Conformity and genetic relatedness estimation in crop species having a narrow genetic base: the case of cucumber (Cucumis sativus L.)*

A set of 155 SSR (107) and SCAR (48) markers were used to evaluate 53 cucumber (Cucumis sativus L.) accessions of diverse origin to characterize genetic relationships and to define a standard marker array that was most effective in detecting genetic differences in this germplasm array. A multivariate marker‐based analysis of diverse germplasm using this standard marker array (17 SSR and 5 SCAR markers) was compared with results from a set of 70 previously reported RAPD markers, and then used to explore the potential value of these genetic markers for plant variety protection (PVP) and the establishment of essential derivation (ED) threshold values in this species using elite lines and hybrids and backcross progeny. Diversity analysis allowed identification of distinctly different lines that were used for the construction of three sets of backcross families (BC₁‐BC₃). While general genetic relationships among accessions were similar in SSR/SCAR analyses (r_s= 0.65) using two genetic distance (GD) estimators, differences in accession relationships were detected between RAPD and SSR/SCAR marker evaluations regardless of the estimator used. The GDs among elite germplasm with known pedigrees were relatively small (0.06‐0.23 for any pairwise comparison). GD values decreased and degree of fixation (at three to seven loci depending on the mating) increased with increased backcrossing such that recurrent parent allelic fixation occurred in least one family of each of the BC₃ families. In many instances the degree of fixation of loci was not uniformly achieved in the BC₃. Although the level of genetic polymorphisms will likely restrict the use of molecular markers for PVP and the establishment of ED values, the use of single nucleotide differences will likely provide opportunities to define specific functional distances that have potential for PVP in cucumber. Nevertheless, without an expanded, genetically robust standard marker array (e.g. 50 codominant markers), ED threshold values will be difficult to define in this species, and perhaps will require the appraisal of single nucleotide polymorphisms as discriminators of difference in this species.     

Evaluation of resistance to melon yellow spot virus in a cucumber germplasm collection

Melon yellow spot virus (MYSV), a member of the genus Tospovirus, is a serious thrips‐transmitted virus of cucurbits in Japan. Resistant cultivars provide an effective means for reducing the impact of the disease; however, no MYSV‐resistant cucumber has been reported. Susceptibilities of 398 cucumber accessions originating from 26 countries were evaluated by mechanical inoculation of MYSV. Thirteen accessions from South Asia, South East Asia and unknown origin had low disease severity indices (DSIs), and 10 of them showed no necrotic lesions on inoculated leaves. No or little positive reaction was detected by DAS‐ELISA analysis of inoculated leaves of these accessions. 27028930 showed resistance to a melon isolate (MYSV‐S) of MYSV, and ‘Yamakyuri‐1’ showed moderate resistance to a cucumber isolate (FuCu05P).     

Analyses of genetic diversity in Cuban rice varieties using isozyme, RAPD and AFLP markers

A survey of the genetic diversity among the major cuban rice cultivars was conducted using isozyme, RAPD and AFLP markers. Polymorphisms were detected for esterases, peroxidases, alcohol dehydrogenases and polyphenoloxidases systems; 21 RAPD primers and four AFLP primer combinations. Heterozygosity arithmetic mean value (H_av(p)), the effective multiplex ratio (EMR) and the marker index (MI), were calculated for isozyme, RAPD and AFLP markers. The mean value of genetic similarity among the different varieties was 0.92 for isozyme, 0.73 for RAPD and 0.58 for AFLP analyses. Thus, AFLP were able to detect polymorphisms with higher efficiency than RAPD (+15%) and isozyme (+34%). Data from the isozyme, RAPD and AFLP analyses were used to compute matrices of genetic similarities. The efficiency of the UPGMA for the estimation of genetic relatedness among varieties was supported by cophenetic correlation coefficients. The resulting values indicated that the distortion level for the estimated similarities was minimal. 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 isozyme and RAPD markers (r = 0.89), as well as for AFLP and RAPD markers (r = 0.82) were properly related. However, AFLP and isozyme data showed only moderate correlation (r = 0.63). Although the genetic variability found among the different cultivars was low, both RAPD and AFLP markers proved to be efficient tools in assessing the genetic diversity of rice genotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Morphological traits and high resolution RAPD markers for the identification of the main strawberry varieties cultivated in Argentina

Eight genotypes of the main Fragaria×ananassa cultivars grown in Argentina were analysed using the random amplified polymorphic DNA (RAPD) technique combined with electrophoresis in polyacrylamide gels. The high resolution of this procedure allowed the detection, with only 13 random primers, of 37 genotype‐specific bands that can be used as markers for verifying the identity of cultivars. By using this approach, three different accessions of the cultivar ‘Pájaro’ exhibited differences in amplification profiles, confirming the need for DNA analysis to prevent misidentification of cultivars. In addition, RAPD bands and morphological traits were used to assess genetic relatedness among cultivars. Comparison of both dendrograms revealed that there is no correlation between the clustering obtained with molecular and morphological characters.     

QTL mapping of seedling traits in cucumber using recombinant inbred lines

Seedling traits are important for development, flower bud differentiation, fruit production and fruit quality of cucumber (Cucumis sativus L.). In this study, 160 recombinant inbred lines (RILs), derived from crossing wild cucumber inbred line PI 183967 (C. sativus var. hardwickii) with ‘931’ northern China cultivated cucumber inbred line 931, were employed to identify quantitative trait loci (QTLs) of cotyledon length (Cl), cotyledon width (Cw), hypocotyl length (Hl), first true leaf length (Fll), first true leaf width (Flw), aboveground fresh biomass (Afb) and aboveground dry biomass (Adb) at seedling stage. A genetic map including 307 SSR markers was developed which spanned 993.3 cM, with an average genetic distance of 3.23 cM between adjacent markers. 36 QTLs associated with the seven traits were detected on chromosomes 1, 2, 3, 5 and 6 in four environments (spring and autumn of 2012 and 2013), explaining 6.1 to 23.6% of the observed phenotypic variations. Among the 36 QTLs, 21 were responsible for more than 10% of observed phenotypic variations. We obtained 2, 2, 1 and 3 QTL loci for the traits of Fll, Flw, Afw and Adw, respectively. In addition, genes in the genetic region spanned by SSR15321‐SSR07711 on chr. 5 may contribute to Flw, Afw and Adw.     

Direct plant regeneration from leaf explants in cucumber (Cucumis sativus L.) is free of stable genetic variation

A new procedure is described for rapid and efficient plant regeneration from leaf explants of Cucumis sativus and C. anguria. The following factors were most important: young leaves, the NO₃: NH₄ ratio in the plant induction medium, small explants and the growth regulator combination. After about 4 weeks of culture under optimal growth conditions, the frequency of regeneration was 10–100% of explants. Six to 7 weeks were required to obtain well-rooted plants, which were mostly able to survive after transfer into soil. From a single young leaf of C. sativus cv. ‘Borszezagowski’, 135 plants could be regenerated. All plants transferred to a greenhouse were free of morphological or physiological abnormalities, flowered normally and bore fruits. The analysis of R₀ plants showed no genetic variation, whereas in the R₁ two new phenotypes, which were not transmittable to the R₂, were observed. This procedure is recommended for its production of homogeneous cucumber plants.     

Molecular analysis of Lathyrus sativus L. (grasspea) and related Lathyrus species

Eight Lathyrus sativus L. accessions from a variety of geographic origins were used to study intraspecific genetic diversity using RAPD analysis. Fourteen decamer primers produced 64 amplification products, 50% of which were polymorphic between the samples. Jaccard's coefficient of genetic similarity was calculated between samples and a dendrogram was constructed by an unweighted pair-group method with arithmetical averages (UPGMA). The dendrogram showed that most of the L. sativus plants clustered into accessions or common geographical areas. The average genetic similarity coefficient within accessions was 0.12 and between accessions was 0.20, indicating a low level of intraspecific genetic variation. Interspecific genetic diversity and phylogenetic relationships of eight Lathyrus species, including L. sativus and Pisum sativum L. (field pea) were examined using 14 decamer primers which produced 283 amplification products. All amplification products were polymorphic across the nine species. In the dendrogram the Lathyrus species clustered into three distinct groups which correlated with the Sections Lathyrus, Clymenum and Linearicarpus. This supports traditional taxonomic classifications of the genus Lathyrus which are based on morphological traits. Of the species from Section Lathyrus, L. gorgoni and L. cicera were the most similar to L. sativus. The results suggest that a strategy of breeding for producing lines of L. sativus with increased genetic variation would be effectively achieved through hybrid production between accessions from wide geographic areas particularly the Mediterranean area and the Indian subcontinent. However, the most effective method would be introgression of germplasm from other species in Section Lathyrus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of QTLs controlling low‐temperature tolerance during the germination stage in cucumber (Cucumis sativus L.)

Poor seed germination of cucumber at suboptimal temperatures is a great concern for growers wishing to take advantage of the early market. The development of low‐temperature‐tolerant varieties would be aided by understanding the inheritance of the trait and mapping its locus. In this study, a set of 140 recombinant inbred lines (RILs) derived from a cross between 65G (low‐temperature‐tolerant) and 02245 (low‐temperature‐sensitive) was used to identify the QTLs linked with low‐temperature tolerance. A linkage map was developed using 135 simple sequence repeat (SSR) markers and five insertion–deletion (Indel) markers, and three QTLs were identified, qLTG1.1, qLTG2.1 and qLTG4.1. qLTG1.1, the major one for germination rate, germination energy and germination index, explained more than 50% of the observed phenotypic variability. The major QTL for radicle length, qLTG4.1, explained 13.8% of the phenotypic variability. The results showed that qLTG1.1 and qLTG4.1 play an important role in low‐temperature tolerance during seed germination of cucumber and provide a basis for further fine mapping to determine the molecular mechanism for this trait.     

Construction of a genetic map of melon with molecular markers and horticultural traits, and localization of genes associated with ZYMV resistance

Abstract: A partial linkage map of melon was constructed from a cross between PI414723 and Dulce. Twenty-two SSR, 46RAPD, 2 ISSR markers and four horticultural markers [female flower form (a), Fusarium resistance, striped epicarp (st), and fruit flesh pH (pH)] were analyzed in an F₂/F₃ population to produce a map spanning 14 linkage groups. We report for the first time map positions for the st, a, and pH genes. One SSR marker was tightly linked to pH. Mapping the a gene for the female flower form to molecular linkage group 4 enabled the merging of the map of horticultural traits with the of molecular markers in this region. Using the 22 SSR markers of this map, two of the three postulated ZYMV resistance genes were located using a BC₁ population (PI414723 recurrent parent). One SSR marker was tightly linked to a ZYMV resistance gene, designated Zym-1. This revised version was published online in August 2006 with corrections to the Cover Date.     

Estimation of between and within accession variation in selected Spanish melon germplasm using RAPD and SSR markers to assess strategies for large collection evaluation

The population structure of 15 Spanish melon (C. melo L.)accessions, mostly of Group Inodorus, was assessed by the analysis of 16individuals of each accession using 100 random amplified polymorphic DNA (RAPD) bands produced by 36 primers, and allelic variation at 12microsatellite (SSR) loci (23 alleles). A relatively high level of polymorphism (25.6%) was detected using RAPD markers, and eight SSR loci (66.7%) were useful in discriminating accessions. Cluster analysis using RAPD- and SSR-based genetic distance estimates resulted in similar and consistent groupings of most of the accessions studied. The mean genetic distance and standard error among accessions estimated by RAPD variation was 0.421 ± 0.099, and mean SSR-based genetic distance estimate was 0.285 ± 0.141. Albeit many dominant markers examined were fitted to a 3:1 test ratio, deviation from this ratio and from Hardy-Weinberg expectations for many SSR loci suggests that some populations were in genotypic disequilibrium. Moreover, a higher level of genetic variation was observed between Cassaba market classes than within accessions, suggesting that, depending upon the accession, allelic fixation has occurred in these populations. The relatively high level of heterogeneity observed (different band morphotypes and cluster grouping within a particular market class), however, indicates that the Spanish melons examined possess a relatively broad genetic background. An appraisal of accession population structure such as the one reported herein indicates that bulk sampling techniques coupled with molecular analysis techniques that employ a unique array of discriminating markers can provide information leading to effective strategies for diversity analyses of large collections. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evaluation of random amplified polymorphic DNA (RAPD) markers in Hevea brasiliensis

The applicability of random amplified polymorphic DNA (RAPD) markers in the cultivated rubber tree, Hevea, was evaluated using 43 decamer oligonucleotide primers in a set of 24 clones selected in different South-East Asian countries. A total of 220 0.35–3.5 kb DNA fragments were amplified, of which 111 were polymorphic. Of these, 80 fragments (RAPD markers) which were repeatable and clearly scorable across all genotypes were used to estimate genetic distances among the clones tested. The estimated genetic distances ranged from 0.05 (RRII 308 and PB 5/51) to 0.75 (RRIC 100 and SCATC 88–13). A mean genetic distance of 0.5 indicates a rather high genetic variability among the tested clones. As expected, because of the breeding history of Hevea, UPGMA cluster analysis and Principal Coordinate Analysis (PCoA) indicated the absence of a distinct geographical grouping. The possible application of RAPD markers for clone identification and also for analysis of genetic relationships among Hevea clones is discussed.     

Relationship between molecular marker heterozygosity and hybrid performance in intra- and interspecific hybrids of cotton

The present study was conducted to investigate the relationship between parental molecular marker diversity and hybrid performance in both intra‐ and interspecific hybrids of cotton to evaluate the feasibility of predicting hybrid performance using molecular markers. Three cytoplasmic male sterile (CMS) lines were crossed with 10 restorer lines to produce 22 F₁ hybrids during 2003. Of 22 F₁s, 14 hybrids were intraspecific (Gossypium hirsutum × G. hirsutum) and eight interspecific (G. hirsutum × G. barbadense). These 22 F₁ hybrids and their parents were evaluated for yield and fibre quality traits at Zhejiang University, Hangzhou, China during 2004 and 2005. Genetic distances (GD) among the parents were calculated from 56 random‐amplified polymorphic DNAs (RAPD) and 66 simple sequence repeat (SSR) marker data, and their correlation with hybrid performance and heterosis were analysed. The parents could be discriminated into G. hirsutum and G. barbadense clusters by cluster analysis based on both RAPD and SSR markers data. The correlation (r = 0.503, P ≤ 0.05) was calculated between GD_rapd (GD based on RAPD markers) and GD_ssr (GD based on SSR markers). Correlation of GD with hybrid performance and heterosis differed considerably between intra‐ and interspecific hybrids. The correlation between GD and hybrid performance was non‐significant for most of traits within the hybrids of G. hirsutum species. However, it was significantly and positively correlated for fibre length, fibre strength and elongation in interspecific hybrids. The relationship between GD and heterosis was observed to be positively significant for boll weight within hybrids of G. hirsutum with significant and negative correlations for fibre length and elongation. In conclusion, the power of predicting hybrid performance using molecular markers in cotton is low. But, the relationship between SSR marker heterozygosity and hybrid performance can be used to predict fibre length during interspecific hybrid cotton breeding.     

Linkage of random amplified polymorphic DNA markers to downy mildew resistance in cucumber (Cucumis sativus L.)

Marker assisted selection (MAS) may improve the efficiency of breeding downy mildew resistant cucumber cultivars. A study was conducted to identify random amplified polymorphic DNA (RAPD) markers linked to the downy mildew resistance gene (dm) which would be suitable for MAS. A total of 145 F₃ families from two populations (55 from the WI 1983G × Straight 8 population and 90 from the Zudm1 × Straight 8 population) were evaluated over five locations in North America and Europe. Resistant and susceptible F₃ families were identified and mean family resistance ratings were used to type individual F₂ plants. No evidence for race differences in the pathogen (Psuedoperonospora cubensis (Berk. & Curt.) Rostow) between North America and Europe was found. Phenotypic correlations between locations ranged from 0.3 to 0.7. Of the 135 polymorphic RAPD markers identified from 960 primers, five were linked to dm - G14₈₀₀, X15₁₁₀₀, AS5₈₀₀, BC519₁₁₀₀, and BC526₁₀₀₀. In the WI 1983G × Straight 8 population, G14₈₀₀ was linked to dm at 16.5 cm, AS5₈₀₀ at 32.8 cm, BC519₁₁₀₀ at 9.9 cm, and BC526₁₀₀₀ at 19.2 cm. In the Zudm1 ×Straight 8 population, G14₈₀₀ was linked at 20.9 cm, X15₁₁₀₀at 14.8 cm, AS5₈₀₀ at 24.8 cm, and BC526_{_1000} at 32.9 cm. MarkersG14₈₀₀ and BC519₁₁₀₀ were linked in repulsion to the dm allele, and X15₁₁₀₀, AS5₈₀₀, and BC526₁₀₀₀ were linked in coupling phase. These genetic markers may be exploited to develop an efficient MAS strategy for breeding resistant cucumber cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of genetic relationships among 22 European barley varieties based on two PCR markers

Two long primers of 19 (F17) and 20 (F13) nucleotides, respectively,were used in polymerase chain reactions to amplify DNA from differentcultivated barley accessions. These primers can distinguish closely relatedvarieties and, with a unique primer, all the barley accessions analysedshowed a characteristic fingerprint. Sixty per cent and 76% of thefragments generated using F13 and F17, respectively, were polymorphic.The genetic similarity values between accessions were estimated from F13and F17 data. The dendrogram and principal coordinate analysis performedwith F13 data revealed a clear separation of these varieties in accord withtheir pedigree relationships.     

Application of RAPD markers in the characterisation of Chrysanthemum varieties and the assessment of somaclonal variation

Characterisation of fifteen commercial varieties of Chrysanthemum was carried out through RAPD analysis. Varieties could be distinguished from each other and the level of similarity between varieties seemed to be not very high. In vitro cultures were establish from four varieties and were subjected to different proliferation conditions. Five individuals from each variety and treatment were analysed using RAPD at the beginning of the treatment and after a month of culture. Variation was detected at both stages of the culture period. The rate of variation found showed differences between varieties, but no significant difference was found between culture conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Assessment of genetic diversity within and among Basmati and non-Basmati rice varieties using AFLP,ISSR and SSR markers

Molecular markers provide novel tools to differentiate between the various grades of Basmati rice, maintain fair-trade practices and to determine its relationship with other rice groups in Oryza sativa. We have evaluated the genetic diversity and patterns of relationships among the 18 rice genotypes representative of the traditional Basmati, cross-bred Basmati and non-Basmati (indica and japonica) rice varieties using AFLP, ISSR and SSR markers. All the three marker systems generated higher levels of polymorphism and could distinguish between all the 18 rice cultivars. The minimum number of assay-units per system needed to distinguish between all the cultivars was one for AFLP, two for ISSR and five for SSR. A total of 171 (110 polymorphic), 240 (188 polymorphic) and 160 (159 polymorphic) bands were detected using five primer combinations of AFLP, 25 UBC ISSR primers and 30 well distributed, mapped SSR markers, respectively. The salient features of AFLP, ISSR and SSR marker data analyzed using clustering algorithms, principal component analysis, Mantel test and AMOVA analysis are as given below: (i) the two traditional Basmati rice varieties were genetically distinct from indica and japonica rice varieties and invariably formed a separate cluster, (ii) the six Basmati varieties developed from various indica × Basmati rice crosses and backcrosses were grouped variably depending upon the marker system employed; CSR30 and Super being more closer to traditional Basmati followed by HKR228, Kasturi, Pusa Basmati 1 and Sabarmati, (iii) AFLP, ISSR and SSR marker data-sets showed moderate levels of positive correlation (Mantel test, r = 0.42–0.50), and (iv) the partitioning of the variance among and within rice groups (traditional Basmati, cross-bred Basmati, indica and japonica) using AMOVA showed greater variation among than within groups using SSR data-set, while reverse was true for both ISSR and AFLP data-sets. The study emphasizes the need for using a combination of different marker systems for a comprehensive genetic analysis of Basmati rice germplasm. The high-level polymorphism generated by SSR, ISSR and AFLP assays described in this study shall provide novel markers to differentiate between traditional Basmati rice supplies from cheaper cross-bred Basmati and long-grain non-Basmati varieties at commercial level.The first two authors have equal contribution     

Assessment of genetic variability of olive varieties by microsatellite and AFLP markers

Fourteen developed microsatellite markers were characterized for their use in genotyping and diversity studies of olive varieties. After optimisation of microsatellite assay and allele sizing, ninety-six alleles were found in nineteen varieties, with an average of 6.8 alleles per locus. The characteristics of the microsatellite markers were used to identify markers that can be reliably applied for variety genotyping. Such features were the generation of complex banding patterns supported by underlying allele sequences, `short allele dominance', an unstable repeat structure and a low number of alleles. AFLP analysis was performed on the same set of olive varieties using eight primer pair combinations. The genetic relationships among nineteen olive varieties were compared on the basis of microsatellite and AFLP polymorphisms. Genetic distances between all pairwise combinations of the varieties were calculated using Jaccard's coefficient of similarity and dendrograms were constructed by the UPGMA method. The results of clustering analysis with both molecular systems showed the common genetic background of Tuscan varieties, and genetic divergence within Slovene olive germplasm. Slovenian varieties ‘Buga’, ‘Štorta’ and ‘Samo’ might represent regionally selected olives, while ‘Zelenjak’ and ‘Črnica’ are probably derived from the Central Italian region. The predominant local ‘Istrska belica’ was introduced to Slovenia independently from the other regional varieties and showed the lowest genetic similarity with the other regional varieties. This revised version was published online in August 2006 with corrections to the Cover Date.     

Assessment of natural and induced genetic variation in Alstroemeria using random amplified polymorphic DNA (RAPD) markers

Summary We have used random amplified polymorphic DNA (RAPD) markers to study genetic variation in Alstroemeria. The first objective was to examine the discriminatory power of RAPD markers in different genotypes of Alstroemeria obtained by traditional breeding. All genotypes examined, including commercial Alstroemeria varieties, could be distinguished on the basis of their RAPD profiles. Progeny plants could be distinguished from their parents. A second objective of this study was to investigate whether RAPD markers can be used as a routine tool to detect mutant plants, as an alternative to glasshouse testing. To address this objective, we analysed Alstroemeria plants that carried phenotypically visible mutations that either were induced by irradiation using X-rays or were the result of somaclonal variation. In eight out of a total of 13 mutant Alstroemeria plants obtained after irradiation or tissue culture we detected no polymorphisms when compared to control plants that were considered to be non-mutated. Only in five of the mutant plants analysed we detected one to two polymorphisms. These results suggest that frequent genome rearrangements had not occurred in the mutant plants analysed. These results also demonstrate that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It seems probable that this conclusion would be equally applicable in other plant genera in which induced variation has occurred. However, the RAPD technique is a simple and effective tool for genetic fingerprinting of Alstroemeria varieties, provided their differences are due to sexual propagation.