QTLs associated with chlorimuron ethyl sensitivity in soybean: Effects on seed yield and related traits

 Soybean, Glycine max (L.) Merr., genotypes are known to differ in chlorimuron ethyl sensitivity (CS). Earlier we have reported two putatively independent marker loci linked to two quantitative trait loci (QTLs) controlling CS in a soybean population derived from a cross of PI97100 (sensitive to chlorimuron ethyl) and ‘Coker 237’ (tolerant to chlorimuron ethyl). The objective of the present study was to quantify the association of the two marker loci with seed yield and related traits in this soybean population following application of chlorimuron ethyl. Phenotypic data were collected for 111 F₂-derived lines of the cross grown in replicated plots at Athens, G.A., in 1994 and 1995, and at Blackville, S.C., in 1995. The two CS marker loci explained as much as 50% of the genetic variation in seed yield and seed number m^-2, but had no association with seed weight, plant height, lodging, seed protein, and seed oil. There were no epistatic interactions between the two marker loci for any of the traits. The marker locus (cr168-1 on USDA linkage group E) linked to the major CS QTL explained between 13 and 23% of the variation in seed yield. The Coker 237 allele at this locus was associated with decreased CS and increased seed yield. The marker locus (Blt015-2 on an unknown linkage group) linked to the minor CS QTL accounted for a maximum of 11% of the variation in seed yield. The Coker 237 allele at this locus was associated with an increase in CS and a decrease in seed yield. The association of the two marker loci with seed number m^-2 strongly resembled their association with seed yield. Seed yield had a strong positive correlation (r=0.74 – 0.94) with seed number m^-2, and the effect of chlorimuron ethyl on seed yield was due mainly to its effect on seed number m^-2 rather than seed weight. Received: 6 August 1996 / Accepted: 28 February 1997     

Identification of molecular markers linked to quantitative trait loci for soybean resistance to corn earworm

 One hundred and thirty nine restriction fragment length polymorphisms (RFLPs) were used to construct a soybean (Glycine max L. Merr.) genetic linkage map and to identify quantitative trait loci (QTLs) associated with resistance to corn earworm (Helicoverpa zea Boddie) in a population of 103 F₂-derived lines from a cross of ‘Cobb’ (susceptible) and PI229358 (resistant). The genetic linkage map consisted of 128 markers which converged onto 30 linkage groups covering approximately 1325 cM. There were 11 unlinked markers. The F₂-derived lines and the two parents were grown in the field under a plastic mesh cage near Athens, Ga., in 1995. The plants were artificially infested with corn earworm and evaluated for the amount of defoliation. Using interval-mapping analysis for linked markers and single-factor analysis of variance (ANOVA), markers were tested for an association with resistance. One major and two minor QTLs for resistance were identified in this population. The PI229358 allele contributed insect resistance at all three QTLs. The major QTL is linked to the RFLP marker A584 on linkage group (LG) ‘M’ of the USDA/Iowa State University public soybean genetic map. It accounts for 37% of the total variation for resistance in this cross. The minor QTLs are linked to the RFLP markers R249 (LG ‘H’) and Bng047 (LG ‘D1’). These markers explain 16% and 10% of variation, respectively. The heritability (h²) for resistance was estimated as 64% in this population. Received: 15 October 1997 / Accepted: 4 November 1997     

Seed quality QTLs identified in a molecular map of early maturing soybean

This study identified QTLs influencing seed quality characters in a cross of two early maturing soybean (Glycine max [L.] Merr.) cultivars (Ma.Belle and Proto) adapted to the short growing seasons of Central Europe. A molecular linkage map was constructed by using 113 SSR, 6 RAPD and 1 RFLP markers segregating in 82 individuals of an F₂ population. The map consists of 23 linkage groups and corresponds wellto previously published soybean maps. Using phenotypic data of the F₂-derived lines grown in five environments, four markers for protein content, three for oil content and eight for seed weight were identified. Four from fifteen seed quality QTL-regions identified in the present study were also found by other authors. Markers associated with seed weight QTLs were consistent across all environments and proved to have effects large enough to be useful in a marker-assisted breeding program, whereas protein and oil QTLs showed environmental interactions. Received: 9 October 2000 / Accepted: 26 February 2001     

Relationships between nuclear DNA content and seed and leaf size in soybean

 A correlation between genome size and agronomically important traits has been observed in many plant species. The goal of the present research was to determine the relationship between genome size, seed size, and leaf width and length in soybean [Glycine max (L.) Merr.] Twelve soybean strains, representing three distinct seed size groups, were analyzed. Flow cytometry was used to estimate their 2C nuclear DNA contents. Data on seed size and leaf size of the 12 strains were obtained from 1994 and 1995 field experiments. Variation of 2C nuclear DNA among the 12 soybean strains was 4.6%, ranging from 2.37 pg for a small-seed strain to 2.48 pg for a large-seed strain. Strain seed size was positively associated with leaf width (r=0.92) and leaf length (r=0.93). Genome size was highly correlated with seed size (r=0.97), leaf width (r=0.90) , and leaf length (r=0.93). The results of our study indicate that there is a significant correlation between genome size and leaf and seed size in soybean. It is possible that selection for greater seed size either leads to, or results from, greater genome size. If so, this relationship might be worth exploring at a more fundamental level. Received: 5 April 1997 / Accepted: 9 January 1998     

DNA methylation and AFLP marker distribution in the soybean genome

Amplified fragment length polymorphisms (AFLPs) have become important markers for genetic mapping because of their ability to reliably detect variation at a large number of loci. We report here the dissimilar distribution of two types of AFLP markers generated using restriction enzymes with varying sensitivities to cytosine methylation in the soybean genome. Initially, AFLP markers were placed on a scaffold map of 165 RFLP markers mapped in 42 recombinant inbred (F_6:7) lines. These markers were selected from a map of over 500 RFLPs analyzed in 300 recombinant inbred (F_6:7) lines generated by crossing BSR101×PI437.654. The randomness of AFLP marker map position was tested using a Poisson-model distribution. We found that AFLP markers generated using EcoRI/MseI deviated significantly from a random distribution, with 34% of the markers displaying dense clustering. In contrast to the EcoRI/MseI AFLP markers, PstI/MseI-generated AFLP markers did not cluster and were under represented in the EcoRI/MseI marker clusters. The restriction enzyme PstI is notably sensitive to cytosine methylation, and these results suggest that this sensitivity affected the distribution of the AFLP markers generated using this enzyme in the soybean genome. The common presence of one EcoRI/MseI AFLP cluster per linkage group and the infrequent presence of markers sensitive to methylation in these clusters are consistent with the low recombination frequency and the high level of cytosine methylation observed in the heterochromatic regions surrounding centromeres. Thus, the dense EcoRI/MseI AFLP marker clusters may be revealing structural features of the soybean genome, including the genetic locations of centromeres. Received: 5 November 1998 / Accepted: 20 February 1999     

RFLP tagging of QTLs conditioning specific leaf weight and leaf size in soybean

 Selection for high specific leaf weight (SLW) in soybean [Glycine max (L) Merr.] may increase apparent photosynthetic rate per unit leaf area (AP), which in turn may improve seed yield. In general, the SLW and leaf size are negatively correlated in soybean. To maximize total photosynthetic performance, and perhaps the seed yield, of a soybean cultivar, it would be necessary to establish a large leaf area rapidly while maintaining a high SLW. The objective of the present study was to identify quantitative trait loci (QTLs) conditioning SLW and leaf size in soybean. One hundred and twenty F₄-derived lines from a ‘Young’×PI416937 population were evaluated using restriction fragment length polymorphism (RFLP) markers. The genetic map consisted of 155 loci on 33 linkage groups (LGs) covering 973 cM of map distance. The phenotypic data were collected from two different environments – a greenhouse at Athens, Ga. and a field site at Windblow, N.C. The SLW and leaf-size measurements were made on leaves from the 8th and 9th node of soybean plants at the V12 stage of development. Combined over environments, six putative independent RFLP markers were associated with SLW, and four of these loci were consistent across environments. Individually, the six markers each explained between 8 and 18% of the phenotypic variation among lines for SLW. The Young alleles contributed to a greater SLW at four of the six independent marker loci, and transgressive segregation occurred among the progeny for SLW. Three putative independent RFLP markers were associated with leaf size, each explaining between 6 to 11% of the phenotypic variation in the trait, and one of these markers was identified in both environments. There was no correlation between SLW and leaf size in this population. Similarly, none of the six QTLs conditioning SLW were linked to any of the three QTLs for leaf size. In this soybean population, it is possible to select for progeny lines with greater SLW than either parent perhaps without affecting the leaf size. It is feasible to pyramid all of the desirable alleles for greater SLW and large leaf size in a single genetic background. Received: 16 August 1997 / Accepted: 20 October 1997     

Relationship between single-cross performance and molecular marker heterozygosity

Summary In studies involving isozymes or restriction fragment length polymorphisms (RFLPs), correlations of parental molecular marker diversity with grain yield of maize (Zea mays L.) single-crosses have been too low to be of any predictive value. The relationship of molecular marker heterozygosity (D_ij) with hybrid performance ( _ij) and combining ability was examined. For a simple genetic model involving uncorrelated parental allele frequencies and complete coverage of quantitative trait loci (QTL) by molecular markers, the correlations between _ij and D_ij were 0.25. _ij and D_ij were partitioned into general and specific effects. The expected correlation between specific combining ability and specific molecular marker heterozygosity is high. Expected correlations between general combining ability and general molecular marker heterozygosity are either positive or negative, depending on allele frequencies in the tester lines. Computer simulation was used to investigate a more complex but more realistic genetic model involving incomplete coverage of QTL by molecular markers. All of the following conditions are necessary for effective prediction of hybrid performance based on molecular marker heterozygosity: (1) dominance effects are strong; (2) allele frequencies at individual loci in the parental inbreds are negatively correlated; (3) trait heritability is high; (4) average parental allele frequencies vary only within a narrow range; (5) at least 30–50% of the QTL are linked to molecular markers; and (6) not more than 20–30% of the molecular markers are randomly dispersed or unlinked to QTL.A contribution from Limagrain Genetics, a Groupe Limagrain company     

QTLs conditioning early growth in a soybean population segregating for growth habit

 There are both economic and environmental reasons for reducing the use of herbicides for weed control in soybean [Glycine max (L.) Merr.] fields. Optimizing crop competitiveness can reduce reliance on chemical weed control. Fast and vigorous early growth and rapid canopy development can be effective in suppressing weed infestation of crop plants. The purposes of this study were to identify and molecularly map the quantitative trait loci (QTLs) conditioning soybean plant height and canopy width during the early vegetative stages of soybean growth. A restriction fragment length polymorphism (RFLP) linkage map was created using 142 markers and 116 F₂-derived lines from a cross of ‘S100’בTokyo’. The parents and the 116 F₂-derived lines were evaluated in the greenhouse and in the field at Athens, Ga., in 1996 and 1997. Combined over environments, Tokyo averaged 41 and 17% taller plants than S100 at the V7 and V10 stages of development. Transgressive segregation was observed among the progeny at both stages. Based on single-factor analysis of variance (ANOVA), three and four independent RFLP loci were associated with plant height at the V7 and V10 stages, respectively. All three loci detected [on linkage groups (LGs) C2 and F, and unlinked] at the V7 stage were also detected at the V10 stage along with one additional independent locus on LG E. The Tokyo allele contributed to increased plant height at all loci except at the unlinked locus. Three QTLs (on LGs C2, E, and F) were consistent across environments, three (on LGs C2 and F, and unlinked) were consistent across stages of plant development, and two (on LGs C2 and F) were consistent both across environments and stages of plant development. Within each stage of development, there was no interaction among the independent loci, and the respective loci together explained most of the variation in the traits. Three independent RFLP loci were associated with canopy width at the V10 stage, of which one was unique to the trait, while the remaining loci (on LGs C2 and F) were in common with the independent loci for plant height. Canopy width had a strong correlation (r=0.87) with plant height at the V10 stage. However, mature plant height, lodging, or seed weight had no phenotypic or QTL association with early plant height or canopy width. Received: 10 May 1998 / Accepted: 13 July 1998     

Selection for high seed oil content in soybean families derived from plants regenerated from protoplasts and tissue cultures

Recurrent selection for high seed oil content was carried out with 2,008 progeny of 28 plants regenerated via embryogenesis, 95 via organogenesis and 25 from protoplasts via organogenesis from five different soybean cultivars. Two lines derived from plants regenerated from the cultivar Jack with small increases in seed oil content emerged after three selection cycles in the field but in both cases the protein content was decreased and the seed yield of one of the lines was also decreased. Apparently somaclonal variation for seed oil content can arise, but on the basis of the decreases in protein and yield found in this study, this small change is not useful for soybean improvement. Received: 22 July 2000 / Accepted: 28 July 2000     

Inbreeding and true seed in tetrasomic potato. II. Selfing and sib-mating in heterogeneous hybrid populations of Solanum tuberosum

 Inbreeding depression may affect the performance of consecutive generations of potatoes propagated by true potato seed (TPS). The effect of inbreeding was established using selfed and sib-mated generations of five TPS families. Correlation coefficients were calculated between the level of inbreeding and different traits. Inbreeding depression was expressed mainly by pollen viability, as measured by its stainability (r=−0.912, P<0.01), and tuber yield (r=−0.837, 0.01<P<0.05). The results also indicated that without unavoidable selection inbreeding depression is expected to be more evident. Furthermore, the TPS families responded quite differently to inbreeding depression. They did not show the same amount of depression for yield as they did for the characters concerning fertility. The high tuber-yielding families displayed a greater inbreeding depression for tuber yield than the lower-yielding families. Received: 10 November 1997 / Accepted: 3 March 1998     

Inheritance of reduced linolenic acid content in soybean seed oil

 Linolenic acid is the unstable component of soybean [Glycine max (L.) Merr.] oil that is responsible for the undesirable odors and flavors commonly associated with poor oil quality. Two mutants, M-5 and KL-8, have been identified that have lower linolenic acid levels in the seed oil than the ‘Bay’ cultivar. Our objective was to determine the relationships between the genetic systems controlling linolenic acid in these mutants. Reciprocal crosses were made between the mutants and ‘Bay’, and between the two mutants. No maternal effect for linolenic acid content was observed from the analysis of F₁ seeds in any of the crosses. The data for linolenic acid content in F₂ seeds of M-5בBay’ and KL-8בBay’ crosses satisfactorily fit a 1 : 2 : 1 and 3 : 1 ratio, respectively. For the M-5×KL-8 cross, segregation observed from the analysis of F₂ seeds for linolenic acid content satisfactorily fit a ratio of 3 more than either mutant: 12 within the range of the two mutants: 1 less than either mutant. The segregation ratio of F₂ seeds and the segregation of F₃ seeds from F₂ plants indicated that M-5 and KL-8 have alleles at different loci that control linolenic acid content. The allele in KL-8 has been designated as fanx (KL-8) to distinguish it from fan (M-5). The low linolenic acid segregates with the genotype fanfanfanxfanx provide additional germplasm to reduce the linolenic acid content from the seed oil of soybean. Received: 18 December 1995 / Accepted: 12 July 1996     

Susceptibility to Agrobacterium tumefaciens and cotyledonary node transformation in short-season soybean

 Short-season adapted soybean [Glycine max (L.) Merrill] genotypes (maturity group 0 and 00) were susceptible to Agrobacterium tumefaciens in tumor-formation assays with A. tumefaciens strains A281, C58 and ACH5. The response was bacterial-strain and plant-cultivar dependent. In vitro Agrobacterium-mediated transformation of cotyledonary node explants of these genotypes with A. tumefaciens EHA105/pBI121 was inefficient but resulted in a transgenic AC Colibri plant carrying a linked insertion of the neomycin phosphotransferase and β-glucuronidase (gus) transgenes. The transgenes were transmitted to the progeny and stable gus expression was detected in the T₇ generation. The low rate of recovery of transgenic plants from the co-cultured cotyledonary explants was attributed to inefficient transformation of regenerable cells, and/or poor selection or survival of such cells and not to poor susceptibility to Agrobacterium, since, depending on the cultivar, explants were transformed at a rate of 27–92%, but transformation events were usually restricted to non-regenerable callus. Received: 8 January 1998 / Revision received: 30 June 1999 / Accepted: 12 July 1999     

Two Erysiphe species associated with recent outbreak of soybean powdery mildew: results of molecular phylogenetic analysis based on nuclear rDNA sequences

 Serious outbreaks of powdery mildew by a fungus belonging to the mitosporic genus Oidium subgenus Pseudoidium have been reported on soybean (Glycine max) in a wide area of eastern Asia since 1998. The taxonomic and phylogenetic placement of the causal fungus has not yet been determined because of lack of the perfect stage. We found ascomata having mycelioid appendages on a single leaf of soybean infested by powdery mildew. Molecular phylogenetic analysis was conducted based on a total of 14 sequences of the rDNA internal transcribed spacer (ITS) region from 13 soybean and wild soybean (Glycine soja) materials collected in Japan, Korea, Vietnam, and the United States, combined with 47 sequence data obtained from the DNA databases. It was revealed that two Erysiphe species were associated with the outbreak of soybean powdery mildew. There was 16% difference between the two species in genetic divergence of the ITS sequence. One species with perfect stage has an ITS sequence identical to that of Erysiphe glycines on Amphicarpaea and is identified as Erysiphe glycines based on the ITS sequence and morphology of ascomata. The second species, without the perfect stage, is likely to be Erysiphe diffusa (= Microsphaera diffusa), known as the fungus causing soybean powdery mildew in the United States, because the ITS sequences are identical to those from materials collected in the United States. However, we need materials having ascomata of E. diffusa to confirm the species name. Received: March 15, 2002 / Accepted: May 22, 2002     

Allele-specific hybridization markers for soybean

 Soybean (Glycine max) is one of the world’s most important crop plants due to extensive genetic improvements using traditional breeding approaches. Recently, marker-assisted selection has enhanced the ability of traditional breeding programs to improve soybeans. Most methods of assessing molecular markers involve electrophoretic techniques that constrain the ability to perform high-throughput analyses on breeding populations and germplasm. In order to develop a high-capacity system, we have developed allele-specific hybridization (ASH) markers for soybean. As one example, restriction fragment length polymorphism (RFLP) locus A519-1 (linkage group B) was converted into an ASH marker by (1) sequencing the pA519 cloned insert, (2) designing locus-specific PCR amplification primers, (3) comparative sequencing of A519-1 amplicons from important soybean ancestors, and (4) designing allele-specific oligonucleotide probes around single nucleotide polymorphisms (SNPs) among soybean genotypes. Two SNPs were identified within approximately 400 bp of the sequence. Allele-specific probes generated a 100-fold greater signal to target amplicons than to targets that differed by only a single nucleotide. The A519-1 ASH marker is shown to cosegregate with the A519-1 RFLP locus. In order to determine ASH usefulness, we genotyped 570 soybean lines from the Pioneer Hi-Bred soybean improvement using both A519-1 SNPs. Combined haplotype diversity (D) was 0.43 in this adapted germplasm set. These results demonstrate that ASH markers can allow for high-throughput screening of germplasm and breeding populations, greatly enhancing breeders’ capabilities to do marker-assisted selection. Received: 10 August 1998 / Accepted: 17 September 1998     

Effects of UV-B radiation on soybean yield and seed quality: a 6-year field study

Two soybean [ Glycine max (L.) Merr.] cultivars, Essex and Williams, were grown in the field for 6 consecutive seasons under ambient and supplemental levels of ultravio-Set-B radiation to determine the potential for alterations in yield or seed quality with a reduction in the stratospheric ozone column. The supplemental UV-B fluences simulated a 16 or 25% ozone depletion. The data presented here represent the first field experiment conducted over multiple seasons which assesses the effects of increased UV-B radiation on seed yield. Overall, the cultivar Essex was found to be sensitive to UV-B radiation (yield reductions of 20%) while the cultivar Williams was tolerant. However, the effectiveness of UV-B radiation in altering yield was strongly influenced by the seasonal microclimate, and the 2 cultivars responded differently to these changing factors. Yield was reduced most in Essex during seasons in which water availability was high and was reduced in Williams only when water was severely limiting. The results of these experiments demonstrate the necessity for multiple-year experiments and the need to increase our understanding of the interaction between UV-B radiation and other environmental stresses in order to assess the potential consequences of stratospheric ozone depletion.     

Genetic mapping of soybean cyst nematode race-3 resistance loci in the soybean PI 437.654

Resistance to the soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is difficult to evaluate in soybean [Glycine max (L.) Merr.] breeding. PI 437.654 has resistance to more SCN race isolates than any other known soybean. We screened 298 F₆₇ recombinant-inbred lines from a cross between PI 437.654 and BSR101 for SCN race-3 resistance, genetically mapped 355 RFLP markers and the I locus, and tested these markers for association with resistance loci. The Rhg ₄ resistance locus was within 1 cM of the I locus on linkage group A. Two additional QTLs associated with SCN resistance were located within 3cM of markers on groups G and M. These two loci were not independent because 91 of 96 lines that had a resistant-parent marker type on group G also had a resistant-parent marker type on group M. Rhg ₄ and the QTL on G showed a significant interaction by together providing complete resistance to SCN race-3. Individually, the QTL on G had greater effect on resistance than did Rhg ₄, but neither locus alone provided a degree of resistance much different from the susceptible parent. The nearest markers to the mapped QTLs on groups A and G had allele frequencies from the resistant parent indicating 52 resistant lines in this population, a number not significantly different from the 55 resistant lines found. Therefore, no QTLs from PI 437.654 other than those mapped here are expected to be required for resistance to SCN race-3. All 50 lines that had the PI 437.654 marker type at the nearest marker to each of the QTLs on groups A and G were resistant to SCN race-3. We believe markers near to these QTLs can be used effectively to select for SCN race-3 resistance, thereby improving the ability to breed SCN-resistant soybean varieties.     

Composite and clinal distribution of Glycine soja in Japan revealed by RFLP analysis of mitochondrial DNA

 Wild soybean (Glycine soja Sieb. et Zucc.), regarded as the progenitor of cultivated soybean [G. max (L.) Merr.], is widely distributed in East Asia. We have collected 1097 G. soja plants from all over Japan and analyzed restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) in them. Based on the RFLPs detected by gel-blot analysis, using coxII and atp6 as probes, the collected plants were divided into 18 groups. Five mtDNA types accounted for 94% of the plants examined. The geographic distribution of mtDNA types revealed that, in many regions, wild soybeans grown in Japan consisted of a mixture of plants with different types of mtDNA, occasionally even within sites. Some of the mtDNA types showed marked geographic clines among the regions. Additionally, some wild soybeans possessed mtDNA types that were identical to those widely detected in cultivated soybeans. Our results suggest that the analysis of mtDNA could resolve the maternal lineage among plants of the genus Glycine subgenus Soja. Received: 16 June 1997/Accepted: 5 August 1997     

Relationship between heterosis and heterozygosity at marker loci: a theoretical computation

Summary In this paper we have studied the linear correlation between a genetic distance index between two parent lines (based on marker loci information) and the heterosis observed in the F₁ hybrid from the two lines, for a quantitative character (determined by several loci, or QTL). Theoretical computations of the correlation coefficient () between the distance index and the heterosis were made, assuming the biallelic model (defined by Fisher). When the alleles at both marker loci and QTL are equally distributed among the whole population of considered lines, the coefficient is a function of the squares of linkage disequilibria between alleles at marker loci and alleles at QTL. The QTL that are not marked by marker loci and marker loci that do not mark any QTL play symmetrical roles and can decrease greatly. We conclude that the prediction of F₁ hybrid heterosis based on marker loci would be more efficient if these markers were selected for their relationship to the alleles implicated in the heterotic traits considered.     

Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein

 Embryogenic soybean [Glycine max (L.) Merrill.] suspension cultures were bombarded with five different gene constructions encoding the jellyfish (Aequorea victoria) green fluorescent protein (GFP). These constructions had altered codon usage compared to the native GFP gene and mutations that increased the solubility of the protein and/or altered the native chromophore. All of the constructions produced green fluorescence in soybean cultures upon blue light excitation, although a soluble modified red-shifted GFP (smRS-GFP) was the easiest to detect based on the brightness and number of foci produced. Expression of smRS-GFP was visible as early as 1.5 h after bombardment, with peak expression at approximately 6.5 h. Large numbers of smRS-GFP-expressing areas were visible for 48 h postbombardment and declined rapidly thereafter. Stably transformed cultures and plants exhibited variation in the intensity and location of GFP expression. PCR and Southern hybridization analyses confirmed the presence of introduced GFP genes in stably transformed cultures. Received: 23 September 1998 / Revision received: 4 January 1999 / Accepted: 15 January 1999     

Reproducibility of the differential amplification between leaf and root DNAs in soybean revealed by RAPD markers

 Random amplified polymorphic DNA (RAPD) was used to determine whether such markers can be employed for detecting genomic modification during plant development or under certain stress environments. Pairwise comparisons in RAPD patterns of leaf and root DNA amplifications were studied for 11 soybean accessions representing different origins. Hydroponic culture was used for the ease of harvesting roots. From a total of 40 primers screened, it was found that 16 can detect leaf DNA polymorphism, 19 for root DNA polymorphism, while 10 show a greater consistency for detecting polymorphism between leaf and root (L/R) DNAs. Nevertheless, problems were encountered when the newly synthesized oligo-primers and different thermal cyclers were used to check the data. Several factors were then tested for their reproducibility. The results indicated that the amplified differences between root and leaf DNAs are mostly not affected by template DNA concentrations. The addition of DMSO (dimethyl sulphoxide) or TMAC (tetramethyl-ammonium chloride) also did not mask the L/R differences. However, DNA polymerase and oligo-primers synthesized from different manufacturers, as well as the thermal cyclers, reacted differently sometimes. Regardless of the general problems of reproducibility in RAPD patterns, some amplified differences remain between the L/R DNAs. The most distinct patterns involve differences in the relative intensity of amplified bands. Differential amplification might have occurred during plant leaf and root development. Southern hybridization of the eluted polymorphic bands against restriction digestion of total genomic DNA confirms their being homologous to soybean DNA fragments. Polymorphism of these specific L/R differences also exists among varieties. RAPD should be a useful tool in detecting genomic alterations during plant development or under certain stress environments, as long as the factors affecting the reproducibility of RAPD patterns can be properly controlled. An additional cycle of selection would be possible if such a type of polymorphism is proved to be correlated with certain developmental characters. Received: 7 October 1996 / Accepted: 20 May 1997