Amplified Fragment Length Polymorphism Primer Pairs Research Articles

Long-Term Grazing Effects on Genetic Variability in Mountain Rough Fescue

Festuca campestris Rydb. (mountain rough fescue) is a dominant grass species in the montane grasslands of western Canada. Little is known about the genetic diversity of this plant and the effects of long-term grazing on the genetics of populations. The amplified fragment length polymorphism (AFLP) technique was applied to compare the genetic diversity of fescue plants at adjacent grazed and protected areas for 3 populations spread across a longitudinal range: Stavely in the foothills, Milroy in the Rocky Mountain trench, and Goose Lake on the interior plateau. Five AFLP primer pairs were used to screen the tiller samples of about 39 plants in each grazed (or ungrazed) area, and 139 polymorphic AFLP bands were scored for each individual sample. These scored bands had frequencies ranging from 0.03 to 0.98 with an average of 0.56. About 81% of the total AFLP variation resided within the populations. The Goose Lake population had the lowest level of AFLP variation, but genetically was the most distinct. Four AFLP bands were possibly associated with chromosomal segments significant for grazing resistance. Comparisons of AFLP variation between grazing and nongrazing samples revealed variable and relatively small impacts of the long-term grazing on the genetic diversity of the grazed populations. The AFLP variation of grazed samples was 1.5% lower at Goose Lake, 2.2% higher at Milroy, and not different at Stavely. If developing diverse germplasm for rangeland seedings is desired, one should sample across geographic space rather than combining materials with and without historical grazing pressure.

Assessment of genetic diversity in bread wheat genotypes for tolerance to drought using AFLPs and agronomic traits

Moisture stress greatly limits the productivity of wheat in many wheat-growing regions of the world. Knowledge of the degree of genetic diversity among parental materials for key selection traits will facilitate the development of high yielding, stress tolerant wheat cultivars. The objectives of this study were to: (i) use amplified fragment length polymorphisms (AFLPs) to assess genetic diversity among bread wheat lines and cultivars with different responses to drought stress in two distinct environments and, (ii) compare genetic diversity estimated by AFLPs with diversity evaluated on agronomic performance under drought stress. Twenty-eight genotypes, 14 from Iran and 14 developed or obtained by CIMMYT, were evaluated in the study. Phenotypic data on the 14 Iranian lines were obtained in Iran, and data on the 14 CIMMYT lines were collected in Mexico. Ten AFLP primer pairs detected 335 polymorphic bands among the 28 cultivars. At the 5th fusion level of the resulting dendrogram, 6 genotype clusters were identified. Thirteen of the 14 CIMMYT genotypes grouped into one cluster while 4 of the remaining groups were comprised only of Iranian genotypes. When the agronomic performance of the Iranian materials was compared with the AFLP diversity analysis, 5 of the 6 drought susceptible genotypes clustered together in the agronomic dendrogram, and were located in the same cluster in the AFLP dendrogram. However, the drought tolerant Iranian materials did not show the same degree of relationship. The CIMMYT materials did not demonstrate a significant association between agronomic performance and genetic diversity determined using AFLPs. Clearly these data show that there are genotypes with similar agronomic performance and different genetic constitutions in this study that can be combined in a breeding program to potentially improve tolerance to drought stress.

Genetic Diversity in Natural Populations and Corresponding Seed Collections of Little Bluestem as Revealed by AFLP Markers

Little bluestem [Schizachyrium scoparium (Michx.) Nash] is one of the most widespread native grasses in North America. Little is known about the genetic diversity of this species in natural populations and in seed collections. The amplified fragment length polymorphism (AFLP) technique was applied to assess the comparative genetic diversity of six natural populations of little bluestem in Manitoba and Saskatchewan and their corresponding seed collections. Five AFLP primer pairs were employed to screen a total of 180 samples representing about 15 tillers per population and 15 seeds per collection, and 158 polymorphic AFLP bands were scored for each sample. Analyses of these scored bands revealed that >91% of the total AFLP variation was present within the natural populations and within the seed collections. The among-population and among-collection variation components, although relatively small (7-9%), were statistically significant from zero. Comparisons of AFLP profiles between the seed and tiller samples revealed the seed samples had fewer polymorphic bands, higher average band frequencies, and more bands with extremely high or low frequencies. A significant association of AFLP variation with geographical origin was detected in the seed, but not the tiller samples. These results indicate collecting seeds may not be as effective as collecting tillers in sampling genetic diversity from natural populations for the improvement of little bluestem germplasm for rangeland seeding.

Genetic diversity and biogeography of Cunninghamia konishii (Cupressaceae), an island species in Taiwan: a comparison with Cunninghamia lanceolata, a mainland species in China

Luanta-fir ( Cunninghamia konishii), an endemic to Taiwan, is an outcrossing, long-lived conifer. Populations of C. konishii are generally fragmented due to a once high intensity of timber exploitation. C. konishii and Cunninghamia lanceolata are two sibling taxa constituting derivative-progenitor species relationship. The amount of genetic variations within and between 11 and 10 populations of C. konishii and C. lanceolata, respectively, were assessed using amplified fragment length polymorphism (AFLP) markers in this report. Three AFLP primer pairs generated a total of 357 and 226 markers for C. konishii and C. lanceolata samples, of which 56.1 and 65.3% are polymorphic, respectively. Analysis of molecular variance indicates a 4.78% variation between C. konishii and C. lanceolata. A relatively high value of genetic variation (24.60%) was apportioned between the populations of C. konishii. In contrast, a lower divergence value (12.21%) between populations was found for C. lanceolata. The population with the highest genetic diversity was found in Nantou County, which concurred with the results of many other tree species investigated in Taiwan. The estimates of the number of migrants between populations ( Nm), obtained from population pair-wise Φ ST, suggest that gene flow in C. konishii is efficient in some adjacent populations but is restricted in the rest. Individual UPGMA tree, generated based on AFLP markers, suggests six evolutionary lineages for C. konishii. All evolutionary lineages of C. konishii were derived from C. lanceolata. In conclusion, the migration patterns of Cunninghamia from mainland China may have been established following multiple sources, migrant-pools, long-distance dispersal events, and via different directions.

AFLP Variation in Four Blue Grama Seed Sources

Blue grama [Bouteloua gracilis (Willd. ex Kunth) Lag. ex Griffiths] is one of the most widespread native grasses in western North America. Several blue grama seed sources are currently used for rangeland seeding, but little is known about the genetic diversity of these seed sources. Amplified fragment length polymorphism (AFLP) technique was applied to compare the genetic diversity among four blue grama seed sources (a precultivar germplasm [balanced multisite composite, BMSC], the ecotype Bad River, a Minnesota ecotype, and a native Manitoba seed collection) and to assess the genetic shift during two generations of BMSC seed multiplication. Germplasm BMSC was a balanced multisite composite of 99 clones selected from 495 live plants collected from 11 sites across Manitoba. Six AFLP primer pairs were employed to screen a total of 176 individual plants sampled from both the first three generations of BMSC and the other three seed sources and 167 polymorphic AFLP bands were scored for each plant. Large AFLP variation was observed within the four seed sources. Greater AFLP variation was detected in the BMSC than Bad River, Minnesota ecotype, and the Manitoba native harvest. No genetic shift in the BMSC was found across the two seed multiplications. These results indicate a balanced composite of multisite blue grama germplasm can maintain high genetic diversity with little genetic shift in a few generations of seed multiplication.

Genetic diversity inNarcissus poëticusL. andN. radiiflorusSalisb. (Amaryllidaceae) in two different populations: AFLP and karyological studies.

The two taxa, Narcissus poeticus and N. radiiflorus, although apparently quite morphologically distinct, grow together and there has been some debate as to whether they should be considered as a single or two separate species. In order to throw some light on this debate, a survey of amplified fragment length polymorphism (AFLP) was conducted to examine genetic diversity/similarity among 120 individuals of N. poeticus and N. radiiflorus ob- tained from two populations coming from sites separated by more than 100 km. In addition, a karyological study of the two taxa was carried out to determine chromosome number and morphology, the number of nucleoli and band- ing pattern with the fluorochromes chromomycin A3 and DAPI. Using two AFLP primer pairs, a total of 103 clearly scorable fragments were amplified; of which 91 (88%) were polymorphic (78% if polymorphism is described as the most common band not present in more than 95% of individuals sampled). Cluster analysis and principal coordi- nates analysis of the calculated similarity matrix revealed there to be only a low level of genetic diversity between the entities constituting the populations and species. The genetic distance between the individual belonging to the dif- ferent populations of the same species was slightly greater than that between species. Comparative chromosome analysis showed no differences in the two taxa at the cytological level.

Amplified fragment length polymorphism analysis of 96 Canadian oat cultivars released between 1886 and 2001

Canadian oat breeders have developed and released more than 130 cultivars since 1886, but no systematic analyses of the genetic diversity of Canadian oat have been made. Ninety-six Canadian oat cultivars released between 1886 and 2001 were examined for genetic diversity and relationships using amplified fragment length polymorphism (AFLP) markers. Ten AFLP primer pairs were applied and over 442 polymorphic bands were generated for each cultivar. Most of the cultivars were found to be interrelated, although a few genetically distinct cultivars were also identified. The genetic variation observed among these cultivars was low. Only 42.8% of the total scorable bands were polymorphic, of which 59 polymorphic bands were observed frequently (f ≥ 0.90) and 130 bands were detected infrequently (f ≤ 0.01) among the 96 cultivars. The mean proportion of fixed recessive bands for all cultivars was high (60.2%) and ranged from 52.3% in cultivar ‘Erban’ to 65.2% in cultivar ‘Glen’. The genetic variability did not change significantly in the Canadian oat breeding programs; only about 1% of the AFLP variation might have been fixed during the 115 years of oat breeding. These findings demonstrate the narrowness of the Canadian oat gene pool and the urgent need to broaden the gene pool for sustainable oat improvement in Canada. Key words: Amplified fragment length polymorphism (AFLP), oat, Avena sativa, genetic diversity, genetic relationship

AFLP analysis of Solanum phureja DNA introgressed into potato dihaploids

AbstractIntrogression of Solanum phureja DNA into S. tuberosum dihaploids was analysed by the use of amplified fragment length polymorphism (AFLP) markers. Five dihaploids, derived from crosses between S. tuberosum cv. Pentland Crown and two different S. phureja pollinators (IVP48 and EC90) were investigated by use of 17 AFLP primer pairs. Also 30 dihaploids, derived from pollination of five different S. tuberosum seed parents with S. phureja IVP101, were investigated for the presence of S. phureja‐specific markers. In total approximately 680 and 850 AFLP products were detected in the diploid S. phureja clones and in the tetraploid S. tuberosum genotypes, respectively. A total of 68 S. phureja IVP48‐specific markers were detected, while the total number of S. phureja IVP101‐specific markers was in the range of 72‐96, depending on the S. tuberosum seed parent. Introgression of DNA in the S. tuberosum cv. Pentland Crown dihaploids, after pollination with S. phureja IVP48 and S. phureja EC90, was demonstrated by the detection of 14 of 68 IPV48‐specific markers in the dihaploids. However, no DNA introgression was found in any of the 30 S. tuberosum dihaploids derived from S. phureja IVP101. Hence, S. phureja IVP101 is regarded as an excellent pollinator in the production of S. tuberosum dihaploids in potato breeding programmes because of the high yield of dihaploids per 100 berries, and because no introgression of DNA into the S. tuberosum dihaploids was evidenced.

The genetic diversity of the Vigna angularis complex in Asia.

A selected set of accessions of components of the azuki bean (Vigna angularis) complex comprising 123 cultivated accessions and 23 wild or weedy accessions from Bhutan, China (including Taiwan), India, Japan, Korea, and Nepal was analyzed using amplified fragment length polymorphism (AFLP) methodology. Using 12 AFLP primer pairs, 580 unambiguous bands were generated, 313 (53.9%) of which were polymorphic among azuki bean accessions. All 580 bands were used to assess phenotypic (band) and genetic (nucleotide) diversity among the 146 azuki bean accessions. The results indicate five major groups of azuki bean germplasm primarily associated with geographic origin of accessions and their status: wild, weedy, or cultivated. These five groups are (i) Himalayan wild, (ii) Nepal-Bhutan cultivated, (iii) Chinese wild, (iv) Taiwan wild - Bhutan cultivated, and (v) northeast Asian accessions. Within the northeast Asian accessions, three subgroups are present. These consist of (v1) Japanese complex - Korean cultivated, (v2) Japanese cultivated, and (v3) Chinese cultivated accessions. The results suggest domestication of azuki bean occurred at least twice, once in the Himalayan region of southern Asia and once in northeast Asia. The remarkable diversity of azuki bean germplasm in the Himalayan region compared with other regions suggests this is a rich source of germplasm for plant breeding. The results suggest there are important gaps in the germplasm collections of azuki bean and its close relatives from various parts of Asia and that specific collecting missions for Vigna germplasm related to azuki bean in the highlands of subtropical Asia are needed.

Molecular detection of cell line cross-contaminations using amplified fragment length polymorphism DNA fingerprinting technology.

We have tested amplified fragment length polymorphism (AFLP) technology, in comparison with isoenzyme analysis, for the simultaneous detection of inter- and intraspecific cell line cross-contaminations (CCCs) in the cell line collection held at the Istituto Zooprofilattico della Lombardia e dell'Emilia Romagna. Isoenzyme analysis identified four cases of interspecific CCCs. In a single experiment, AFLP was able to identify the species of origin of all cell lines for which a reference genomic deoxyribonucleic acid was available and to detect five interspecific contaminations. Four CCCs confirmed data on isoenzymes, whereas the fifth CCC was detected in a species for which isoenzyme analysis was noninformative. In addition, AFLP was able to identify the putative source of the contaminations detected. The utility of the technology in the detection of intraspecific cell line contaminations depends on the number of cell lines that have to be distinguished in a specific species and on the availability of highly informative fingerprinting systems. In mice, a single AFLP primer pair produced 16 polymorphisms and distinguished all the 15 strains of mouse cell lines analyzed. In humans, 18 AFLPs identified 83 different profiles in the 159 cell lines analyzed. Amplified fragment length polymorphism can conveniently be applied for cell line fingerprinting in species for which hypervariable markers are not available. In species for which a highly informative multiplex of microsatellite markers is available, AFLP can still provide a useful and cheap tool for simultaneously testing inter- and intraspecific contaminations.

Identification of a sequence-tagged site (STS) marker linked to a restorer gene for Ogura cytoplasmic male sterility in radish ( Raphanus sativus L.) by non-radioactive AFLP analysis

To identify DNA markers linked to a fertility restorer (Rf) genefor Ogura cytoplasmic male sterility in radish (Raphanus sativus L.),a non-radioactive, amplified fragment length polymorphism (AFLP) analysiswas performed on bulked DNA samples from male-sterile and male-fertileradishes. Ten male-fertile and 10 male-sterile plants selected arbitrarilyfrom an F2 population made by selfing of F1 plant from a crossbetween a male-sterile (`MS-Gensuke') plant and a restorer (`Comet') plantwere used as material. Using 32 AFLP primer pairs, one AFLP fragment(AFLP190) which is specific to the bulked DNA samples from male-fertileF2 plants was identified. AFLP190 was characterized by molecularcloning and nucleotide sequencing, and was converted to a sequence-taggedsite (STS) marker, STS190. A linkage analysis performed in 126individuals of two independent F2 populations showed tight linkageof STS190 to the Rf gene. The rate of recombination between themarker and Rf was estimated to be less than 1%, making STS1901.2 cM from the gene.

Genetic information of three pure lines of Porphyra yezoensis (Bangiales, Rhodophyta) obtained by AFLP analysis

: Porphyra (Bangiales, Rhodophyta), which includes several valuable marine crops, has recently received great interest as a model plant for fundamental and applied studies in marine sciences. Amplified fragment length polymorphisms (AFLPs) are a robust and efficient means for genetic mapping, linkage analysis of genetic characters for breeding and population studies in land plant genomes. To examine whether AFLPs are applicable as genetic markers in the present study, we detected AFLP markers with three pure lines in order to promote genetic analysis in Porphyra yezoensis. The following five sets of AFLP primer pairs (E-AA, M-CAA) (E-AA, M-CAC) (E-AA, M-CAG) (E-AA, M-CAT) (E-AA, M-CTA) were tested with template DNAs from three pure lines and they showed a total of 227 bands. This suggests that AFLP markers are promising tools for genetic analysis in Porphyra.

DNA Markers Linked to Fusarium Wilt Race 1 Resistance in Pea

Dry pea (Pisum sativum L.) production in many areas of the world may be severely diminished by soil inhabiting pathogens such as Fusarium oxysporum f. sp. pisi race 1, the causal organism of fusarium wilt race 1. Our objective was to identify closely linked marker(s) to the fusarium wilt race 1 resistance gene (Fw) that could be used for marker assisted selection in applied pea breeding programs. Eighty recombinant inbred lines (RILs) from the cross of Green Arrow (resistant) and PI 179449 (susceptible) were developed through single-seed descent, and screened for disease reaction in race 1 infested field soil and the greenhouse using single-isolate inoculum. The RILs segregated 38 resistant and 42 susceptible fitting the expected 1:1 segregation ratio for a single dominant gene (χ2 = 0.200). Bulk segregant analysis (BSA) was used to screen 64 amplified fragment length polymorphism (AFLP) primer pairs and previously mapped random amplified polymorphic DNA (RAPD) primers to identify candidate markers. Eight AFLP primer pairs and 15 RAPD primers were used to screen the RIL mapping population and generate a linkage map. One AFLP marker, ACG:CAT_222, was within 1.4 cM of the Fw gene. Two other markers, AFLP marker ACC:CTG_159 at 2.6 cM linked to the susceptible allele, and RAPD marker Y15_1050 at 4.6 cM linked to the resistant allele, were also identified. The probability of correctly identifying resistant lines to fusarium wilt race 1, with DNA marker ACG:CAT_222, is 96% percent. These markers will be useful for marker assisted breeding in applied pea breeding programs.

Genetic diversity and strain-typing in cultivated strains of Lentinula edodes (the shii-take mushroom) in Japan by AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis is based on selective PCR (polymerase chain reaction) amplification of genomic DNA restriction fragments. The present study was performed to: (1) assess the extent of AFLP variation among major cultivated strains of Lentinula edodes (the shii-take mushroom) in Japan; (2) evaluate the usefulness of AFLP as genetic marker for typing the cultivated strains; and (3) infer the genetic relatedness among them. Six AFLP primer pairs detected a total of 304 DNA fragments in a sample of 13 cultivated strains for wood log cultivation and 2 strains for sawdust cultivation, of which 179 DNA fragments (58.9% of detected fragments) were polymorphic between two or more strains. These polymorphic DNA fragments could differentiated all of the cultivated strains. Cluster analysis and principle coordinate analysis based on AFLP data showed two distinct groups; one group was composed of the strains in which fruiting occurred during the middle to low temperature period from autumn to next spring under outdoor wood log cultivation, and the other strains in which fruiting could be induced in the summer under outdoor cultivation and strains for sawdust cultivation under indoor air-controlled condition.

AFLP diversity within and between populations of the Caribbean seagrass Thalassia testudinum (Hydrocharitaceae)

Genetic variation was assessed in the seagrass Thalassia testudinum from three regions of the Caribbean and north Atlantic using allozyme electrophoresis and amplified fragment length polymorphism (AFLP) analysis. Very low allozyme variability was detected among the 196 shoots analyzed from a range of sites in the San Blas region of Panama. AFLP markers detected high similarity (0.87) among the population samples surveyed from Bermuda and Panama across six AFLP primer pairs and over 260 banding positions. High levels of gene flow were detected between all the sites analyzed (N m>1.7). Significantly complete genotypic similarity was observed between samples from Bermuda and Panama, indicating that long distance vegetative fragment dispersal is highly probable. Very low genetic differentiation between all sites, even Bermuda and Panama, some 2,700 km apart, agrees with other studies and is further evidence of a highly uniform gene pool in T. testudinum. High levels of genetic uniformity in T. testudinum may be related to long-term environmental change over its geographic range. While AFLP analysis proved useful in determining genetic variation in this seagrass, the application of co-dominant markers such as microsatellites will be more informative in determining the nature of genetic uniformity and its adaptive significance in T. testudinum.

Amplified Fragment Length Polymorphism in a Selected Sample of Soft Red Winter Wheat

AFLP (Amplified Fragment Length Polymorphism) markers were investigated for assessing diversity and fingerprinting of a selected sample of soft red winter wheat (SRWW) genotypes that differed for pre-harvest sprouting (PHS) resistance and for the presence of awns. Sixty AFLP primer pairs generated 2944 polymorphic bands out of a total of 6778 bands scored among 15 SRWW genotypes. The mean polymorphism information content was 0.37 and the average marker index was 15.1, indicating that AFLP generated more information in wheat than other molecular markers. Forty-two primer pairs that contained no missing data points were used to create a dendrogram to estimate the genetic distance among genotypes. A total of 4761 loci, of which 1915 were polymorphic, were included in the genetic similarity matrix. Primer pairs showed a high degree of correlation between four arbitrarily divided primer subsets (r=0.82). The SRWW genotypes investigated did not group together regarding resistance to PHS or the presence of awns. The mean genetic similarity values among the 15 genotypes ranged from 0.83 to 0.94. Although the genetic similarity among these soft red winter wheat genotypes was high, differences were found that could be used for assessing genetic diversity as well as for plant variety protection use.

Molecular mapping of a new gene for resistance to frogeye leaf spot of soya bean in ‘Peking’

AbstractAmplified fragment length polymorphism (AFLP) and microsatellite (simple sequence repeat, SSR) techniques were used to map the _RGSpeking gene, which is resistant to most isolates of Cercospora sojina in the soya bean cultivar ‘Peking’. The mapping was conducted using a defined F2 population derived from the cross of ‘Peking’(resistant) בLee’(susceptible). Of 64 EcoRI and MseI primer combinations, 30 produced polymorphisms between the two parents. The F2 population, consisting of 116 individuals, was screened with the 30 AFLP primer pairs and three mapped SSR markers to detect markers possibly linked to RcsPeking. One AFLP marker amplified by primer pair E‐AAC/M‐CTA and one SSR marker Satt244 were identified to be linked to ResPeking. The gene was located within a 2.1‐cM interval between markers AACCTA178 and Satt244, 1.1 cM from Satt244 and 1.0 cM from AACCTA178. Since the SSR markers Satt244 and Satt431 have been mapped to molecular linkage group (LG) J of soya bean, the ResPeking resistance gene was putatively located on the LG J. This will provide soya bean breeders an opportunity to use these markers for marker‐assisted selection for frogeye leaf spot resistance in soya bean.

From Dampier to DNA: the 300-year-old mystery of the identity and proposed allopolyploid origin of Conostylis stylidioides (Haemodoraceae)

The tetraploid Conostylis stylidioides (n= 16) has been proposed to be a stabilised hybrid between the diploid (n = 8) species C. prolifera and C. candicans because of morphological and geographical intermediacy, as well as a polyploid chromosome number. To test this hybrid-origin hypothesis, we used the DNA-fingerprinting technique amplified fragment length polymorphism (AFLP) and measured genetic variation within these taxa, as well as the putative outgroups C. robusta and C. aculeata. One AFLP primer pair generated 192 dominant markers for 36 samples from these species, of which 189 were polymorphic. Polymorphism within populations was uniformly high for all species, with 66–86% of all fragments polymorphic and estimates of heterozygosity ranging from 0.36 to 0.41. Ordination, UPGMA and maximum parsimony analyses of these genetic data consistently clustered species, supporting the current species’ level taxonomy. The intermediate placement of C. stylidioides between C. proliferaand C. candicans on the maximum parsimony tree supports the hybrid-origin hypothesis, although other interpretations are possible. The phenetic results for AFLP data, in which C. stylidioides is not strictly intermediate between C. prolifera and C. candicans, are either concordant with recent research suggesting that rapid intra- and inter-genomic rearrangements occur with the origin of polyploid taxa, or indicate an ancient hybridisation event. While our results do not reject the hybrid origin hypothesis, the extremely high levels of genetic variation detected with AFLP within these populations, in combination with extensive genomic reorganisation with the origin of C. stylidioides and the possibility of independent origins for different populations, make it difficult to confidently exclude other scenarios.

Isozyme and amplified fragment length polymorphisms fromCephalosporium maydis in Egypt

Isoenzyme and amplified fragment length polymorphisms (AFLP) variation within a set of 48 isolates ofCephalosporium maydis was characterized. These isolates included ten cultures that have served as standards in the Egyptian maize resistance breeding program and 38 additional strains collected from 11 governates in Egypt during the 1997 growing season. Eight isozymes also were tested, but only five (acid phosphatase, fumerase, gtucose 4-phosphate isomerase, isocitrate dehydrogenase, and malate dehydrogenase) produced identifiable bands and all five of these enzymes were monomorphic. Sixty-eight AFLP primer-pair combinations were used and 865 bands were scored, of which 288 (33%) were polymorphic and could be used to discriminate four distinct subgroups, or lineages. Representatives from only two of the four lineages are included in the set of ten strains that has been used to challenge new lines in the Egyptian maize breeding program. From among these 68 primer-pair combinations, we identified a set of four AFLP primer-pairs that were strongly correlated (Pearson‘sr > 0.85) with the full data set that can be used as markers to determine the distribution of these lineages and to identify new lineages in field populations.

049 Development of Molecular Markers Linked to High Pigment (hp) and Dark Green (dg) Loci in Tomato

The carotenoids have an important influence on tomato fruit quality and enhance the fruit contribution to human nutrition. Expression of the high pigment (hp) locus in tomato results in increased total carotenoids and increased efficiency of utilization of the polyenes. A similar mutant, dark green (dg), contains higher level of chlorophyll in immature fruit and results in darker red pigmentation, both externally and internally in ripe fruit. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses were performed using two pairs of near isogenic lines (NILs) designed to be isogenic at the hp and dg loci. Sixty-four AFLP primer pairs and more than 1000 RAPD 10-mer primers were screened for polymorphism between each pair of the NILs. One RAPD marker was identified to be linked to the hp gene, and two AFLP primer pairs showed polymorphic fragments which distinguished the dg NILs. The markers identified in this study will be converted to allele specific SCAR (sequence characterized amplified region) markers, which are more useful in marker-assisted selection breeding programs.