FRET hybridization probes for the rapid detection of disease resistance alleles in plants: detection of corky root resistance in lettuce

We describe the development of a non-electrophoresis PCR-based assay for allele discrimination at a disease resistance locus. The assay is based on the emission of light by fluorescence resonance energy transfer (FRET) upon annealing of two hybridization probes. The analysis of melting curve profiles of the probes and templates allowed the detection of single nucleotide polymorphisms. The assay was applied to the detection of alleles at the cor locus in lettuce (Lactuca sativa) that confers recessive resistance to corky root disease. Probes and primers for the assay were designed after the characterization of a single nucleotide polymorphism between alleles of PCR products amplified using a linked marker. That polymorphism was validated in a collection of lettuce varieties representing different genetic backgrounds. The FRET hybridization probes approach provided fast and accurate genotyping of breeding material directly in a one-tube reaction. The absence of electrophoresis makes this approach suitable for applications that require automation and high-throughput genotyping analyses such as marker-assisted selection programs.     

Two simple sequence repeat markers to select for soybean cyst nematode resistance coditioned by the rhg1 locus

The soybean cyst nematode (SCN) (Heterodera glycines Inchinoe) is the most economically significant soybean pest. The principal strategy to reduce or eliminate damage from this pest is the use of resistant cultivars. Identifying resistant segregants in a breeding program is a difficult and expensive process which is complicated by the oligogenic nature of the resistance and genetic variability in the pathogen. Fortunately, resistance at one SCN-resistance locus, rhg1, is generally accepted as a necessity for the development of resistant genotypes using any source of resistance and when challenged by any SCN race. Thus, the development of SCN resistant cultivars would be expedited if an effective and rapid system were available to identify breeding lines carrying a resistance allele at the rhg1 locus. In this study we report two simple sequence repeat (SSR) or microsatellite loci that cosegregate and map 0.4 cM from rhg1. Allelic variation at the first of these loci, BARC-Satt309, distinguished most, if not all, SCN-susceptible genotypes from those carrying resistance at rhg1 derived from the important SCN-resistance sources ’Peking’, PI 437654, and PI 90763. BARC-Satt309 was also effective in distinguishing SCN resistance sources PI 88788 and PI 209332 from many, but not all, susceptible genotypes. BARC-Satt309 cannot be used in marker-assisted selection in populations developed from typical southern US cultivars crossed with the important resistance sources PI 88788 or PI 209332 because these genotypes all carry the identical allele at the BARC-Satt309 locus. A second SSR locus, BARC-Sat_168, was developed from a bacterial artificial chromosome (BAC) clone that was identified using the primers to BARC-Satt309. BARC-Sat_168 distinguished PI 88788 and PI 209332 from southern US cultivars such as ’Lee’, ’Bragg’ and ’Essex’. Both BARC-Satt309 and BARC-Sat_168 were used to assay lines from SCN-susceptible×SCN-resistant crosses and proved to be highly effective in identifying lines carrying rhg1 resistance from those carrying the allele for SCN susceptibility at the rhg1 locus. Received: 5 November 1998 / Accepted: 3 February 1999     

Identification of a codominant amplified polymorphic DNA marker linked to the verticillium wilt resistance gene in tomato

Resistance to verticillium wilt, a vascular disease causing yield losses in many crops, is conferred in tomato by a single dominant allele, Ve. A population segregating for the Ve allele was generated using near-isogenic tomato lines. Analysis of the parental tomato DNA using the polymerase chain reaction and 400 random primers, each 10 deoxyribonucleotides in length, produced 1,880 amplified DNA fragments. Of the four polymorphisms observed between the resistant and susceptible parental genotypes, only one was linked to the Ve gene. No recombination was observed between this DNA marker and the Ve locus, indicating that the linkage is less than 3.5±2.7 cM. The marker detected both the susceptible and resistant alleles, producing amplified DNA fragments of approximately 1,300 and 1,350 bp, respectively. The sequence of the primer, determined from cloned amplified products, was 5 CTCACATGCA 3 instead of the expected 5 CTCACATGCC 3. The marker will be of value to tomato breeding programs because of the tight linkage, Codominant nature, and analytical procedure utilized.     

Development of a Simple PCR-based Marker for the Determination of Growth Habit in Vicia faba L. using a Candidate Gene Approach

We have developed the first molecular marker suitable for the selection of determinate growth habit in faba bean using the candidate gene approach. We obtained the sequences of TFL1/CEN like genes from public databases and designed primers on conserved domains. We used three cultivars with determinate growth habit and four accessions (two cultivars and two lines) with indeterminate growth habit. All these genotypes are used in our faba bean breeding program. A single monomorphic PCR fragment was obtained. A set of restriction enzymes was assayed. The enzyme Hind1II produced a clear polymorphism between determinate and indeterminate genotypes. This new cleaved amplified polymorphism (CAP) marker was tested using an F₂ population contrasting for growth habit derived from the cross ‘Verde Bonita’ × 2N52. This marker showed 100% efficiency in discriminating both types of genotypes. Moreover, the codominancy of this marker allows the detection of heterozygous individuals facilitating the breeding process when pyramiding different genes. The perfect cosegregation of the marker with the trait indicates that an orthologue of TFL1/CEN controls the growth habit in faba bean. This marker has been tested in all the genotypes used in our faba bean breeding program as donors of the determinate growth habit. Therefore, it is expected to work well in all the crosses performed with these parental lines as happens in the F₂ tested. The CAPS marker developed in this work will be useful for Marker Assisted Selection programs. In addition, this marker is useful for quality control to determinate the percentage of outsider seeds in commercial seed lots. Moreover, it is a valuable tool to breeders when submitting new faba bean varieties for registration since the method allows guaranteeing that outsider plants remains under the requested limit for registration.     

Divergence and allelomorphic relationship of a soybean virus resistance gene based on tightly linked DNA microsatellite and RFLP markers

The use of genetically diverse resistance sources is important in breeding for durable disease resistance. Detection and evaluation of resistance genes by conventional inheritance experiments, however, often require laborious screening and genetic testing. In the present study, a marker-assisted screening for resistance sources was initiated in soybean [Glycine max (L.) Merr] using one DNA microsatellite and two RFLP markers tightly linked to a soybean mosaic virus (SMV) resistance gene (Rsv1). The three marker loci were used to screen 67 diverse soybean cultivars, breeding lines, and plant introductions. Five variants were found at the microsatellite locus (HSP176L), and the two RFLP loci (pA186 and pK644a) near Rsv1 show a remarkably higher level of restriction polymorphism than Rsv1-independent RFLP loci. Several specific variants at the three marker loci were found to be correlated with virus resistance, among which HSP176L-2 can be detected by PCR, thus may be useful for germplasm screening. The grouping of the 67 accessions according to their multilocus marker variants agrees with the available pedigree information. When all, or most, of the cultivars within a given group with the same Rsv1-linked marker variant are resistant, their SMV resistance is most likely conferred by Rsv1. These putatively Rsv1-carrying groups contain a total of 38 SMV-resistant lines including six differential cultivars that are known to carry Rsv1. The remaining seven resistant accessions (Columbia, Holladay, Peking, Virginia, FFR-471, PI 507403, and PI 556949) do not carry resistance marker variants, and at least some of them could be sources of resistance genes independent of Rsv1.     

Allele-specific amplification of polymorphic sites for the detection of powdery mildew resistance loci in cereals

Primers for the polymerase chain reaction (PCR) were tailored to selectively amplify RFLP marker alleles associated with resistance and susceptibility for powdery mildew in cereals. The differentiation between marker alleles for susceptible and resistant genotypes is based on the discrimination of a single nucleotide by using allele-specific oligonucleotides as PCR primers. The PCR assays developed are diagnostic for RFLP alleles at the loci MWG097 in the barley genome and Whs350 in the wheat genome. The first marker locus is closely linked to MlLa resistance in barley, while the latter is linked to Pm2 resistance locus in wheat. PCR analysis of 31 barley and 30 wheat cultivars, with some exceptions, verified the presence or absence of the resistance loci investigated. These rapid PCR-based approaches are proposed as an efficient alternative to conventional procedures for selecting powdery mildew-resistant genotypes in breeding programs.     

Development of a simple functional marker for fragrance in rice and its validation in Indian Basmati and non-Basmati fragrant rice varieties

Fragrance development in rice has been reported due to a 8-bp deletion in the exon 7 of badh2 gene located on Chromosome 8S. Multiplex markers targeting the functional InDel polymorphism was earlier reported for genotyping fragrance trait, but the marker was observed to be inconsistent and difficult to use. We have developed a simple, co-dominant, functional marker for fragrance trait, which can be resolved in an agarose gel and validated in Basmati and non-Basmati aromatic rice varieties and in a mapping population segregated for fragrance trait. The marker targets the InDel polymorphism in badh2 gene and amplifies 95 and 103 bp fragments in fragrant and non-fragrant genotypes, respectively. The newly developed marker was highly efficient in discriminating all fragrant and non-fragrant genotypes and showed perfect co-segregation with the trait of fragrance in the mapping population. We recommend the use of this simple, low-cost marker in routine genotyping for fragrance trait in large scale breeding materials and germplasm.     

Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes

Experiments were conducted to produce transgenic barley plants following infection of immature embryos with Agrobacterium tumefaciens. Transformed callus was obtained using hygromycin resistance as a selectable marker and either green fluorescent protein (GFP) or -glucuronidase (GUS) as a reporter. Significantly reduced plant transformation frequencies were obtained with the GFP gene compared to GUS. However, GFP proved to be an excellent reporter of early transformation events and was used to compare four barley cultivars for efficiency in two phases of transformation: the generation of stably transformed barley callus and the regeneration of plantlets from transformed callus. Transformed callus was generated at a high frequency (47–76%) in all four cultivars. Regeneration of transformed plantlets was also achieved for all four cultivars although the frequency was much higher for Golden Promise than for the other three genotypes, reiterating that genotype is an important determinant in the regenerative ability of barley. This study has demonstrated for the first time that Agrobacterium-mediated transformation can be used to transform the Australian cultivars Sloop and Chebec.Communicated by W. Harwood     

Mapping and diagnostic marker development for Soil-borne cereal mosaic virus resistance in bread wheat

Monogenically-inherited resistance to Soil-borne cereal mosaic virus (SBCMV) in hexaploid bread wheat cultivars ‘Tremie’ and ‘Claire’ was mapped on chromosome 5D. The two closest flanking markers identified in the Claire-derived mapping population, Xgwm469-5D and E37M49, are linked to the resistance locus at distances of 1 and 9 cm, respectively. Xgwm469-5D co-segregated with the SBCMV resistance in the Tremie-derived population and with the recently identified Sbm1 locus in the cv. Cadenza. This suggested that Tremie and Claire carry a resistance gene allelic to Sbm1, or one closely linked to it. The diagnostic value of Xgwm469-5D was assessed using a collection of SBCMV resistant and susceptible cultivars. Importantly, all susceptible genotypes carried a null allele of Xgwm469-5D, whereas resistant genotypes presumably related to either Claire and Tremie or Cadenza revealed a 152 or 154 bp allele of Xgwm469-5D, respectively. Therefore, Xgwm469-5D is well suited for marker assisted selection for SBCMV resistance.     

Combination of newly developed SNP and InDel markers for genotyping the <Emphasis Type="Italic">Cf-9</Emphasis> locus conferring disease resistance to leaf mold disease in the tomato

The Cf-9 gene in the tomato is known to confer resistance against leaf mold disease caused by Cladosporium fulvum, and a gene-based marker targeted to the Cf-9 allele has been widely used as a crop protection approach. However, we found this marker to be misleading in genotyping. Therefore, we developed new single-nucleotide polymorphism (SNP) and insertion and deletion (InDel) markers targeted to the Cf-9 allele in order to increase genotyping accuracy and facilitate high-throughput screening. The DNA sequences of reported Cf-9, cf-9, Cf-0, and closely related Cf-4 alleles were compared, and two functional and non-synonymous SNPs were found to distinguish the Cf-9 resistance allele from the cf-9, Cf-0, and Cf-4 alleles. An SNP marker including these two SNPs was developed and applied to the genotyping of 33 tomato cultivars by high-resolution melting analysis. Our SNP marker was able to select all three Cf-9 genotypes (resistant, heterozygous, and susceptible alleles). Interestingly, two cultivars were grouped separately from these three genotypes. To further examine this outgroup, we preformed polymerase chain reaction (PCR) on two InDel regions identified by sequence comparison of the Cf-9 and Cf-4 genes. The band patterns revealed that these two cultivars carried Cf-4 rather than Cf-9 alleles and that three cultivars classified in the Cf-9 resistance group actually carried both Cf-9 and Cf-4 genes. To determine whether these genotyping results were consistent with disease resistance phenotypes, we examined the induction of a hypersensitive response by transiently expressing the corresponding effector genes, and found that the results matched perfectly with the genotyping results. These findings indicate that the combination of our SNP and InDel markers allows resistant Cf-9 alleles to be distinguished from cf-9 and Cf-4 alleles, which will be useful for marker-assisted selection of tomato cultivars resistant to C. fulvum.     

Introgression of diverse genes for resistance to rusts into an improved wheat variety, Kalyansona

Breeding for resistance to the three rusts of wheat usually requires incorporation of genetically independent factors conferring resistance to each rust. Linked resistance genes in some alien translocation stocks permit concurrent transference of resistance for more than one rust. Alien derived resistances, however, are often reported to be associated with reduced yield and other undesirable characters. In our experience, backcross breeding when given a limited number of backcrosses (3–6) and with suitable selection procedures has resulted in lines giving yields higher or comparable to the recurrent wheat parent Kalyansona and resistance to one, two or all three rusts without any adverse effects. Some of the rust resistant derivatives also show resistance to Neovossia indica (Karnal bunt). The derivatives thus developed when used as parents in a breeding programme have produced several improved cultivars with high yields, superior grains and diversity for resistance to rust pathogens. One of the cultivars, named Vaishali (DL784-3), has been officially released for cultivation in the country.     

Rust mite resistance in apple assessed by quantitative trait loci analysis

The aim of this study was to assess the genetic basis of rust mite (Aculus schlechtendali) resistance in apple (Malus × domestica). A. schlechtendali infestation of apple trees has increased as a consequence of reduced side effects of modern fungicides on rust mites. An analysis of quantitative trait loci (QTLs) was carried out using linkage map data available for F₁ progeny plants of the cultivars ‘Fiesta’ × ‘Discovery’. Apple trees representing 160 different genotypes were surveyed for rust mite infestation, each at three different sites in two consecutive years. The distribution of rust mites on the individual apple genotypes was aggregated and significantly affected by apple genotype and site. We identified two QTLs for A. schlechtendali resistance on linkage group 7 of ‘Fiesta’. The AFLP marker E35M42-0146 (20.2 cM) and the RAPD marker AE10-400 (45.8 cM) were closest positioned to the QTLs and explained between 11.0% and 16.6% of the phenotypic variability. Additionally, putative QTLs on the ‘Discovery’ chromosomes 4, 5 and 8 were detected. The SSR marker Hi03a10 identified to be associated to one of the QTLs (AFLP marker E35M42-0146) was traced back in the ‘Fiesta’ pedigree to the apple cultivar ‘Wagener’. This marker may facilitate the breeding of resistant apple cultivars by marker assisted selection. Furthermore, the genetic background of rust mite resistance in existing cultivars can be evaluated by testing them for the identified SSR marker. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular identification of powdery mildew resistance genes in common wheat (Triticum aestivum L.)

RFLP markers for the wheat powdery mildew resistance genes Pm1 and Pm2 were tagged by means of near-isogenic lines. The probe Whs178 is located 3 cM from the Pm1 gene. For the powdery mildew resistance gene Pm2, two markers were identified. The linkage between the Pm2 resistance locus and one of these two probes was estimated to be 3 cM with a F₂ population. Both markers can be used to detect the presence of the corresponding resistance gene in commercial cultivars. Bulked segregant analysis was applied to identify linkage disequillibrium between the resistance gene Pm18 and the abovementioned marker, which was linked to this locus at a distance of 4 cM. Furthermore, the RAPD marker OPH-11₁₉₀₀ (5-CTTCCGCAGT-3) was selected with pools created from a population segregating for the resistance of Trigo BR 34. The RAPD marker was mapped about 13 cM from this resistance locus.     

A fluorescent PCR assay for the codominant interpretation of a dominant SCAR marker linked to the virus resistance allele bc-1 2 in common bean

Our objective was to develop a rapid and accurate procedure to genotype common bean plants for the bc-1 ² allele, which conditions resistance to bean common mosaic and bean common mosaic necrosis viruses. A segregating F₂ population was derived from the cross between pinto bean breeding lines P94207-43 (bc-1 ²//bc-1 ²) and P94207-189 (bc-1//bc-1). A quantitative PCR assay based on the detection of fluorescent labeled amplicons was developed to distinguish between homozygous (bc-1 ²//bc-1 ²), heterozygous (bc-1 ²//bc-1) and null (bc-1//bc-1) F₂ genotypes. Remnant F₁ plants were used as a comparative reference sample. PCR results among this sample fit a normal distribution, and 99% and 95% confidence intervals for heterozygotes were determined. F₂ plants for which no amplification was detected were classified as null (bc-1//bc-1) genotypes. F₂ plants that fell within the confidence intervals for heterozygotes were classified as heterozygotes (bc-1 ²//bc-1), while plants that fell outside the right tail of the heterozygote confidence intervals were classified as homozygotes (bc-1 ²//bc-1 ²). F₂ plants were also genotyped for the bc-1 ² allele by performing F₃ family progeny tests for virus resistance. Agreement between the two methods for genotyping plants was 100% (59/59) when PCR genotyping was based on a 99% heterozygote confidence interval, and 98.3% (58/59) when based on a 95% heterozygote confidence interval. This assay will accelerate breeding for virus resistance in bean by facilitating discrimination among plants that are heterozygous or homozygous for the bc-1 ² allele. The experimental design may be generally applicable towards developing other assays for the codominant interpretation of dominant markers in diploid plants.     

RFLP patterns of gliadin alleles in Triticum aestivum L.: implications for analysis of the organization and evolution of complex loci

A correspondence between RFLP patterns and gliadin alleles at the Gli-1 and Gli-2 loci was established in a set of 70 common wheat (T.aestivum L.) cultivars using -gliadin (K32) and -gliadin (pTU1) specific probes. All Gli-B1 and Gli-D1 alleles which differed in encoded -gliadins showed definite RFLP patterns after hybridization with the K32 probe. Two groups of Gli-B1 alleles, Gli-B1b-like and Gli-B1e-like, were identified, and these could originate from distinct genotypes of the presumptive donor of the B-genome. Intralocus recombination and/or gene conversion as well as small deletions, gene silencing and gene amplification were assumed to be responsible for the origin of new gliadin alleles. Silent -gliadin sequences were shown to exist in all of the genotypes studied. K32 also differentiated Gli-A1a from all other Gli-A1 alleles as well as the Gli-B11 allele in cultivars carrying the 1B/1R (wheat/rye) translocation. PTU1 was shown to recognize several Gli-A2 alleles, but not the Gli-B2 or Gli-D2 alleles. Moreover, this probe hybridized to chromosome 1R sequences suggesting the existence of rye gene(s), probably silent, for -gliadin-like proteins on chromosome 1R.     

A vapor pressure deficit effect on crop canopy photosynthesis

Canopy CO₂-exchange rates (CER), air temperatures, and dew points were measured throughout ten days during the 1987 growing season for cotton (Gossypium hirsutum L.), grain sorghum [Sorghum bicolor (L) Moench], and five soybean [Glycine max (L) Merr.] cultivars, and throughout seven days in 1988, on maize (Zea maize L.). The objective was to determine if the decline in CER per unit light during the afternoon is associated with a vapor pressure deficit (VPD) increase. Some of the soybean and maize plots were kept as dry as possible. A VPD term significantly contributed (P0.05) to a canopy CER regression model in 54 of 80 data sets in 1987. Grain sorghum was less sensitive than the well-watered soybean genotypes to an increasing VPD (P0.05) on three of the ten measurement days and less sensitive than cotton (P0.05) on only one day. Cotton demonstrated less VPD sensitivity than soybean (P0.05) on one day. The moisture stressed soybean plots showed a greater CER sensitivity to VPD (P0.05) than the well-watered soybean plots. In 1988, the frequently irrigated maize plots were less sensitive to VPD (P0.05) than the rain-fed plots early in the season, before the rain-fed plots were excessively damaged by moisture stress. These results indicate that the afternoon declines in canopy CER found in a number of different species are associated with increases in the VPD; recent work of others suggests that this may be due to partial stomatal closure.Abbreviations CER carbon dioxide exchange rate - VPD vapor pressure deficit - PPFD photosynthetic photon flux density - DAP days after planning     

Measurements of genetic variability for symbiotic dinitrogen fixation in fieldgrwon fababean (Vicia faba L.) using a low level15N-tracer technique

Before starting a breeding program aimed at improving the nitrogen nutrition ofVicia faba, the authors tried an alternative technique to the acetylene reduction assay, to measure some genetic variability in the plant material. The quantity of dinitrogen fixed by several cultivars ofVicia faba was estimated using a low enrichment¹⁵N tracer method and high precision¹⁵N mass spectrometry. The fababeans were cultivated for two years in two different soils. The percentage of fixed dinitrogen in the seed varied between genotypes from 40 to 83% of the total nitrogen and was positively correlated with the total seed nitrogen (r=0.64 to 0.86). A highly significant positive correlation was also found between the total seed nitrogen and the quantity of fixed dinitrogen in the seed (r=0.95 to 0.99). The technique used to measure dinitrogen fixation proved to be useful and reliable enough to discriminate between various genotypes, grown over a period of two years in two different soils. However, several non-fixing control plants showed significant differences in their¹⁵N enrichment and the problem of choosing a good reference plant was raised and discussed.     

Eyespot resistance gene Pch-1 from Aegilops ventricosa is associated with a different chromosome in wheat line H-93-70 than the resistance factor in “Roazon” wheat

Summary The hexaploid wheat line H-93-70 carries a gene (Pch-1) that has been transferred from the wild grass Aegilops ventricosa and confers a high degree of resistance to eyespot diesease, caused by the fungus Pseudocercosporella herpotrichoides. Crosses of the resistant line H-93-70 with the susceptible wheat Pané 247 and with a 7D/7Ag wheat/Agropyron substitution line were carried out and F2 kernels were obtained. The kernels were cut transversally and the halves carrying the embryos were used for the resistance test, while the distal halves were used for genetic typing. Biochemical markers were used to discriminate whether the transferred Pch-1 gene was located in chromosome 7D, as is the case for a resistance factor present in Roazon wheat. In the crosses involving Pané 247, resistance was not associated with the 7D locus Pln, which determines sterol ester pattern (dominant allele in H-93-70). In the crosses with the 7D/7Ag substitution line, resistance was neither associated with protein NGE-11 (7D marker), nor alternatively inherited with respect to protein C-7 (7Ag marker). It is concluded that gene Pch-1 represents a different locus and is not an allele of the resistance factor in Roazon wheat.     

The use of bulk segregant analysis to identify a RAPD marker linked to leaf rust resistance in barley

An F₂ population from a cross between barley accession Q21861 and the Australian barley variety Galleon was used to develop RAPD markers for resistance to barley leaf rust (Puccinia hordei). Resistant and susceptible DNA bulks were constructed following the classification of F₂ plants by leaf rust infection type. Bulked segregant analysis was then used to identify a 2.7-kb marker, designated OU02₂₇₀₀ and located approximately 12cM from RphQ, a leaf rust resistance gene in Q21861. The marker was generated by PCR with the oligonucleotide primer OPU-02 (Operon). Infection types of F₃ progeny were used to confirm assignment of F₂ genotypes. OU02₂₇₀₀ was shown, retrospectively, to be useful in the identification of individual F₂ plants that had been originally misclassified as having susceptible infection types. Both the RAPD marker and RphQ will be potentially useful in the development of new barley cultivars.     

Differential responses of soybean genotypes to excess manganese in an acid soil

To determine the tolerance of soybean genotypes to Mn toxicity, a green house study was conducted. Hayesville sandy loam (clayey, oxidic, mesic, Typic Hapludult), high in manganese, was used for the experiment. The experimental design was split-plot with three replications. Forty-one different soybean genotypes were planted in pots at two different pH levels: 5.2 (original soil pH) and 6.4 (amended with lime). Soybean genotypes were allowed to grow to the dry pod stage.Soil pH levels affected the soybean genotypes yields significantly (p < 0.01). Tolerant genotypes showed a higher or similar seed yield at pH 5.2 compared to pH 6.4. Sensitive genotype yields were lower at pH 5.2 than at pH 6.4. In general, Mn in leaves was higher at pH 5.2 than at pH 6.4. Some of the sensitive genotypes at pH 5.2. showed severe chlorosis and crinkle leaf symptoms as a result of Mn toxicity. Excess available Mn at pH 5.2. induced Ca deficiency. Soybean genotypes PI423758, PI417440, Aoda, Kingston, Rokusum and some others were tolerant to Mn toxicity, whereas PI417288, Verde, Wilson 5, Sango, Funk Delicious and some others were sensitive to Mn toxicity. The genotypes found to be tolerant can be recommended to plant breeders for development of Mn-tolerant cultivars.