Combination Of Amplified Fragment Length Polymorphism Research Articles

Genetic structure of wild raspberry populations in the Central Balkans depends on their location and on their relationship to commercial cultivars

The red raspberry (Rubus idaeus L.) is one of the most important fruit species in Serbia; there are currently approximately 11000 ha under cultivation. Wild red raspberry populations can be an important source of genes for breeding new raspberry varieties. This study was carried out to determine the genetic variability of wild populations, as well as their relationship with the most common cultivated cultivars using amplified fragment length polymorphism (AFLP) markers. In this study were included 128 individuals from seven wild populations and seven cultivar specimens from a nursery ('Mekeer', 'Willamette', 'Polka', 'Polana', 'Loganberry', 'Tayberry', 'Black Jewel'). Four AFLP combinations yielded a total of 247 polymorphic bands in 135 R. idaeus specimens. Analysis of AFLP markers showed that the cultivars are clearly distinct from wild populations. Population P4 (Mt. Ozren) had the highest gene diversity, the highest number of private markers and the highest rarity index. Analysis of molecular variance among and within wild populations showed that the most of the genetic diversity was attributable to differences between individuals within populations. A Mantel test showed that differences in altitude play the crucial role in structuring of the genetic diversity of red raspberry populations. Altitude contributed more to the population differentiation than spatial distance. Populations of wild raspberry from the territory of Serbia are characterized by high diversity, and should be protected and used as a resource in selection and breeding processes for the production of new varieties, with improved properties in terms of better yield and stronger resistance to external factors.

Analysis of Genetic Diversity Among ‘Misket’ Apple Clones Using AFLP, SSR and RAPD Markers

Knowledge of genetic relationship among genotypes is an important concern for utilization of germplasm resources in breeding. The need for knowledge in local apple cultivars is increasing and molecular marker analysis has contributed to understanding of the apple genetic diversity and phylogenetic relationship between genotypes. Three different DNA-based techniques, random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) were used for detecting genetic variation among the 30 local ‘Misket’ apple genotypes. A total of 423 bands amplified and 205 polymorphic band were obtained with three marker techniques. Thirty RAPD primers amplified 207 bands, of which 91 were polymorphic (40.1%), 10 SSR primers produced 33 bands and 26 of them were polymorphic (78.78%) and 5 AFLP combinations amplified 183 bands of which 88 were polymorphic (48.08%). The average numbers of polymorphic marker per primer combination were 17.6, 3.03 and 2.6 for AFLP, RAPD and SSR respectively. Our results indicate that SSR was less informative in characterizing closely related ‘Misket’ apple genotypes, compared with RAPD and AFLP markers. AFLP seemed to best suited for evaluating the genetic relationship among the apple genotypes. Closely related genotypes may require more than one marker technique. The findings will be useful to identify and evaluate the local growing apple genetic resources.

<b>AFLP analysis of genetic diversity in determinate and indeterminate snap bean accessions

The present study aimed to estimate and characterize the genetic divergence between determinate and indeterminate snap bean accessions from the Universidade Estadual de Londrina (UEL) germplasm bank based on amplified fragment length polymorphism (AFLP) markers. A total of 40 and 32 accessions with determinate and indeterminate growth habits, respectively, were characterized for this purpose. Seven combinations of primers corresponding to EcoR1 and Mse1 were tested for the AFLP analysis, and the combinations E-AAG/M-CTC, E-ACT/M-CTT and E-ACC/M-CTT were selected. These selective AFLP combinations revealed 485 informative loci in total, and the combination E-ACC/M-CTT detected the greatest number of informative loci (49%). The analysis of dissimilarity frequency distribution showed that the distribution was uniform, ranging from 0.1285 to 0.7310 with a mean of 0.4801, and the accessions with indeterminate growth habits exhibited greater variability than the accessions with determinate growth habits. The clustering UPGMA, PCoA and Bayesian analyses showed the formation of two large clusters, wherein there is a possible association between snap bean growth habit and gene pool. The determinate accessions may be more closely associated with the Andean gene pool, while the indeterminate ones may be associated with the Mesoamerican gene pool. The Bayesian analysis showed accessions intermediate to both groups, suggesting introgression between the Andean and Mesoamerican gene pools.

Analisis Keragaman Genetik Temulawak (Curcuma xanthorrhiza Roxb.) Menggunakan Penanda Amplified Fragment Length Polymorphism (AFLP)

Curcuma xanthorrhiza Roxb, which is well-known as Java turmeric, has been extensively used in pharmaceutical industries in Indonesia. In spite of this commercial value, the identity of this species is commonly mistaken from other similar orange rhizomes Curcuma. Correct identity of these species is vital in pharmaceutical industries. The objective of the study was to determine genetic diversity of 32 accession Curcuma xanthorrhiza Roxb. Genomic DNA was extracted from leaf using Sodium Dodesyl Sulphate (SDS) modification. Amplified fragment length polymorphism (AFLP) was carried out according to the protocol ofAFLPTM plant mapping kit and the final polymerase chain reaction (PCR) products were separated using The Agilent 2100 Bioanalyzer. The number of fragment produced by 12 pairs primer combination of AFLP ranged from 42 to 60 with an average of 52. Data obtained was analyzed by the NTSys program. From the AFLP amplification on 32 DNA samples, it was proven that the accession of Curcuma xanthorrhiza Roxb. had a high degree of diversity. Based on analysis of AFLP and unweighted pair group with arithme average (UPGMA) it was shown that the accession of Curcuma xanthorrhiza Roxb. could be grouped into two cluster at relative ecludian distance of 0.10 (10%). Cluster I for accession from Palembang, Pacitan and Ciamis 2. Cluster II for accession from Makale, Pontianak, Kulonprogo, Mataram, Boyolali, Salatiga, Sumberejo, Bali, P. Seram, Sentolo, Purworejo, Samas Bantul, Ciamis1, Blora, Semarang, Poso, Kalsesl, Tagari, Merapi Farm, Salakaria, NTB, Menoreh, Karang Anyar, Mangunan, Medan, Toraja, dan Solok.

Assessment of genetic diversity and relationships based on RAPD and AFLP analyses in Miscanthus genera landraces

Qin, Y., Kabir, M. A., Wang, H. W., Lee, Y. H., Hong, S. H., Kim, J. Y., Yook, M. J., Kim, D. S., Kim, C. S., Kwon, H. and Kim, W. 2013. Assessment of genetic diversity and relationships based on RAPD and AFLP analyses in Miscanthus genera landraces. Can. J. Plant Sci. 93: 171–182. In this study, molecular markers, random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) as well as combined RAPD and AFLP analysis were used to assess genetic diversity in a reference set of 38 Miscanthus accessions of which 32 were collected from South Korea and 6 from foreign countries. Using 30 selected RAPD primers, 197 amplified products were generated with an average of 6.6 bands, of which 135 bands were polymorphic (68.6%). A total of 1150 bands were detected by four-primer AFLP combinations with an average of 287.5 bands, out of which 923 bands were polymorphic (80.3%) across all the accessions. Analysis of molecular variance (AMOVA) indicated that a high proportion of the genetic variation (56% for RAPD and 58% for AFLP) was found among the Miscanthus species in South Korea. Genetic relationship was estimated using the Jaccard's coefficient values between different accessions, ranging from 0.23 to 0.93 in RAPD and 0.34 to 0.94 in AFLP. The un-weighted pair group method with arithmetic mean (UPGMA) analysis demonstrated less difference between RAPD and AFLP when alternative similarity coefficient was applied. The principal coordinates (PCO) analysis also revealed the significant geographic structure in the tested accessions. Among the accessions, SNU-M-040, 074 and 157 were highly divergent. Pattern of isolation by distance was observed in Miscanthus accessions, indicating that significant genetic differentiation among accessions might be due to the geographic distance.

Identification and mapping of a major dominant quantitative trait locus controlling seeds per silique as a single Mendelian factor in Brassica napus L.

One putative quantitative trait locus (QTL) for seeds per silique (SS), cqSS.A8, was identified using a double haploid (DH) population in Brassica napus, and near-isogenic lines (NILs; BC(3)F(1)) for cqSS.A8 were developed. However, the flanking markers from cqSS.A8 showed no significant difference using single-marker analysis, even though the frequency distribution of SS in the BC(3)F(1) was bimodal, suggesting that one novel locus existed. In this study, we characterized the effects of this locus in the NILs and used a published linkage map to determine its location. A three-step approach was designed for mapping the locus in the NILs (BC(3)F(2)): (1) determining the individual BC(3)F(2) genotype at the locus using a progeny test; (2) identifying amplified fragment length polymorphism (AFLP) markers linked to the locus using a combination of AFLP and bulked segregant analysis; and (3) determining the location and effects of this locus. QTL analysis in the BC(3)F(2) revealed that this locus explained 85.8 and 55.7 % of phenotypic variance for SS and SL, respectively. Its additive and dominant effects on SS were 6.1 and 5.7, respectively. The locus was validated using a DH population by composite interval mapping and located to linkage group C9 (designated as qSS.C9). Mapping qSS.C9 was undertaken using 230 extremely low-SS plants of a BC(4)F(1) population containing 807 plants. We found that qSS.C9 delimited a 1.005-Mb interval including 218 predicted genes in the reference Brassica rapa (Chiifu-401). These results will greatly facilitate map-based cloning of qSS.C9 and seed yield improvement in rapeseed.

Comparative analysis of genetic diversity in Tunisian apricot germplasm using AFLP and SSR markers

Eighty-one accessions representing apricot germplasm in Tunisia were collected from different areas of cultivation and fingerprinted using amplified fragment length polymorphism (AFLP) and microsatellites (SSR) markers. A total of 339 polymorphic markers were revealed using 5 AFLP primers combinations and 24 SSR loci. AFLP and SSR markers expressed a high level of polymorphism allowing the distinction of the accessions with an efficiency coefficient of discrimination of 100% for AFLP and 97% for SSR markers. Genetic diversity structure was assessed with AFLPs and SSRs markers separately then with combined matrix data by the help of hierarchical clustering elaborated using Wards method based on Nei and Li (1979) distances. Comparison of the obtained dendrograms revealed a phylogeographic structure into two major groups with significant conservation between the observed subgroups in relation with the geographic origin of the accessions. The relative efficiency of the markers in determining the genetic relationships among apricot accessions has been assessed and a combination of AFLPs and SSRs markers was the most effective. In addition, Mantel test based on genetic distances indicated highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with Pearson correlation values of r = 0.873 and r = 0.692, respectively, revealing the higher efficiency of the combination of both molecular techniques (AFLP and SSR) to estimate the levels of genetic variability among apricot germplasm.

Comparative analysis of genetic variation among Xanthomonas axonopodis pv manihotis isolated from the western states of Nigeria using RAPD and AFLP

Xanthomonas axonopodis pv manihotis is the causal agent of cassava bacterial blight (CBB) worldwide. CBB disease is a major constraint to cassava cultivation, and losses can be extremely severe in regions where highly susceptible cultivars are grown. To develop an efficient disease management policy, the genetic diversity of the pathogens population must be known. There is dearth of information on the genetic diversity of X. axonopodis pv manihotis population in Nigeria. We used RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism), a PCR-based technique, to characterize the X. axonopodis pv manihotis isolates from the western States of Nigeria. Thirteen strains Xam and 2 reference strains were tested with eight primers combination of AFLP and 4 RAPD primers. RAPD amplified DNA fragment data showed four major clusters at 80 % similarity coefficient level and two strains were not clustered by this analysis. Strains Kwa76A and Ond48A were also separated in the principal component analysis of the same data. Numerical analysis differentiated the AFLP patterns into four distinct clusters and grouped two strains separately at 66 % similarity. PCA assembly grouped the bacterial strains into 4 and one of the strains was singled out from the others. The two DNA analyses techniques seem to be complimentary to one another and informative on the genomic structure of Xam population in Western Nigeria. The genetic analysis presented here contributes to understanding of the Xam population structure in Western Nigeria.

CONSERVATION GENETICS OF THE SONOMA TREE VOLE (ARBORIMUS POMO) BASED ON MITOCHONDRIAL AND AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS

We used a comparative, multimarker approach to investigate the conservation genetics of an arboreal vole (the Sonoma tree vole [Arborimus pomo]) in the Pacific Northwest of North America. We compared geographic patterns and overall levels of genetic diversity based on 55 amplified fragment length polymorphism (AFLP) loci with those based on a single, commonly used mitochondrial locus, the control region. Although examination of the control region data revealed the presence of 2 distinct mitochondrial DNA (mtDNA) clades within A. pomo (1 in the north and 1 in the south of the species' range), the nuclear perspective provided by AFLP did not reveal a similar geographic division within the species, supporting instead that A. pomo consists of a single panmictic population. Genetic diversity estimates based on the mtDNA data (gene diversity = 0.79 and 0.80 for the 2 clades) were much greater than those based on AFLP (gene diversity = 0.31 and 0.19 for the 2 clades). These contrasting results reflect inherent differences between mitochondrial and nuclear loci in mutation rate, effective population size, expected time to monophyly, and mode of inheritance, and highlight the utility of using the combination of AFLP and mtDNA when assessing the genetic status of wild populations and species of mammals, especially those of conservation concern. In the case of A. pomo, our combined AFLP and mtDNA data support the recognition of the southern Sonoma tree voles as a distinct management unit within the species.

Microsatellite and AFLP markers in the Prunus persica [L. (Batsch)]×P. ferganensis BC1linkage map: saturation and coverage improvement

A set of 146 single sequence repeats (SSRs) and 14 amplified fragment length polymorphism (AFLP) primer combinations were used to enrich a previously developed linkage map obtained from a (Prunus persicaxP. ferganensis)xP. persica BC(1) progeny. Forty-one SSR primer pairs gave polymorphic patterns detecting 42 loci. The restriction/selective primer AFLP combinations produced a total of 79 segregating fragments. The resulting map is composed of 216 loci covering 665 cM with an average distance of 3.1 cM. Novel regions were covered by the newly mapped loci for a total of 159 cM. Eight linkage groups were assembled instead of the earlier 10 as two small groups (G1a and G8b), previously independent, were joined to their respective major groups (G1b and G8a). Several gaps were also reduced resulting in an improved saturation of the map. Twelve gaps >or=10 cm are still present. A comparative analysis against the Prunus reference map (71 anchor loci) pointed out an almost complete synteny and colinearity. Six loci were not syntenic and only two were not colinear. Genetic distances were significantly longer in our map than in the reference one.

A combination of AFLP and SSR markers provides extensive map coverage and identification of homo(eo)logous linkage groups in a sugarcane cultivar

Sugarcane varieties are complex polyploids carrying in excess of 100 chromosomes and are derived from interspecific hybridisation between the domesticated Saccharum officinarum and the wild relative S. spontaneum. A map was constructed in Denotes variety covered by Australian plant breeding rights., an Australian cultivar, from a segregating F1 population, using 40 amplified fragment length polymorphism (AFLP) primer combinations, five randomly amplified DNA fingerprints (RAF) primers and 72 simple sequence repeat (SSR) primers. Using these PCR-based marker systems, we generated 1,365 polymorphic markers, of which 967 (71%) were single-dose (SD) markers. Of these SD 967 markers, 910 were distributed on 116 linkage groups (LGs) with a total map length of 9,058.3 cM. Genome organisation was significantly greater than observed in previously reported maps for Saccharum spp. With the addition of 123 double-dose markers, 36 (3:1) segregating markers and a further five SD markers, 1,074 markers were mapped onto 136 LGs. Repulsion phase linkage detected preferential pairing for 40 LGs, which formed 11 LG pairs and three multi-chromosome pairing groups. Using SSRs, double-dose markers and repulsion phase linkage, we succeeded in forming 127 of the 136 LGs into eight homo(eo)logy groups (HG). Two HGs were each represented by two sets of LGs. These sets of LGs potentially correspond to S. officinarum chromosomes, with each set aligning to either end of one or two larger LGs. The larger chromosomes in the two HGs potentially correspond to S. spontaneum chromosomes. This suggestion is consistent with the different basic chromosome number of the two species that are hybridised to form sugarcane cultivars, S. spontaneum (x=8) and S. officinarum (x=10), and illustrates the structural relationship between the genomes of these two species. The discrepancy of coverage between HGs highlights the difficulty in mapping large parts of the genome.

Cloning of AFLP markers linked to resistance to Peronosclerospora sorghi in maize.

Genetic mapping of resistance genes for sorghum downy mildew (SDM) in maize revealed multiple-locus inheritance. A combination of AFLP (amplified fragment length polymorphism) technique with bulked segregant analysis (BSA) was applied to map the genes involved in the resistance to SDM (Peronosclerospora sorghi) in a recombinant inbred population. Three AFLP markers were identified and mapped to chromosomes 1 and 9, in regions previously associated with SDM resistance. One other AFLP marker was found to be associated with disease susceptibility but could not be linked to any chromosome. These four AFLP fragments were isolated, cloned and sequenced. A BLAST search of the GenBank database showed that none of these four sequences was closely related to resistance genes that have been reported previously. Sequence-characterized amplified regions (SCARs) were produced and used to assess the presence of SDM resistance genes and characterize specific genotypes. These markers may be useful in marker-assisted breeding programs.

Detection of quantitative trait loci for carcass traits in the pig by using AFLP

For evaluation of the suitability of Amplified Fragment Length Polymorphism (AFLP) for detection of quantitative trait loci in farm animals, a combination of AFLP and selective genotyping has been applied as a rapid screening method for marker-QTL associations. Focusing on loci affecting eye muscle area, six extreme discordant sib pairs were selected from a Duroc x Berlin Miniature Pig F2 experimental cross and examined by using 48 AFLP primer combinations. Two prominent AFLP markers were converted into simple codominant PCR markers ( STS-Bo1 and STS-Bo3) and assigned to Sscr4 by physical and linkage mapping. Single marker analysis indicated association of the STS markers with a putative QTL influencing eye muscle area. Interval mapping confirmed the presence of a significant QTL for eye muscle area (Pgenomewide < 0.01) on the Sscr4, with STS-Bo1 being the closer marker. At the same location, significant effects (Pgenomewide < 0.01) on carcass length and backfat thickness were also detected. Our results demonstrate the capability of the combination of AFLP analysis and selective genotyping as a method for detection of genome regions containing QTL in livestock.