RFLP Loci Research Articles

Molecular Marker Analysis of the Approach to Homozygosity by Selfing in Diploid Alfalfa

Although inbreeding is used extensively in alfalfa breeding and genetics, the actual approach to homozygosity during inbreeding is unknown and may differ from that predicted by the inbreeding coefficient (F) because of the need to select for vigor and fertility when inbreeding. Observed and expected values of heterozygosity were compared by means of 40 co‐dominant RFLP loci for two inbred series each consisting of four independent lines derived by selfing two diploid alfalfa genotypes. Selfing was continued with selection for fertility until self‐sterility was reached at the S2, S3, or S4 generation. Additional S1 progenies were also evaluated to identify the heterozygons RFLP loci. The observed percentage heterozygosity, calculated as the percentage of the 40 RFLP loci still heterozygous in each plant, declined less than expected based on the inbreeding coefficient in each inbred series. The lowest observed percentage heterozygosity was 30% in one inbred series and 40% in the other inbred series despite expectations as low as 6%. Tests of genotype and allele ratios indicated that the deviations may be due to selection favoring hetcrozygotes. Our results show that inbreeding in diploid alfalfa proceeds more slowly than expected, based on F, and that inbreeding depression may limit the level of homozygosity obtainable.

The genetic structure of Beta vulgaris ssp. maritima (sea beet) populations. II. Differences in gene flow estimated from RFLP and isozyme loci are habitat-specific

In a previous paper, isolation by distance at RFLP loci was demonstrated in 10 populations of sea beet growing on the Dorset (U.K.) coast. In the same populations there was no evidence of isolation by distance at isozyme loci. Here, the data are reanalysed to reduce biases resulting from nonrandom mating within the populations; the contrast between RFLPs and isozymes remains very clear. Separate analysis of cliff-top populations shows strong isolation by distance at both RFLP and isozyme loci, whereas in populations on sea walls around the perimeter of Poole Harbour there is isolation by distance at RFLP loci but not isozyme loci. Thus, whatever the cause of the difference between RFLP and isozyme loci, it occurs in the harbour populations only. There was no evidence that founder effects are a significant source of variation between populations on the perimeter of the harbour. This is very different from a previous finding of strong founder effects between populations on Furzey Island (in Poole Harbour).

The genetic structure of Beta vulgaris ssp. maritima (sea beet) populations. II. Differences in gene flow estimated from RFLP and isozyme loci are habitat-specific

In a previous paper, isolation by distance at RFLP loci was demonstrated in 10 populations of sea beet growing on the Dorset (U.K.) coast. In the same populations there was no evidence of isolation by distance at isozyme loci. Here, the data are reanalysed to reduce biases resulting from nonrandom mating within the populations; the contrast between RFLPs and isozymes remains very clear. Separate analysis of cliff-top populations shows strong isolation by distance at both RFLP and isozyme loci, whereas in populations on sea walls around the perimeter of Poole Harbour there is isolation by distance at RFLP loci but not isozyme loci. Thus, whatever the cause of the difference between RFLP and isozyme loci, it occurs in the harbour populations only. There was no evidence that founder effects are a significant source of variation between populations on the perimeter of the harbour. This is very different from a previous finding of strong founder effects between populations on Furzey Island (in Poole Harbour).

RFLP-based estimates of parental contribution to F2- and BC1-derived maize inbreds

Selection and genetic drift during inbreeding may cause differences between the actual and expected proportions of the genome derived by an inbred from each of its parents. We used 70 RFLP loci to determine the frequency and magnitude of deviations from the expected parental contribution among F2- and BC1-derived maize (Zea mays L.) inbreds. Assuming inbreds i and j were the parents of inbred k, the parental contribution of i to k was estimated as p=(Sik−Sij)/ (1−Sij), where Sik and Sij were the average proportions, across the ten linkage groups in maize, of RFLP loci with alleles common to the inbreds in subscript. Bootstrap confidence intervals (CIs) were obtained for p by re-sampling RFLP similarity for each linkage group. Among 62 F2-derived inbreds, 13 had estimates of p that deviated significantly from the expected value of 0.5. One F2-derived inbred obtained p=0.801 of its genome from a parent. Among 34 BC1-derived inbreds, eight had estimates of p that deviated significantly from the expected contribution of 0.75 from the recurrent parent. Two inbreds, both from the same BC1 population, had an estimated p?0.94. The results suggested that selection during backcrossing generally favored the recurrent parent over the donor parent. Among the inbreds with significant deviations from the expected p, the width of 95% CIs with 70 RFLP loci was >0.20. Inbreds selfed from the same F2 or BC1 population varied in p, indicating that coefficients of co-ancestry calculated from pedigree records may give erroneous estimates of genetic relationship.

Location of the self-incompatibility locus in an RFLP and RAPD map of Brassica oleracea.

A single locus controlling self-incompatibility (SI) was located on an RFLP and RAPD linkage map of Brassica oleracea by assaying for SI phenotype and segregation of an RFLP locus detected by the cloned St gene SLG6. The map was developed using an F2 population derived from a cabbage x broccoli cross and included 112 RFLP and 47 RAPD loci arranged into nine main linkage groups covering 921 cM. A portion of the population was assayed for St reaction by pollinating with the broccoli parent. The SI reactions cosegregated precisely with the RFLP locus detected by SLG6, and this locus mapped to linkage group 2.

Genome Mapping of Soybean Cyst Nematode Resistance Genes in ‘Peking’, PI 90763, and PI 88788 Using DNA Markers

Several sources of soybean cyst nematode (Heterodera glycines Ichinohe; SCN) resistance have been identified in soybean [Glycine max (L.) Merrill]; yet, the genetic basis of this economically important trait remains poorly characterized. This study was conducted to identify quantitative trait loci that control disease response in three commonly used sources of SCN resistance, Using genetic markers, we analyzed three segregating soybean F2 populations [‘Evans’ × ‘Peking’, Evans × Plant Introduction (PI) 90763, and Evans × P188788] and compared the results with those of a previous study involving PI 209332. for the Peking, PI 90763, and PI 209332 populations, races 1, 3, and 6 were used as SCN inoculum; for the PI 88788 population, only races 3 and 6 were used. To uncover putative resistance loci, F2 DNA marker genotypes at between 63 and 99 loci in each population were contrasted with cyst indices averaged from 12 F2:3 progeny individuals. Four independent partial SCN resistance loci were uncovered at P < 0.0002 (probability per locus). One of these loci, located at the top of linkage group ‘G’ near RFLP locus C006V, was significant at P < 0.0001 in all populations and races tested. Other significant loci included one near RFLP A378H at the opposite end of linkage group ‘G’ from C006V, another on linkage group ‘J’ near marker B032V‐1, and a fourth on linkage group ‘N’ near marker A280Hae‐1. Comparisons between different SCN races indicated that some of the putative resistance loci behave in a race‐specific manner. These results may serve as a resource for SCN researchers and soybean breeders by summarizing a wide range of genetic data on the soybean‐SCN interaction.

Soybean pedigree analysis using map-based molecular markers: recombination during cultivar development

An analysis of the genome structure of soybean cultivars was conducted to determine if cultivars are composed of large regions of chromosomes inherited intact from one parent (indicative of minimal recombination) or if the chromosomes are a mixture of one parent's DNA interspersed with the DNA from the other parent (indicative of maximal recombination). Twenty-one single-cross-derived and 5 single-backcross-derived soybean cultivars and their immediate parents (47 genotypes) were analyzed at 89 RFLP loci to determine the minimal number and distribution of recombination events detected. Cultivars derived from single-cross and single-backcross breeding programs showed an average of 5.2 and 8.0 recombination events per cultivar, respectively. A homogeneity Chi-square test based upon a Poisson distribution of recombination events across 13 linkage groups indicated that the number of recombinations observed among linkage groups was random for the single-cross cultivars, but not for the single-backcross-derived cultivars. A twotailed t-test demonstrated that for some linkage groups, the number of recombinations per map unit exceeded the confidence interval developed from a t-distribution of recombinations standardized for map unit distance. Paired t-tests of the number of recombinations observed between linkage-group ends and the mid-portion of the linkage groups indicated that during the development of the cultivars analyzed in this study more recombinations were associated with the ends of linkage groups than with the middle region. Detailed analysis of each linkage group revealed that large portions of linkage groups D, F, and G were inherited intact from one parent in several cultivars. A portion of linkage group G, in contrast, showed more recombination events than expected, based on genetic distance. These analyses suggest that breeders may have selected against recombination events where agronomically favorable combinations of alleles are present in one parent, and for recombination in areas where agronomically favorable combinations of alleles are not present in either parent.

Molecular-mapping analysis in Brassica napus using isozyme, RAPD and RFLP markers on a doubled-haploid progeny

We have undertaken the construction of a Brassica napus genetic map with isozyme (4%), RFLP (26.5%) and RAPD (68%) markers on a 152 lines of a doubled-haploid population. The map covers 1765 cM and comprises 254 markers including three PCR-specific markers and a morphological marker. They are assembled into 19 linkage groups, covering approximatively 71% of the rapeseed genome. Thirty five percent of the studied markers did not segregate according to the expected Mendelian ratio and tended to cluster in eight specific linkage groups. In this paper, the structure of the genetic map is described and the existence of non-Mendelian segregations in linkage analysis as well as the origins of the observed distortions, are discussed. The mapped RFLP loci corresponded to the cDNAs already used to construct B. napus maps. The first results of intraspecific comparative mapping are presented.

Short Communication Comparison between QTL analysis of powdery mildew resistance in barley based on detached primary leaves and on field data

AbstractA quantitative trait loci (QTL) analysis of quantitative powdery mildew resistance was performed on 216 doubled haploid lines derived from a cross between the winter‐barley varieties ‘Igri’ and ‘Danilo’ using 67 RFLP loci. Resistance to powdery mildew was determined in the field with natural infection and on detached primary leaves with a specific isolate. The major QTL found in both sets of analysis mapped to the same chromosomal region. No further QTL could be found in the analysis based on detached leaves and one additional minor QTL was found in the analysis based on field data.

Quantitative trait loci influencing grain protein content in tetraploid wheats

AbstractSeed storage protein content of durum wheat (Triticum turgidum var. durum) has an important effect on nutritional value and pasta‐making characteristics. The objective of this study was to determine by association with genetic markers the number, chromosomal location, and magnitude of effect of quantitative trait loci (QTLs) controlling protein concentration in kernels. A set of 65 recombinant inbred lines (RIs) was developed by single seed descent from a cross between cultivated durum wheat cv. ‘Messapia’ (low protein content) and accession MG4343 of the wild tetraploid wheat var. dicoccoides (high protein content). This population was characterized for eight morphological, six storage protein, one isozyme and 124 RFLP loci. Field trials were conducted in one location in 1993 and two locations in 1994. QTLs were mapped by regression analysis on each marker locus for each location and for the average across environments. A total of six putative QTLs were located on chromosome arms 4BS, SAL, 6AS, 6BS and 7BS. The number and size of QTLs detected varied across environments. The marker with the highest r2 value per QTL in each environment and across environments was chosen for a multiple linear regression analysis, which explained 49.2‐ 56.4% of the phenotypic variation for protein content. Only some of the markers were found to be negatively associated with plant grain yield and/or seed weight in one or two of the environments.

A physical map of theThinopyrum-derivedLr19translocation

Twenty-nine lines with deletions in the Lr19 ('Indis') translocated chromosome segment were used to physically map three Thinopyrum RFLP loci as well as the Sr25 and Sd1 loci. From the data, the relative locations of marker loci on the translocation were determined as: Sd1, Xpsr165, Xpsr105, Xps129, Lr19, Wsp-D1, Sr25/Y. The data confirmed the reported homoeology between the Lr19 segment and chromosome arm 7DL of wheat. Also, it seems that the Lr19 translocation in 'Indis' is very similar to the Lr19 segment in the T4 source and that the former may not derive from Thinopyrum distichum. Key words : deletion mapping, leaf rust resistance.

The genetic structure of Beta vulgaris ssp. maritima (sea beet) populations: RFLPs and isozymes show different patterns of gene flow

Genetic variation in 12 populations of sea beet was assessed at nine isozyme and seven RFLP loci. Mean observed heterozygosity, diversity index and number of alleles per locus were not significantly different between the two classes of marker. The genetic structure of 10 of the populations was analysed using seven of the isozymes and six of the RFLPs. FST values between all pairs of populations were calculated separately for the isozymes and RFLPs. FST values were converted to amounts of gene flow (Nm) between populations under the assumptions of an island model. A regression of log Nm against log distance was used to test for isolation by distance. Mantel tests showed a highly significant decrease in Nm with distance for RFLPs but not for isozymes. It is suggested that uniform balancing selection may operate to maintain approximately equal allele frequencies among populations at the isozyme loci. If this is true then isozymes may be unsuitable for modelling the spread of neutral transgenes.

The genetic structure of Beta vulgaris ssp. maritima (sea beet) populations: RFLPs and isozymes show different patterns of gene flow

Genetic variation in 12 populations of sea beet was assessed at nine isozyme and seven RFLP loci. Mean observed heterozygosity, diversity index and number of alleles per locus were not significantly different between the two classes of marker. The genetic structure of 10 of the populations was analysed using seven of the isozymes and six of the RFLPs. FST values between all pairs of populations were calculated separately for the isozymes and RFLPs. FST values were converted to amounts of gene flow (Nm) between populations under the assumptions of an island model. A regression of log Nm against log distance was used to test for isolation by distance. Mantel tests showed a highly significant decrease in Nm with distance for RFLPs but not for isozymes. It is suggested that uniform balancing selection may operate to maintain approximately equal allele frequencies among populations at the isozyme loci. If this is true then isozymes may be unsuitable for modelling the spread of neutral transgenes.

Development of a PCR-based allele-specific assay from an RFLP probe linked to resistance to cereal cyst nematode in wheat.

The RFLP locus Xglk605 identified by the probe Tag605 maps to a proximal position on the long arm of wheat chromosome 2B about 7 cM away from a gene conditioning resistance to cereal cyst nematode in the wheat line AUS10894. The clone Tag605 was partially sequenced and the PCR primer set AWP1 was designed. The 292-bp product, which showed no polymorphism between varieties, was cloned and sequenced. A single base difference was found in the sequence of the AWP1 products amplified and cloned from the wheat lines AUS10894 and "Spear'. PCR primers were designed with 3' termini that corresponded to the two alleles. A dual-PCR system was developed in which the primer sets AWP2 and AWP3 produced allele-specific amplification. The concentration of the oligonucleotide primers and the sequence of the primer-template mismatches were critical to the success of discriminatory allele amplification.

A comparison of RFLP maps based on anther culture derived, selfed, and hybrid progenies ofSolanum chacoense

Comparative RFLP linkage maps were constructed using five segregating populations derived from two self-incompatible lines (termed PI 230582 and PI 458314) of diploid tuber-bearing Solanum chacoense Bitt. The analysis was based on 84 RFLP loci identified by 73 different cDNA clones. Distortion of expected Mendelian segregation ratios was observed; less than 10% of the markers showed a skewed segregation in the gametes forming the F1, hybrid population compared with 30% in the selfed population and 46 and 70% in the two populations produced by anther culture. For the anther culture derived populations, most of the skewed loci were scattered throughout the genome, whereas in the populations derived from selfing, they were found primarily in linkage group 1, around the S locus. In this study, we also found that the rate of meiotic recombination could differ between the male and female gametes produced by our parental lines. Thus, male gametes of line PI 458314 showed significantly less recombination as assessed by the total length of the map (206 cM for male gametes vs. 375 cM for female gametes) and the phenomenon was genome-wide. In contrast, the maps from the gametes of PI 230582 had about the same length, but some linkage groups were longer in the female gametes, while others were longer in the male gametes. Key words : Solanum chacoense, RFLP, anther culture, skewed segregation, self-incompatibility, sex differences in recombination.

Linkage mapping of quantitative trait loci controlling seed weight in pea (Pisum sativum L.)

Quantitative trait loci (QTLs) affecting seed weight in pea (Pisum sativum L.) were mapped using two populations, a field-grown F2 progeny of a cross between two cultivated types ('Primo' and 'OSU442-15') and glasshouse-grown single-seed-descent recombinant inbred lines (RILs) from a wide cross between a P. sativum ssp. sativum line ('Slow') and a P. sativum ssp. humile accession ('JI1794'). Linkage maps for these crosses consisted of 199 and 235 markers, respectively. QTLs for seed weight in the 'Primo' x 'OSU442-15' cross were identified by interval mapping, bulked segregant analysis, and selective genotyping. Four QTLs were identified in this cross, demonstrating linkage to four intervals on three linkage groups. QTLs for seed weight in the 'JI1794' x 'Slow' cross were identified by single-marker analyses. Linkage were demonstrated to four intervals on three linkage groups plus three unlinked loci. In the two crosses, only one common genomic region was identified as containing seed-weight QTLs. Seed-weight QTLs mapped to the same region of linkage group III in both crosses. Conserved linkage relationships were demonstrated for pea, mungbean (Vigna radiata L.), and cowpea (V. unguiculata L.) genomic regions containing seed-weight QTLs by mapping RFLP loci from the Vigna maps in the 'Primo' x 'OSU442-15' and 'JI1794' x 'Slow' crosses.

Identification of genetic factors for Alachlor tolerance in maize by molecular markers analysis.

Genetic factors controlling tolerance to the herbicide Alachlor in maize were localised by means of two different strategies. In the first approach, backcross (BC) plants, derived from pollen which had been subjected to selective pressure for resistance to the herbicide, were analysed for segregation distortion at 47 RFLP loci and compared to BC plants obtained from non-selected pollen. Preferential transmission of five chromosomal regions where putative QTLs (Quantitative Trait Loci) are localised was revealed in the BC plants from selected pollen. A second approach was based on a classical linkage analysis for segregation of the same set of RFLPs and factors controlling the trait, in a BC population of 210 individuals, by means of regression analysis. This study detected seven significant loci in four genomic regions. Overall, two loci revealed both segregation distortion and association with the expression of the trait, indicating linkage to genes expressed in both gametophytic and sporophytic phase. Three chromosomal regions appeared to carry factors involved in plant tolerance to Alachlor which are not expressed in pollen. Conversely, three loci were linked to factors selectable in pollen, but did not reveal significant association with tolerance in the plant in the segregating populations.

Mapping loci controlling the concentrations of erucic and linolenic acids in seed oil of Brassica napus L.

The quality of plant oil is determined by its component fatty acids. Relatively high levels of linolenic acid reduce the oxidative stability of the oil, and high levels of erucic acid in the diet have been associated with health problems. Thus, oilseed Brassica napus cultivars with low linolenic and low erucic acid contents are highly desirable for edible oil production. In order to identify genes controlling the levels of erucic and linolenic acids, we analyzed the oil composition of 99 F1-derived doubled haploid lines from a cross between cv 'Major' (high levels of erucic and linolenic acids) and cv 'Stellar' (low levels of both fatty acids). A molecular marker linkage map of 199 loci for this population was used to identify quantitative trait loci (QTL) controlling oil composition. We identified two regions that accounted for nearly all of the phenotypic variation in erucic acid concentration and one region that accounted for 47% of the variation in linolenic acid concentration. The QTL associated with linolenic acid concentration mapped near a RFLP locus detected by a cDNA clone encoding an omega-3 desaturase, suggesting that the low linolenic acid content of 'Stellar' may be due to a mutation in this gene.

Soybean chromosome painting: a strategy for somatic cytogenetics.

Cytological identification of soybean mitotic metaphase chromosomes (2n = 40) has been severely limited by their small size and uniform karyomorphology. We have developed fluorescent in situ hybridization (FISH), PCR-primed in situ labelling (PCR-PRINS) procedures, and molecular probes for routine cytological identification and for the physical mapping of soybean somatic chromosomes. Chromosome preparation has been achieved by modifications of previous protocols and through the preparation of root-tip protoplasts prior to chromosome spreading. Initially our probe selection focused on highly repeated DNAs that provide very intense localized hybridization signals. Repetitive gene probes that have proven valuable include the rDNA loci (5S and 45S) which are chromosome specific. We have also developed satellite DNA probes for two different sequence families: the SB92 and the STR120 satellites. Both of these are tandemly arranged at multiple chromosomal loci. By using different cloned examples of each family, we have been able to selectively label unique subsets of soybean chromosomes. Double hybridization with biotin and digoxigenin labeled probes has allowed us to determine the chromosomal overlap between different probes. In addition, we have joined portions of the metaphase chromosome painting patterns with the genetic map by single-copy FISH and PCR-PRINS detection of the RFLP loci G8.15, G17.3, and A199a and A199b. Total genomic DNA in situ hybridization (GISH) patterns were also used to characterize the soybean chromosomes.

Theobroma cacao L.: a genetic linkage map and quantitative trait loci analysis

A genetic linkage map of Theobroma cacao (cocoa) has been constructed from 131 backcross trees derived from a cross between a single tree of the variety Catongo and an F1 tree from the cross of Catongo by Pound 12. The map comprises 138 markers: 104 RAPD loci, 32 RFLP loci and two morphologic loci. Ten linkage groups were found which cover 1068 centimorgans (cM). Only six (4%) molecular-marker loci show a significant deviation from the expected 1∶1 segregation ratio.The average distance between two adjacent markers is 8.3 cM. The final genome-size estimates based on two-point linkage data ranged from 1078 to 1112 cM for the cocoa genome. This backcross progeny segregates for two apparently single gene loci controlling (1) anthocyanidin synthesis (Anth) in seeds, leaves and flowers and (2) self-compatibility (Autoc). The Anth locus was found to be 25 cM from Autoc and two molecular markers co-segregate with Anth. The genetic linkage map was used to localize QTLs for early flowering, trunk diameter, jorquette height and ovule number in the BC1 generation using both single-point ANOVA and interval mapping. A minimum number of 2-4 QTLs (P<0.01) involved in the genetic expression of the traits studied was detected. Coincident map locations of a QTL for jorquette height and trunk diameter suggests the possibility of pleiotropic effects in cocoa for these traits. The combined estimated effects of the different mapped QTLs explained between 11.2% and 25.8% of the phenotypic variance observed in the BC1 population.