition, ISSR was also most useful for variety identification since all 30 yardlong beans and dwarf yardlong bean accessions can be effectively distinguished by only four ISSR primers with the highest PIC values.     

杜梨叶绿体基因组分析

利用高通量测序平台对杜梨（Pyrus betulaefolia）进行测序，并对其叶绿体全基因组序列的结构特征进行分析。结果表明，杜梨叶绿体基因组与大多数高等植物一样，具有典型的环状双链四分体结构。其叶绿体基因组大小为160 058 bp，共检测到61 个SSR 位点（58 个单核苷酸重复和3 个二核苷酸重复）和56 个RNA 编辑位点，这些RNA 编辑位点均引起了氨基酸的变化。与砂梨（Pyrus pyrifolia）、川梨（Pyrus pashia）和桃叶梨（Pyrus spinosa）的叶绿体基因组比较，其基因种类和数量都比较保守，表明了梨属植物进化的缓慢性。在4 个梨属植物中共检测到351 个SNP 和217 个InDel。     

Assessment of Genetic Variability in Pistachio (<Emphasis Type="Italic">Pistacia vera</Emphasis> L.) with Nuclear SSR Molecular Markers

Pistachio (Pistacia vera L.) is considered to be the most widely cultivated species in the genus Pistacia and regarded as the most probable ancestral species within the genus. As one of the oldest nut crops in human history, pistachio nuts have a high nutritional value and are commercially important. In the present study, the genetic variation of pistachio genotypes was investigated by nuclear SSR markers. The transferability of the SSR markers was high across genotypes, thus, flanking regions of the SSRs are conserved in the studied genotypes. High level of polymorphism was detected among the studied genotypes. The seven SSR primer pairs generated a total of 18 alleles that 13 of them were polymorphic among the genotypes. The range of the polymorphic alleles was 1 (for Ptms9, Ptms40, Ptms41, and Ptms42) to 5 (for Ptms7 locus) with an average of 2.57. The amplified allele sizes ranged from 120 to 250?bp. Pair-wise genetic similarity coefficients varied from 0.20 to 0.75. The UPGMA dendrogram differentiated the genotypes into two major clusters. The present results may be used for the conservation, core collection and future breeding of the pistachio.     

Molecular characterization and genetic relationship among almond cultivars assessed by RAPD and SSR markers

RAPDs and SSRs were used to study the genetic diversity of Iranian almond cultivars and their relationship to important foreign cultivars and three related species. Eight unidentified almond Shahrodi cultivars and three wild almonds (Prunus communis, Prunus orientalis and Prunus scoparia) were also included. Of the primers tested, 42 (out of 80) RAPD and 18 (out of 26) SSR primers were selected for their reproducibility and high polymorphism. A total of 664 polymorphic RAPD bands were detected out of 729 bands. The number of presumed alleles revealed by the SSR analysis ranged from 3 to 10 alleles per locus with a mean value of 6.64 alleles per locus. Both techniques discriminated the genotypes very effectively, but only RAPDs were able to discriminate the cultivars Monagha and Sefied. Results demonstrated an extensive genetic variability within the tested cultivars as well as the value of SSR markers developed in peach for characterization of almond and related species of Prunus. Dice similarity coefficient was calculated for all pair wise comparisons and was used to construct a UPGMA dendrogram. For both markers a high similarity in dendrogram topologies was obtained although some differences were observed. All dendrograms, including that obtained by the combined use of both the marker data, depicted the phenetic relationships among the cultivars and species, depending upon their geographic region and/or pedigree information. Almond cultivars clustered with accession of P. communis showing their close relationship. P. orientalis and P. scoparia were clustered out of the rest of P. dulcis.     

大花君子兰叶绿体基因组及其特征

采用Illumina MiSeq测序平台对大花君子兰（Clivia miniata）叶片总DNA进行测序，通过组装获得了其叶绿体基因组（cpDNA）全长序列（158 114 bp）。对其cpDNA注释得到135个基因，包含87个蛋白编码基因、40个tRNA基因和8个rRNA基因。采用生物信息学方法对获得的cpDNA进行简单序列重复（SSR）分析和密码子偏好性分析。结果显示：①大花君子兰cpDNA中共有61个SSR位点，其中单核苷酸、二核苷酸、三核苷酸、四核苷酸、五核苷酸和六核苷酸重复数分别为38、9、2、8、3和1个，多数SSR分布在基因间隔区；②大花君子兰cpDNA密码子偏爱以A或U（T）结尾，亮氨酸使用频率最高，半胱氨酸使用频率最低。基于24种植物的cpDNA全长和23种植物的叶绿体ycf2基因序列进行系统发育分析，结果显示大花君子兰与石蒜科植物在同一分支，显示最近的亲缘关系，支持大花君子兰属于石蒜科。基于叶绿体ycf2的系统发育分析结果与基于cpDNA全长的系统发育分析研究结果大部分相同，支持ycf2基因可以代替cpDNA全长用于植物系统发育分析。     

Bioinformatically mined simple sequence repeats in UniGene of Citrus sinensis

Characterization of microsatellites is extremely important for the development of molecular markers. Here, we present the detection and abundance of microsatellites or simple sequence repeats (SSRs) in UniGene sequences of Citrus sinensis. A total of 427 SSRs were mined in 8786 UniGene sequences downloaded from National Center for Biotechnology Information (NCBI). Depending on the repeat units, the length of SSRs ranged from 14 to 21 for mono-, 14 to 48 for di-, 18 to 48 for tri-, 24 to 40 for tetra- and 42 bp for hexa-nucleotide repeats. Average density of SSRs (1SSR/12.92 kb of 5518.71 kb sequences mined) suggests that only 4.43% of sequences contained SSRs. Di-nucleotide repeats were most frequent repeat type (49.41%) followed by tri-nucleotide repeats (41.45%). An attempt was made to design primer pairs for 427 identified SSRs but these were found only for 216 sequences. The positions of SSRs with respect to open reading frame (ORF) detected and annotation of sequences containing SSRs were also carried out to assign function to each of the sequences.     

Characterisation of novel simple sequence repeat (SSR) markers for melon (Cucumis melo L.) and their use for genotype identification

Summary

The objectives of this study were to provide a set of useful simple sequence repeat (SSR) markers, to characterise them, and to demonstrate their ability to detect polymorphisms across diverse genotypes of melon. A total of 183 SSR markers were developed in melon from libraries enriched for (CA)_n, (CT)_n, (AAG)_n, and (ATG)_n repeats. The efficiency of marker development was highest in the CT library. The efficiencies of the CA, AAG, and ATG libraries were similar, and approx. 60% of that of the CT library. Dinucleotide motifs were observed more frequently than trinucleotide motifs. Approx. 50% of CA/TG motifs and approx. 30% of CT/AG motifs exhibited a repeat number greater than ten, while most other motifs showed a repeat number of less than ten. Fifty primer pairs were selected at random and used to evaluate polymorphisms among 19 melon accessions representing seven varieties. All 50 markers were amplified successfully in the majority of accessions, and 42 markers were polymorphic among the 19 melon accessions. All polymorphic markers could detect variation between at least two accessions belonging to the same variety, with polymorphic information content values ranging from 0.10 - 0.96. The SSR markers developed were variable and transferable and will be valuable molecular tools in melon breeding programmes.