Mapping quantitative trait loci associated with chlorophyll a fluorescence parameters in soybean (Glycine max (L.) Merr.)

Chlorophyll a fluorescence parameters can provide qualitative and quantitative information about photosynthetic processes in chloroplasts. JIP-test and modulated fluorescence (MF) parameters are commonly used chlorophyll a fluorescence parameters. This study was conducted to identify quantitative trait loci (QTLs) associated with JIP-test parameters, MF parameters, and photosynthetic rate (P_N), and to examine the relationships among them in soybean (Glycine max (L.) Merr.). Pot and field experiments were performed to evaluate 184 recombinant inbred lines (RILs) for five JIP-test parameters (ABS/RC, TR_o/ABS, ET_o/TR_o, RE_o/ET_o, and PI_ABS), four MF parameters (Fv/Fm, Fv′/Fm′, ΦPSII, and qP), and P_N. Significant correlations were commonly observed among JIP-test parameters, MF parameters, and P_N. QTL mapping analysis identified 13, 9, and 4 QTLs for JIP-test parameters, MF parameters, and P_N, respectively, of which 13 were stable. Four major genomic regions were detected: LG A2 (19.81 cM) for JIP-test parameters, LG C1 (94.31 and 97.61 cM) for P_N and MF parameters, LG M (100.51 cM) for JIP-test and MF parameters, and LG O (30.61–49.91 cM) for P_N, JIP-test, and MF parameters. These results indicate that chlorophyll fluorescence parameters, especially ΦPSII and qP, could play an important role in regulating P_N, and that JIP-test and MF parameters could be controlled by the same or different genes. The QTLs identified in this study will help in the understanding of the genetic basis of photosynthetic processes in plants. They will also contribute to the development of marker-assisted selection breeding programs for photosynthetic capacity in soybean.     

Quantitative trait loci underlying the development of seed composition in soybean (Glycine max L. Merr.).

One hundred and forty-three F2:7 recombinant inbred lines (RILs) developed from the cross of soybean cultivars 'Charleston' and 'Dongnong 594' were analyzed for the quantitative trait loci (QTLs) underlying protein or oil content at 6 different developmental stages by composite interval mapping with a mixed genetic model. The genotype x environment (GxE) interactions of the QTLs were also evaluated. Nineteen (2004) and 33 (2005) unconditional QTLs underlying seed protein or oil content at the different developmental stages were mapped onto 8 and 9 linkage groups, respectively. The proportion of phenotypic variation explained by these QTLs ranged from 6.26% to 30.52% and from 5.38% to 28.47%, respectively. Fourteen (2004) and 21 (2005) conditional QTLs underlying seed protein or oil content were mapped onto 5 and 8 linkage groups, respectively. The proportion of phenotypic variation explained by these QTLs ranged from 2.97% to 29.68% and from 5.42% to 31.96%, respectively. The numbers and types of QTLs and the genetic effect for the two traits were different at each developmental stage. However, several genomic regions that simultaneously control the development of both traits were detected. The genetic effect on protein content and oil content was opposite for loci in the marker interval Satt335-SSatt334, reflecting a negative correlation of protein content and oil content. A G x E interaction effect of some QTLs underlying protein or oil content at different growth periods was observed.     

Identification of quantitative trait loci controlling sucrose content in soybean (Glycine max)

Sucrose is a primary constituent of soybean (Glycine max) seed; however, little information concerning the inheritance of seed sucrose in soybean is available. The objective of this research was to use molecular markers to identify genomic regions significantly associated with quantitative trait loci (QTL) controlling sucrose content in a segregating F₂ population. DNA samples from 149 F₂ individuals were analyzed with 178 polymorphic genetic markers, including RFLPs, SSRs, and RAPDs. Sucrose content was measured on seed harvested from each of 149 F_2:3 lines from replicated field experiments in 1993 and 1995. Seventeen marker loci, mapping to seven different genomic regions, were significantly associated with sucrose variation at P<0.01. Individually, these markers explained from 6.1% to 12.4% of the total phenotypic variation for sucrose content in this population. In a combined analysis these genomic regions; explained 53% of total variation for sucrose content. No significant evidence of epistasis among QTLs was observed. Comparison of our QTL mapping results for sucrose content and those previously reported for protein and oil content (the other major seed constituents in soybean), suggests that seed quality traits are inherited as clusters of linked loci or that `major' QTLs with pleiotropic effects may control all three traits. Of the seven genomic regions having significant effects on sucrose content, three were associated with significant variation for protein content and three were significantly associated with oil content.     

Epistatic expression of quantitative trait loci (QTL) in soybean [Glycine max (L.) Merr.] determined by QTL association with RFLP alleles

Quantitative trait values for seed oil and protein content or for maturity were measured in recombinant inbred lines (RIL) of soybean derived from a cross between two soybean cultivars: Minsoy PI 27890 and Noir 1 PI 290136. Seed oil was found to be inversely correlated to protein content. By analyzing DNA from plants with extreme phenotypes, we were able to identify quantitative trait loci (QTL) for these traits as being linked to several restriction fragment length polymorphism (RFLP) loci, including R183 for oil and protein content and R79 for maturity. Cumulative distributions of trait values were graphed for those RIL with Minsoy alleles and for those with Noir 1 alleles. As already suggested by the alleles found associated with extreme phenotypes, the distributions were consistent with an independent and additive expression of the maturity QTL linked to R79. That is, the cumulative distributions for plants with Minsoy alleles and for plants with Noir 1 alleles were similar in shape, but the entire Noir 1 curve had been shifted to later maturity dates. In contrast, the trait distributions for a locus affecting oil and protein content linked to R183 were not compatible with an additive model. These results suggest that this approach can be used for rapid identification of QTLs with epistatic expression.     

Quantitative trait loci analysis for the developmental behavior of Soybean (Glycine max L. Merr.)

Quantitative trait loci (QTLs) identified so far in soybean were mainly derived in the final stage of plant development, which did not apply to the exploitation of genetic effects that were expressed during a specific developmental stage. Thus, the aim of this study was to identify conditional QTLs associated with yield traits at a specific developmental interval of soybean plant. The 143 recombinant inbred lines developed from the cross of soybean cultivars ‘Charleston’ and ‘Dongnong 594’ were used for the developmental QTLs analysis of pod number in the main stem and plant height by composite interval mapping method combined with mixed genetic model. The results indicated that the number and type of QTLs and their genetic effects for the two agronomic traits were different in a series of measuring stages. A total of 10 unconditional QTLs in 6 linkage groups and 5 conditional QTLs in 3 linkage groups were identified for the pod number of the main stem, while 13 unconditional QTLs in 7 linkage groups and 12 conditional QTLs in 6 linkage groups were identified for plant height. Many QTLs that were detected in the early stages were different from those detected at the later stages. Some QTLs existed only at one stage and others existed across two or three stages. Five marker intervals (satt509-satt251, sat_099-sat_113, sat_113-OPAW19_4, satt457-OPC10_85, sat_095-OPBA08_5) were proven to be associated both with the development of pod number in the main stem and the development of plant height. The present study suggested that the development of pods and plant height in soybean were governed by time-dependent gene expression.     

Determining the linkage of quantitative trait loci to RFLP markers using extreme phenotypes of recombinant inbreds of soybean (Glycine max L. Merr.)

An experimental test is described for linkages between RFLP markers and quantitative trait loci (QTL). Two hundred and eighty-four F₇-derived recombinant inbred lines (RIL) obtained from crossing the soybean cultivars (Glycine max L. Merr.) Minsoy and Noir 1 were evaluated for maturity, plant height, lodging, and seed yield. RIL exhibiting an extreme phenotype for each trait (earliest and latest plants for maturity, etc.) were selected, and two bulked DNA samples were prepared for each trait. A Southern transfer of the digested bulked DNA was hybridized with restriction fragement length polymorphism (RFLP) probes, and linkages with QTL were established by quantitating the amount of radioactive probe that bound to fragments defining alternative parental RFLP alleles. When an RFLP marker was linked to a QTL, one parental allele predominated in the bulked DNA from a particular phenotype; the other allele was associated with the opposite phenotype. When linkage was absent, radioactivity was associated equally with both alleles for a given phenotype (or with both phenotypes for a given allele). These results confirmed RFLP-QTL associations previously discovered by interval mapping on a smaller segregating population from the same cross. New linkages to QTL were also verified.     

Mapping QTLs for tissue culture response in soybean (Glycine max (L.) Merr.)

Hempseed is rich in polyunsaturated fatty acids (PUFAs), which have potential as therapeutic compounds for the treatment of neurodegenerative and cardiovascular disease. However, the effect of hempseed meal (HSM) intake on the animal models of these diseases has yet to be elucidated. In this study, we assessed the effects of the intake of HSM and PUFAs on oxidative stress, cytotoxicity and neurological phenotypes, and cholesterol uptake, using Drosophila models. HSM intake was shown to reduce H₂O₂ toxicity markedly, indicating that HSM exerts a profound antioxidant effect. Meanwhile, intake of HSM, as well as linoleic or linolenic acids (major PUFA components of HSM) was shown to ameliorate Aβ42-induced eye degeneration, thus suggesting that these compounds exert a protective effect against Aβ42 cytotoxicity. On the contrary, locomotion and longevity in the Parkinson’s disease model and eye degeneration in the Huntington’s disease model were unaffected by HSM feeding. Additionally, intake of HSM or linoleic acid was shown to reduce cholesterol uptake significantly. Moreover, linoleic acid intake has been shown to delay pupariation, and cholesterol feeding rescued the linoleic acid-induced larval growth delay, thereby indicating that linoleic acid acts antagonistically with cholesterol during larval growth. In conclusion, our results indicate that HSM and linoleic acid exert inhibitory effects on both Aβ42 cytotoxicity and cholesterol uptake, and are potential candidates for the treatment of Alzheimer’s disease and cardiovascular disease.     

Mapping quantitative trait loci associated with aluminum toxin tolerance in NJRIKY recombinant inbred line population of soybean (Glycine max)

To investigate the genetic mechanism of AI-tolerance in soybean,a recombinant inbred line population (RIL) with 184 F2:7:11 lines derived from the cross of Kefeng No.1 x Nannong 1138-2 (AI-tolerant x AI-sensitive) were tested in pot experimentwith sand culture medium in net room in Nanjing.Four traits,i.e.plant height,number of leaves,shoot dry weight and root dry weight at seedling stage,were evaluated and used to calculate the average membership index (FAi) as the indicator of AI-tolerance.The composite interval mapping (ClM) under WinQTL Cartographer v.2.5 detected five QTLs (i.e.qFAiol,qFAi-2,qFAi-3,qFAi-4 and qFAi-5),explaining 5.20%-9.07% of the total phenotypic variation individually.While with the multiple interval mapping (MIM) of the same software,five QTLs (qFAi-1,qFAi-5,qFAi-6,qFAi-7,and qFAi-8) explaining 5.7%-24.60% of the total phenotypic variation individually were mapped.Here qFAi-1 and qFAi-5 were detected by both CIM and MIM with the locations in a same flanking marker region,GMKF046-GMKF080 on B1 and satt278-sat_95 on L,respectively.While qFAi-2 under CIM and qFAi-6 under MIM both on D1b2 were located in neighboring regions with their confidence intervals overlapped and might be the same locus.Segregation analysis under major gene plus polygene inheritance model showed that Al-tolerance was controlled by two major genes (h2mg =33.05%) plus polygenes (h2pg=52.73%).Both QTL mapping and segregation analysis confirmed two QTLs responsible for Al-tolerance with relatively low heritability,and there might be a third QTL,confounded with the polygenes in segregation analysis.     

Interval mapping of quantitative trait loci for reproductive,morphological, and seed traits of soybean (Glycine max L.)

Quantitative trait loci (QTL) were mapped in segregating progeny from a cross between two soybean (Glycine max (L.) Merr.) cultivars: Minsoy (PI 27.890) and Noir 1 (PI 290.136). The 15 traits analyzed included reproductive, morphological, and seed traits, seed yield and carbon isotope discrimination ratios (¹³C/¹²C). Genetic variation was detected for all of the traits, and transgressive segregation was a common phenomenon. One hundred and thirty-two linked genetic markers and 24 additional unlinked markers were used to locate QTL by interval mapping and one-way analysis of variance, respectively. Quantitative trait loci controlling 11 of the 15 traits studied were localized to intervals in 6 linkage groups. Quantitative trait loci for developmental and morphological traits (R1, R5, R8, plant height, canopy height, leaf area, etc.) tended to be clustered in three intervals, two of which were also associated with seed yield. Quantitative trait loci for seed oil were separated from all the other QTL. Major QTL for maturity and plant height were linked to RFLP markers R79 (31% variation) and G173 (53% variation). Quantitative trait loci associated with unlinked markers included possible loci for seed protein and weight. Linkage between QTL is discussed in relation to the heritability and genetic correlation of the traits.     

Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr)

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD–nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.     

Association mapping of iron deficiency chlorosis loci in soybean (Glycine max L. Merr.) advanced breeding lines

Association mapping is an alternative to mapping in a biparental population. A key to successful association mapping is to avoid spurious associations by controlling for population structure. Confirming the marker/trait association in an independent population is necessary for the implementation of the marker in other genetic studies. Two independent soybean populations consisting of advanced breeding lines representing the diversity within maturity groups 00, 0, and I were screened in multi-site, replicated field trials to discover molecular markers associated with iron deficiency chlorosis (IDC), a major yield-limiting factor in soybean. Lines with extreme phenotypes were initially screened to identify simple sequence repeat (SSR) markers putatively associated with the IDC. Marker data collected from all lines were used to control for population structure and kinship relationships. Single factor analysis of variance (SFA) and mixed linear model (MLM) analyses were used to discover marker/trait associations. The MLM analyses, which include population structure, kinship or both factors, reduced the number of markers significantly associated with IDC by 50% compared with SFA. With the MLM approach, three markers were found to be associated with IDC in the first population. Two of these markers, Satt114 and Satt239, were also found to be associated with IDC in the second confirmation population. For both populations, those lines with the tolerance allele at both these two marker loci had significantly lower IDC scores than lines with one or no tolerant alleles.     

Quantitative trait loci associated with oligosaccharide and sucrose contents in soybean (Glycine max L.)

Oligosaccharides and sucrose are very important nutritional components in soybean seeds. However, little information is available about their inheritance. We used molecular markers to identify the genomic regions significantly associated with the quantitative trait locus (QTL) that controls oligosaccharide and sucrose contents in segregating F_2:10 Rl lines. Two related, but independent, QTLs were identified for oligosaccharides — near marker satt546 on linkage group (LG) D1b+W and satt278 on LG L. Four others, for sucrose content, were located at LG B1 (satt197), D1b+W (satt546), and L (satt523 and satt278). Finally, we found two common QTLs, on LG D1b+W and L, that are associated with both oligosaccharides and sucrose.     

Mapping quantitative trait loci for seed size traits in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.)

Seed size traits in soybean—length, width and thickness—and their corresponding ratios—length-to-width, length-to-thickness and width-to-thickness—play a crucial role in determining seed appearance, quality and yield. In this study, an attempt was made to detect quantitative trait loci (QTL) for the aforementioned seed size traits in F_2:3, F_2:4 and F_2:5 populations from the direct and reciprocal crosses of Lishuizhongzihuang with Nannong 493-1, using multi-QTL joint analysis (MJA) along with composite interval mapping (CIM). A total of 121 main-effect QTL (M-QTL), six environmental effects, eight environment-by-QTL interactions, five cytoplasmic effects and 92 cytoplasm-by-QTL interactions were detected. Fifty-two common M-QTL across MJA and CIM, 21 common M-QTL in more than two populations and 5 M-QTL in all three populations showed the stability of the results. Five M-QTL had higher heritability, greater than 20%. In addition, 28 cytoplasm-by-QTL and 4 environment-by-QTL interactions were confirmed by CIM. Most M-QTL were clustered in eight chromosomal regions. Our results provide a good foundation for fine mapping, cloning and designed molecular breeding of favorable genes related to soybean seed size traits.     

Genome-scale identification of MLO domain-containing genes in soybean (Glycine max L. Merr.)

In plants, powdery-mildew-resistance locus o (Mlo) genes encode proteins that are calmodulin-binding proteins involved in a variety of cellular processes. However, systematic characterization of this gene family in soybean (Glycine max L. Merr.) has not been yet reported. In this study, we identified MLO domain-contained members in soybean and examined their expression under phytohormone treatment and abiotic stress conditions. A total of 20 soybean Mlo genes were identified (GmMlo1-20), which are distributed on 13 chromosomes, and display diverse exon-intron structures. Phylogenetic analysis indicated that the Mlo family can be classified into four subfamilies. Sequence comparison was used to reveal the conserved calmodulin-binding domain (CaMBD) in GmMLO proteins. The expression of GmMlo genes was influenced by various phytohormone treatments and abiotic stresses, suggesting that these Mlo genes have various roles in the response of soybean to environmental stimuli. Promoter sequence analysis revealed an overabundance of stress and/or phytohormone-related cis-elements in GmMlo genes. These data provide important clues for elucidating the functions of genes of the Mlo gene family.     

Translocation of Sulfate in Soybean (Glycine max L. Merr)

Sulfate translocation in soybean (Glycine max L. Merr) was investigated. More than 90% of the sulfate entering the shoot system was recoverable in one or two developing trifoliate leaves. In young plants, the first trifoliate leaf contained between 10 to 20 times as much sulfate as the primary leaves, even though both types of leaf had similar rates of transpiration and photosynthesis. We conclude that most of the sulfate entering mature leaves is rapidly loaded into the phloem and translocated to sinks elsewhere in the plant. This loading was inhibited by carbonylcyanide m-chlorophenylhydrazone and selenate. At sulfate concentrations below 0.1 millimolar, more than 95% of the sulfate entering primary leaves was exported. At higher concentrations the rate of export increased but so did the amount of sulfate remaining in the leaves. Removal of the first trifoliate leaf increased two-fold the transport of sulfate to the apex, indicating that these are competing sinks for sulfate translocated from the primary leaves. The small amount of sulfate transported into the mesophyll cells of primary leaves is a result of feedback regulation by the intracellular sulfate pool, not a consequence of their metabolic inactivity. For example, treatment of plants with 2 millimolar aminotriazole caused a 700 nanomoles per gram fresh weight increase in the glutathione content of primary leaves, but had no effect on sulfate aquisition.     

Mapping quantitative trait loci for root development under hypoxia conditions in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.)

Key message

Greatest potential, QTLs for hypoxia and waterlogging tolerance in soybean roots were detected using a new phenotypic evaluation method.

Abstract

Waterlogging is a major environmental stress limiting soybean yield in wet parts of the world. Root development is an important indicator of hypoxia tolerance in soybean. However, little is known about the genetic control of root development under hypoxia. This study was conducted to identify quantitative trait loci (QTLs) responsible for root development under hypoxia. Recombinant inbred lines (RILs) developed from a cross between a hypoxia-sensitive cultivar, Tachinagaha, and a tolerant landrace, Iyodaizu, were used. Seedlings were subjected to hypoxia, and root development was evaluated with the value change in root traits between after and before treatments. We found 230 polymorphic markers spanning 2519.2 cM distributed on all 20 chromosomes (Chrs.). Using these, we found 11 QTLs for root length (RL), root length development (RLD), root surface area (RSA), root surface area development (RSAD), root diameter (RD), and change in average root diameter (CARD) on Chrs. 11, 12, 13 and 14, and 7 QTLs for hypoxia tolerance of these root traits. These included QTLs for RLD and RSAD between markers Satt052 and Satt302 on Chr. 12, which are important markers of hypoxia tolerance in soybean; those QTLs were stable between 2 years. To validate the QTLs, we developed a near-isogenic line with the QTL region derived from Iyodaizu. The line performed well under both hypoxia and waterlogging, suggesting that the region contains one or more genes with large effects on root development. These findings may be useful for fine mapping and positional cloning of gene responsible for root development under hypoxia.

     

Regeneration of soybean (Glycine max L. Merr.) from cultured primary leaf tissue

A reproducible method for regeneration of plants from primary leaf tissue of 27 varieties of soybean (Glycine max), encompassing maturity groups 00 to VIII, has been developed. Progeny from seeds recovered from regenerated plants appear normal. Best regeneration was from leaf explants (2.1–4.0 mm) obtained from 5 day old seedlings. While 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was demonstrated to be essential for regeneration, addition of benzyladenine (BA) was found to enhance regeneration. Of the 6 other auxins tested, only picloram induced any regenerative response. Using identical volumes of medium and other conditions, regeneration could be obtained in 95 × 25 mm glass culture tubes but not in 60 × 15 mm Petri dishes.The regeneration of soybeans from primary leaf tissue was shown to be greatly enhanced by pyroglutamic acid (5-oxoproline). Stimulatory effects were attained if pyroglutamic acid was added directly to the medium or if it was formed in situ as a result of chemical transformation of glutamine during autoclaving. The active component produced by autoclaving glutamine was not a conjugate of glutamine with inorganic salts or another organic component of the medium. Filter-sterilized glutamine was shown to be inhibitory to regeneration.Murashige and Skoog (MS) and Schenk and Hildebrandt (SH) basal media were compared to Gamborg B5 medium. All contained 0.1 mg/l 2,4,5-T, 40 mg/l adenine sulfate and 10 mM pyroglutamic acid. No regeneration occurred when MS medium was used. Growth and appearance of callus growing on SH and B5 media with the additives were similar. The incidence of regeneration among cultures growing on SH medium was only one third compared to cultures grown on B5 medium.