Analysis of genetic diversity in Portuguese Ceratonia siliqua L. cultivars using RAPD and AFLP markers

Although carob (Ceratonia siliqua L.) is of great economic importance little is still known about the pattern of genetic variation within this species. Morphological characteristics based on 31 fruit and seeds of continuous characters determinant for agro-industrial uses, were compared with RAPD and AFLP markers for assessing genetic distances in 68 accessions of carob trees, from different cultivars, varieties and eco-geographic regions of Algarve. Eighteen selected RAPD primers applied to the 68 accessions produced a total of 235 fragments ranging from 200 to 2000 bp, of which 93 (40%) were polymorphic. Four AFLP selective primer combinations generated a total of 346 amplification fragments of which 110 were polymorphic. The average level of polymorphism based on four primer combinations was 31.8%. The phenetic trees based on RAPD and AFLP analyses gave high co-phenetic correlation values, and were found to be consistent in general with the analysis of morphological data, carried out on the same accessions. A number of RAPD and AFLP markers were found to be diagnostic for ‘Canela’ cultivar and 13 wild ungrafted trees.     

Race-specific genetics of resistance to black rot disease [Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson] and the development of three random amplified polymorphic DNA markers in cauliflower

Summary

In order to understand the genetics of resistance to black rot disease caused by Xanthomonas campestris pv. campestris (Xcc) (Pammel) Dowson in cauliflower (Brassica oleracea var. botrytis L.) and to identify random amplified polymorphic DNA (RAPD) markers that segregate with the resistance genes, susceptible (‘Pusa Himjyoti’, female parent) and resistant (‘BR-161’, pollen parent) plants were crossed. Six generations of plants (30 P₁, 30 P₂,30 F₁, 120 F₂, 90 B₁, and 90 B₂) were evaluated for the presence or absence of black rot disease in a randomised block design with three replications. The pattern of segregation of resistance was tested by the χ² test at the 5% level of significance. All F₁ progeny plants were resistant, and the segregation of resistant and susceptible plants in the F₂ and two backcross generations (B₁ and B₂) showed that a single dominant gene caused resistance to the black rot pathogen in ‘BR-161’. Three polymorphic RAPD markers (OPO-04₈₃₃, OPAW-20₂₅₃₈, and OPG-25₆₂₅) were found by bulk segregant analysis, which produced unique amplicons 833 bp, 2,538 bp, and 625 bp in length, respectively. These markers were associated in coupling phase to the resistance allele. Best fit ratios of 3:1 (resistant:susceptible) in the F₂ plants with the three RAPD markers, suggested that the markers were linked to the single gene controlling black rot resistance. These markers will be useful to identify more closely-linked markers and to develop black rot-resistant hybrid cauliflower varieties.     

Pollinator selection in kiwifruit (Actinidia deliciosa)

SUMMARY

Pollinator performance was evaluated in kiwifruit to select pollinators for cv. Hayward adapted to the cultural conditions of southern Europe. Flowering time was determined in forty male seedlings. From, these, nine were selected for having a flowering period overlapping with ‘Hayward’. In these nine males, as well as in the commercial pollinators‘Matua’ and Tomuri’, the production of pollen was studied over two years, investigating pollen quantity through flower density and pollen production per flower. Likewise, in vitro pollen viability and its in vitro germination were recorded to study pollen quality. Finally, in vivo pollen performance was studied through fruit set and fruit characteristics in controlled hand pollinations. Two males have been selected with a flowering period coincident with ‘Hayward’, which produce more than twice as much germinable pollen than commercial pollinators. Whilst there were no significant differences in pollen quality or in fruit production, clear differences existed for pollen quantity in terms of both flower density and pollen production per flower. As pollen quantity, together with flowering time, can be easily evaluated at an early stage, this is encouraging for future selection ofkiwifruit pollinators.     

Induction of viable 2n pollen in sterile Oriental × Trumpet Lilium hybrids

In order to induce viable 2n pollen from highly sterile diploid Oriental × Trumpet (OT) (Lilium), N₂O was used to treat flower buds of four sterile diploid OT cultivars (‘Nymph’, ‘Gluhwein’, ‘Yelloween’, and ‘Shocking’) at different stages of meiosis. There was no pollen germination in the controls. However, after N₂O treatment at 600 kPa, three of the OT hybrids (‘Nymph’, ‘Gluhwein’, and ‘Yelloween’) not only exhibited fertile flower percentages (4.3–16%), but also higher rates of pollen germination (18.8–72.5%). In addition, both the fertile flower percentages and rates of pollen germination were higher following 48 h N₂O treatment at 600 kPa than the 24 h treatment. Following 72 h N₂O treatment at 600 kPa, most flower buds or plants were damaged and any undamaged flowers showed abnormal anthers at the time of flowering. This indicated that 48 h of N₂O treatment at 600 kPa was optimal to induce viable 2n pollen in three out of four sterile hybrid OT lily cultivars. These results also showed that prophase I – metaphase I was the optimum stage of meiosis at which to induce 2n pollen by N₂O treatment in lily hybrids.     

Characterization of white sapote (Casimiroa edulis Llave & Lex.) germplasm using floral morphology,RAPD and AFLP markers

Floral morphology, random amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) were used to characterize and verify genetic diversity within a white sapote cultivar collection and to develop molecular markers for germplasm identification. On the basis of floral morphology, the cultivars were classified into three types: type I included 23 cultivars with large ovaries and small anthers; type II included 13 cultivars with small ovaries and large anthers; and type III included one cultivar, named ‘Maltby’, with a large ovary and large anthers. DNA was isolated from 39 cultivars of white sapote and subjected to RAPD and AFLP analysis using 24 and 7 primers, respectively. One hundred and sixty-eight RAPD and 286 AFLP bands were used to assess genetic characterization among white sapote. Sixty percent of the RAPD and 77% of the AFLP amplification products were polymorphic among accessions. RAPD or AFLP markers differentiated all white sapote cultivars effectively. Moreover, each flower type was characterized as specially associated with two RAPD bands. UPGMA dendrograms based on RAPD and AFLP data, showed the majority of the cultivars from flower type I and flower type II clustering together. Finally 101 RAPD markers and 220 AFLP markers were used to construct a neighbor-joining dendrogram. This showed that the 37 cultivars could be classified into six distinct clusters, between which the similarity coefficient was as low as 0.00–0.55, even though the cultivars were morphologically very similar. The remaining two cultivars namely ‘Smathers’ and ‘Maltby’ were found genetically very distant from the other cultivars in RAPD, AFLP or combined RAPD and AFLP based dendrograms. The results suggested that the level of genetic variation among white sapote cultivars is diverse and the morphological and molecular data may lead to representation of the cultivar relationships as well as flower type discrimination.     

Genetic variation and differentiation in tea (Camellia sinensis) germplasm revealed by RAPD and AFLP variation

Summary

Genetic variation and resulting variable groupings in tea (Camellia sinensis) and its wild Camelia relatives were assessed using Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphic (AFLP) markers. Numerous polymorphic bands were generated, of which 266 unambiguous ones were scored. The highest level of polymorphism as determined by the expected heterozygosity (H_av) was detected with AFLPs. Three major groups were recognized in the germplasm based on the parsimony method of cluster analysis. Two of the groups corresponded to varieties assamica and sinensis while the third group consisted of a heterogeneous mix of tea cultivars and related wild species. The Taiwan yamacha, unique tea cultivars grown predominantly in the highlands of Taiwan for the production of pan fired semi-fermented (Oolong and Pouchong) tea clustered in this group. Analysis of phenotypic diversity based on the generated RAPD and AFLP profiles revealed that population diversity (H), decreased in the order; Wild Camellia >India > China > Kenya > Sri-lanka > Vietnam > Japan > Taiwan. Analysis of molecular variance (AMOVA) revealed that most variation resided among individuals within populations (72%). Calculation of genetic distances and nested AMOVA further revealed a significant degree of structuring among the populations based on the country of origin.     

Molecular characterisation of Afghan pistachio accessions by amplified fragment length polymorphisms (AFLPs)

Summary

In Afghanistan, pistachio (Pistacia vera L.) is found mainly in the wild in natural forests. In this study, 17 wild Afghan pistachio accessions and three cultivated genotypes were characterised using amplified fragment length polymorphisms (AFLPs). Material was sampled mainly from the Kunduz and Takhar regions. A total of 288 AFLP fragments were generated using eight AFLP primer combinations. The number of amplified fragments varied from 18 – 48 per primer combination, and 136 bands were polymorphic, with an average of 17 polymorphic bands per primer combination. The percentages of polymorphic bands ranged from 26.2 – 79.2 per primer pair. According to UPGMA clustering of the Nei and Li distance matrix, the 17 wild accessions were grouped according to their region of origin and were distinct from the three cultivars. The AFLP technique was found to be effective for characterising wild P. vera material, which was genetically the closest to cultivated pistachio.     

栽培番茄品种指纹图谱的AFLP分析

以62份栽培番茄为材料,利用65对AFLP引物进行选择性扩增,选出5对多态性丰富、带型清晰的引物进行扩增,共获得85个多态性位点,占总扩增位点的35.41%;选出清晰的16个位点构建了62份栽培番茄的AFLP指纹鉴别图谱。结果表明,由引物组合P55M48、P58M68、P21M49、P77M51、P58M60所构建的DNA指纹鉴别图谱可将供试材料完全区分开。     

Detection of variation among clonal selections of grapevine (Vitis vinifera L.) ‘Kishmish Chernyi’ by AFLP analysis

Summary

Clonal selection is an important method for varietal improvement in grapevine. Ampelometric and morphological markers fail to differentiate clones from their parent genotype. Molecular markers offer the opportunity to identify the clonal material. In this study, five clones of the grapevine variety ‘Kishmish Chernyi’ were analysed using microsatellite (SSR) and AFLP markers. These clones differed significantly in their bunch characteristics including berry size, shape, and colour. Microsatellite (SSR) analysis using 24 primers could not distinguish between these clones. The allele profiles of the clones and the parent variety were identical. AFLP analysis using 13 primer pair combinations yielded 592 markers ranging in size from 50 – 500 bp. Of these, 79 markers (13%) were polymorphic. The majority of the polymorphic markers (75/79) were detected in the clone ‘Sharad Seedless’. Three AFLP primer combinations detected unique markers in three clones which could be useful for future identification.     

A PCR-based assessment of genetic diversity,and parentage analysis among commercial mango cultivars and hybrids

Summary

Three different PCR methods [Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeats (ISSR), and Directed Amplification of Minisatellite DNA (DAMD)] were used to analyse genetic diversity and parentage among 20 mango cultivars, including 18 landraces and two hybrids (‘Amrapali’ and ‘Mallika’). These hybrids together with a third hybrid (‘Ratna’), and an out-group species (Mangifera sylvatica) were also analysed for parentage. Fifteen, seven and four primers were used to amplify a total of 158, 69 and 59 distinct DNA fragments by RAPD, ISSR and DAMD, respectively. Of these, approx. 85%, 64% and 90% were polymorphic, respectively. Genetic distances between pairs of mango cultivars were measured separately by each method and depicted graphically as a Neighbor Joining (NJ) tree. The three methods revealed different groupings of cultivars and hybrids. A NJ tree based on the cumulative data from all methods correlated well with the parentage of the mango hybrids, and the grouping of cultivars on a regional basis. Genetic markers likely to be associated with important agronomic traits were identified by further analysing the hybrids, with their respective parents, using all three methods. On the basis of the highest number of polymorphic bands observed (90%), DAMD was judged to be the best method with which to analyse mango germplasm.     

Genetic relationships of gladiolus cultivars inferred from fluorescence based AFLP markers

Gladiolus is one of the important commercial flowers with a large number of cultivars. However, genetic relationships among its genotypes have not been reported. This study analyzed genetic relatedness of 54 gladiolus cultivars using amplified fragment length polymorphism (AFLP) markers. A total of 24 AFLP primer pairs with three samples were initially screened, from which 9 primer sets that showed clear scorable and highly polymorphic bands were selected for AFLP reactions. Fluorescence-labeled amplification products were subjected to electrophoresis and then analyzed using an automated sequencer. A dendrogram was constructed by the unweighted pair group method using the arithmetic average (UPGMA). The number of AFLP fragments generated per primer set ranged from 10 to 151 with fragment sizes varying from 50 to 450 bp. A total of 660 AFLP fragments were detected, of which 658 (99.70%) were polymorphic. All the primers except E-AGG/M-CTA displayed 100% polymorphism. All cultivars were clearly differentiated by their AFLP profiles. The AFLP data were compared with previously obtained RAPD data and combined to generate a common dendrogram. The first cluster was dominated with indigenously bred cultivars while the second was dominated with exotic cultivars. This shows that most of the exotic cultivars as well as indigenous cultivars are closely related with each other. However, two indigenous cultivars viz., Pusa Suhagin and Pusa Archana share genetic similarity with exotic cultivars. Among the genotypes selected for the investigation, Pusa Gunjan was identified as the most distinct genotype. The AFLP markers developed will help future Gladiolus cultivar identification, germplasm conservation and new cultivar development. The assessed genetic relationships among gladiolus cultivars may enhance the efficiency of breeding program by selecting desirable parents with reduced breeding cycle.     

Clone identification and genetic relationship among vine cacti from the genera Hylocereus and Selenicereus based on RAPD analysis

Clones of Hylocereus and of Selenicereus species were distinguished from each other by the banding pattern generated by one to nine 10-mer oligonucleotide primers in the random amplified polymorphic DNA (RAPD) reaction. RAPD analysis was also applied to estimate the genetic relationship among five Hylocereus and nine Selenicereus species. A dendrogram was constructed based on a data matrix of 173 polymorphic bands originated by nine primers. Two groups were identified, one consisting of Hylocereus species and the other consisting of Selenicereus species. These results are consistent with the accepted taxonomic classification of the genera studied. The principal coordinate analysis (PCO), i.e. the plot drawn on the basis of the RAPD data, clearly distinguished between three groups, namely, Hylocereus species, S. megalanthus and the rest of the Selenicereus species studied. PCO thus strongly support the notion that the tetraploid S. megalanthus is an exception among the Selenicereus group. The RAPD results support our hypothesis regarding the allopolyploid (rather than autopolyploid) origin of this species.     

Utility of RAPD,ISSR, IRAP and REMAP markers for the genetic analysis of Citrus spp.

The application and informativeness of RAPD, ISSR, IRAP and REMAP markers to study the genetic diversity and relationship among the Citrus and its relative genotypes were investigated. High levels of polymorphism were observed for all four marker systems. The RAPD technique generated the highest number of polymorphic bands and average number of polymorphic band per assay unit. Average limit of the discriminating power was very close to its actual discriminating power of each marker. The highest and the lowest values of effective number of patterns were obtained from the marker REMAP (5.94) and RAPD (4.48), respectively. Correlation between the genetic similarities matrices were estimated from all four markers using the Mantel matrix correspondence test, and results showed significant correlations among the RAPD, ISSR, IRAP and REMAP similarity matrices. The highest correlations were found comparing RAPD and ISSR markers, whereas RAPD and REMAP (r = 0.143) markers were poorly correlated. To assess the usefulness of the overall information provided by these marker data for establishing phylogenetic relationships and Citrus germplasm classification, cluster analysis was performed. All four techniques, solely or in combination, discriminated the genotypes very efficiently and generated a high similarity in dendrogram topologies, although some differences were observed. The linkage analysis was conducted based on the segregation of 38 RAPD, 13 ISSR, 19 IRAP and 9 REMAP loci in 96 progeny of an intergeneric cross Citrus sinensis and Poncirus trifoliata. Among the 81 studied loci 65 loci distributed on five linkage groups. Comparing the result obtained with RAPD, ISSR, IRAP and REMAP markers in this study, IRAP and REMAP proved to be as a reliable molecular marker for fingerprinting, mapping and diversity study of Citrus and its relatives.     

Malus cv. Baskatong as an indicator of pollen spread in intensive apple orchards

The Malus cv. Baskatong, possessing a dominant gene for red leaf colour, was used to assess the range of a pollinizer in intensive apple orchards. In 1980 in the centre of each of six orchards, a Baskatong pollinizer was planted. All orchards had more cultivars to provide for cross-pollination. For three to seven years, seeds were gathered from trees surrounding the ‘Baskatong’ pollinizer at ten distances from 2.5 to 40 m and in 8-12 directions. After stratification, the seeds were germinated in a glasshouse and green and red seedlings were counted. ‘Baskatong’ pollen was tested for suitability by controlled hand pollinations on six apple cultivars. Flowering periods of ‘Baskatong’ and all of the apple cultivars involved were assessed over three years. ‘Baskatong’ pollen gave good fruit and seed. The ‘Baskatong’ flowering period did not fully cover those of the apple cultivars, because its bloom period was shorter. Nevertheless, red seedlings arose from the seed samples in all orchards, although not in all years. The percentages of red seedlings found were low except, in a few cases, close to the pollinizer, probably because of the adequate cross-pollination. There was no distinct directional effect, but with increasing distance from the pollinizer the percentages of red seedlings declined sharply. Although red seedlings were occasionally found as far as 40 m from the ‘Baskatong’, most occurred within a circle with a radius of 5 m. In one orchard the upper limit of the radius was about 15 m, probably because the pollinizer tree was larger than those in the other orchards. The implications of the findings for orchard lay-outs and pollinizer siting are discussed, taking a circle with a radius of 5 m as an effective range.     

用AFLP分子标记鉴定冬枣自然授粉实生后代杂种的研究

 应用AFLP技术对65株冬枣自然授粉实生后代进行杂种鉴定, 从81对EcoRⅠ和MseⅠ选择性引物中筛选出12对带型清晰且多态性较高的引物。用这12对引物共扩增出517条谱带, 其中多态性带376条, 多态性百分率为72.7%。共扩增出金丝小枣特征带10条, 鉴定出34株冬枣×金丝小枣杂交实生苗;扩增出尖枣特征带7条, 鉴定出冬枣×尖枣杂交实生苗15株; 不能确定的有16株。     

Genetic diversity of Indonesian cauliflower cultivars and their relationships with hybrid cultivars grown in Australia

The objectives of this study were to investigate genetic variation and relationships between Indonesia-, Australian- and European-based cultivars and to evaluate variation within Indonesia cultivars as all cultivars are open-pollinated. Eight cauliflower cultivars collected from three production regions in Indonesia and four F₁ hybrids cultivars grown in Australia were evaluated using RAPD and ISSR markers. DNA polymorphisms generated from 10 polymorphic RAPD primers were used to construct a dendogram using the unweighted pair-group method with arithmetic averages (UPGMA) and to generate a fingerprinting key. ISSR marker analysis using 14 primers were attempted but DNA polymorphisms could not be clearly identified. The RAPD technique indicated that variation occurred both within and between Indonesian cultivars. Comparison between Indonesian-, Australian- and European-based cultivars showed that Indonesian cultivars have unique genotypes and would be good sources of genes for future crop improvement.     

Inter- and intra-specific genetic diversity among cyclamen accessions investigated by RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to characterize genetic diversity of 26 Cyclamen persicum and Cyclamen com accessions. Eighty-four arbitrary primers tested, among which nine primers showed reliable polymorphic banding patterns and yielded 104 polymorphic markers. Jaccard's similarity coefficient among accessions ranged from 0.99 to 0.08. At a similarity of 68%, accessions were divided into three clusters. The cophenetic correlation coefficient between the similarity matrix and the dendrogram was relatively high (r = 0.9) showing the goodness of fit of the dendrogram. The RAPD analysis offered a rapid and reliable tool for the estimation of inter- and intra-specific variability in cyclamens. The wide genetic variation observed for cyclamens within Iran guarantees a promising future of breeding.     

Application of five DNA marker techniques to distinguish between five apple (Malus × domestica Borkh.) cultivars and their sports

Summary

Genetic variation between five apple cultivars (‘Golden Delicious’, ‘Gala’, ‘Jonagold’, ‘?ampion’, and ‘Idared’) and ten of their sports (‘Golden Delicious Reinders’, ‘Goldrosio’, ‘Gala Must’, ‘Gala Schniga Schnitzer’, ‘Jonagored’, ‘Jonagold Excel’, ‘Szampion Arno’, ‘Szampion Reno Malinowy’, ‘Idaredest’, and ‘Red Idared’) was investigated using five types of DNA markers: Inter-Simple Sequence Repeats (ISSR), Simple Sequence Repeats (SSR), Amplified Fragment Length Polymorphism (AFLP), Sequence-Specific Amplified Polymorphism (S-SAP), and Inter-Primer Binding Site (iPBS) amplification. In total, 941 polymorphic amplified fragments were obtained using 12 ISSR, 12 SSR, ten AFLP, 19 iPBS, and 15 S-SAP primers or primer pairs. Four of the above-described techniques (except for SSRs with the primer pairs used in this study) were able to distinguish between the sports and their parental cultivar. The most effective technique to distinguish between the genotypes analysed was S-SAP, which detects variations in DNA regions flanking retrotransposon insertion sites.The combined use of ISSR,AFLP, iPBS, and S-SAP markers identified and distinguished all of the sports tested.     

Further Work on Plant Injection for Diagnostic and Curative Purposes

Summary

Randomly Amplified Polymorphic DNA (RAPD) markers were used to evaluate genetic similarity and inter-relationship among31 acid citrus species and cultivars, including sour oranges (six accessions); ‘Yuzu’ (four accessions) andits relatives (21 accessions). Out of the 60 decamer primers screened, 27 were selected which produced 108 markers; 76 of which were polymorphic. Species or cultivar-specific RAPD markers were also found. A dendrogram based on genetic distance implied that sour oranges were very distinct from ‘Yuzu’ and its relatives. ‘Yuzu’ accessions were very closely linked to each other, however; for the other specimens genetic polymorphism could easily be detected by RAPDs and the genetic variation between accessions was quite high and revealed their different origins. In this study some RAPDs allowed the distinction of very close cultivars, for instance ‘Kabosu’ from ‘Aka kabosu’.     

Morphology and anatomy of pollen grains from male-fertile and male-sterile cultivars of chestnut (Castanea sativa Mill.)

Summary

Pollen morphology and ultrastructure are described for four male-fertile cultivars (‘Firdola’, ‘Karamehmet’, ‘Sar?a?lama’, and ‘Hac?ömer’) and two male-sterile cultivars (‘Osmanog?lu’ and ‘Vakit Kestanesi’) of chestnut (Castanea sativa Mill.) using light microscopy and both scanning and transmission electron microscopy. Pollen grains of both male-fertile and male-sterile chestnut cultivars are tricolporate, the germinal furrow extending almost the full length of the grain axis. Pollen grains have a slightly reticulate exine. Pollen grain length varied from 13.33 – 21.30 µm, and decreased significantly in the male-sterile cultivars. Three different pollen shapes were observed among the cultivars: prolate, perprolate, and sub-prolate. The ultrastructure of the pollen grains did not differ between male-fertile and male-sterile cultivars. Intine, exine and total wall thickness (exine + intine) of pollen grains were determined as: 83.2 – 153.1 nm, 432.8 – 520.0 nm, and 516.0 – 651.6 nm, respectively; and variations were significant (P ≤ 0.05) among cultivars. The percentages of in vitro germination of pollen grains of male-fertile cultivars were between 11 – 78%, and the variations among cultivars were significant (P ≤ 0.05). The percentages of empty pollen grains observed among cultivars ranged from 3 – 32%. The correlation coefficient between the percentage of normal pollen and the germination rate was r = 0.898 (P ≤ 0.01).