Genetic mapping reveals the complex genetic architecture controlling slow canopy wilting in soybean.

In soybean [Glycine max (L.) Merr.], drought stress is the leading cause of yield loss from abiotic stress in rain-fed US growing areas. Only 10% of the US soybean production is irrigated; therefore, plants must possess physiological mechanisms to tolerate drought stress. Slow canopy wilting is a physiological trait that is observed in a few exotic plant introductions (PIs) and may lead to yield improvement under drought stress. Canopy wilting of 130 recombinant inbred lines (RILs) derived from Hutcheson × PI 471938 grown under drought stress was visually evaluated and genotyped with the SoySNP6K BeadChip. Over four years, field evaluations of canopy wilting were conducted under rainfed conditions at three locations across the US (Georgia, Kansas, and North Carolina). Due to the variation in weather among locations and years, the phenotypic data were collected from seven environments. Substantial variation in canopy wilting was observed among the genotypes in the RIL population across environments. Three QTLs were identified for canopy wilting from the RIL population using composite interval mapping on chromosomes (Chrs) 2, 8, and 9 based on combined environmental analyses. These QTLs inherited the favorable alleles from PI 471938 and accounted for 11, 10, and 14% of phenotypic variation, respectively. A list of 106 candidate genes were narrowed down for these three QTLs based on the published information. The QTLs identified through this research can be used as targets for further investigation to understand the mechanisms of slow canopy wilting. These QTLs could be deployed to improve drought tolerance through a targeted selection of the genomic regions from PI 471938.

Genome-Wide Association Analyses Reveal Genomic Regions Controlling Canopy Wilting in Soybean.

Drought stress causes the greatest soybean [Glycine max (L.) Merr.] yield losses among the abiotic stresses in rain-fed U.S. growing areas. Because less than 10% of U.S. soybean hectares are irrigated, combating this stress requires soybean plants which possess physiological mechanisms to tolerate drought for a period of time. Phenotyping for these mechanisms is challenging, and the genetic architecture for these traits is poorly understood. A morphological trait, slow or delayed canopy wilting, has been observed in a few exotic plant introductions (PIs), and may lead to yield improvement in drought stressed fields. In this study, we visually scored wilting during stress for a panel of 162 genetically diverse maturity group VI-VIII soybean lines genotyped with the SoySNP50K iSelect BeadChip. Field evaluation of canopy wilting was conducted under rain-fed conditions at two locations (Athens, GA and Salina, KS) in 2015 and 2016. Substantial variation in canopy wilting was observed among the genotypes. Using a genome-wide association mapping approach, 45 unique SNPs that tagged 44 loci were associated with canopy wilting in at least one environment with one region identified in a single environment and data from across all environments. Several new soybean accessions were identified with canopy wilting superior to those of check genotypes. The germplasm and genomic regions identified can be used to better understand the slow canopy wilting trait and be incorporated into elite germplasm to improve drought tolerance in soybean.

The importance of slow canopy wilting in drought tolerance in soybean.

Slow canopy wilting (SW) is a water conservation trait controlled by quantitative trait loci (QTLs) in late maturity group soybeans [Glycine max (L.) Merr.]. Recently, two exotic (landraces) plant introductions (PI 567690 and PI 567731) were identified as new SW lines in early maturity groups. Here, we show that the two PIs share the same water conservation strategy of limited maximum transpiration rates as PI 416937. However, in contrast to PI 416937, the transpiration rates of these PIs were sensitive to an aquaporin inhibitor, indicating an independence between limited maximum transpiration and the lack of silver-sensitive aquaporins. Yield tests of selected recombinant inbred lines from two elite/exotic crosses provide direct evidence to support the benefit of SW in drought tolerance. Four SW QTLs mapped in a Pana×PI 567690 cross at multiple environments were found to be co-located with previous reports. Moreover, two new SW QTLs were mapped on chromosomes 6 and 10 from a Magellan×PI 567731 cross. These two QTLs explain the observed relatively large contributions of 20-30% and were confirmed in a near-isogenic background. These findings demonstrate the importance of SW in yield protection under drought and provide genetic resources for improving drought tolerance in early maturity group soybeans.

Unraveling the genetic architecture for carbon and nitrogen related traits and leaf hydraulic conductance in soybean using genome-wide association analyses.

BACKGROUND: Drought stress is a major limiting factor of soybean [Glycine max (L.) Merr.] production around the world. Soybean plants can ameliorate this stress with improved water-saving, sustained N2 fixation during water deficits, and/or limited leaf hydraulic conductance. In this study, carbon isotope composition (δ13C), which can relate to variation in water-saving capability, was measured. Additionally, nitrogen isotope composition (δ15N) and nitrogen concentration that relate to nitrogen fixation were evaluated. Decrease in transpiration rate (DTR) of de-rooted soybean shoots in a silver nitrate (AgNO3) solution compared to deionized water under high vapor pressure deficit (VPD) conditions was used as a surrogate measurement for limited leaf hydraulic conductance. A panel of over 200 genetically diverse soybean accessions genotyped with the SoySNP50K iSelect BeadChips was evaluated for the carbon and nitrogen related traits in two field environments (Athens, GA in 2015 and 2016) and for transpiration response to AgNO3 in a growth chamber. A multiple loci linear mixed model was implemented in FarmCPU to perform genome-wide association analyses for these traits. RESULTS: Thirty two, 23, 26, and nine loci for δ13C, δ15N, nitrogen concentration, and transpiration response to AgNO3, respectively, were significantly associated with these traits. Candidate genes that relate to drought stress tolerance enhancement or response were identified near certain loci that could be targets for improving and understanding these traits. Soybean accessions with favorable breeding values were also identified. Low correlations were observed between many of the traits and the genetic loci associated with each trait were largely unique, indicating that these drought tolerance related traits are governed by different genetic loci. CONCLUSIONS: The genomic regions and germplasm identified in this study can be used by breeders to understand the genetic architecture for these traits and to improve soybean drought tolerance. Phenotyping resources needed, trait heritability, and relationship to the target environment should be considered before deciding which of these traits to ultimately employ in a specific breeding program. Potential marker-assisted selection efforts could focus on loci which explain the greatest amount of phenotypic variation for each trait, but may be challenging due to the quantitative nature of these traits.

Genome-wide association mapping of canopy wilting in diverse soybean genotypes.

KEY MESSAGE: Genome-wide association analysis identified 61 SNP markers for canopy wilting, which likely tagged 51 different loci. Based on the allelic effects of the significant SNPs, the slowest and fastest wilting genotypes were identified. Drought stress is a major global constraint for crop production, and slow canopy wilting is a promising trait for improving drought tolerance. The objective of this study was to identify genetic loci associated with canopy wilting and to confirm those loci with previously reported canopy wilting QTLs. A panel of 373 maturity group (MG) IV soybean genotypes was grown in four environments to evaluate canopy wilting. Statistical analysis of phenotype indicated wide variation for the trait, with significant effects of genotype (G), environment (E), and G × E interaction. Over 42,000 SNP markers were obtained from the Illumina Infinium SoySNP50K iSelect SNP Beadchip. After filtration for quality control, 31,260 SNPs with a minor allele frequency (MAF) ≥5% were used for association mapping using the Fixed and random model Circulating Probability Unification (FarmCPU) model. There were 61 environment-specific significant SNP-canopy wilting associations, and 21 SNPs that associated with canopy wilting in more than one environment. There were 34 significant SNPs associated with canopy wilting when averaged across environments. Together, these SNPs tagged 23 putative loci associated with canopy wilting. Six of the putative loci were located within previously reported chromosomal regions that were associated with canopy wilting through bi-parental mapping. Several significant SNPs were located within a gene or very close to genes that had a reported biological connection to transpiration or water transport. Favorable alleles from significant SNPs may be an important resource for pyramiding genes to improve drought tolerance and for identifying parental genotypes for use in breeding programs.

Enhanced seed viability and lipid compositional changes during natural ageing by suppressing phospholipase Dα in soybean seed.

Changes in phospholipid composition and consequent loss of membrane integrity are correlated with loss of seed viability. Furthermore, phospholipid compositional changes affect the composition of the triacylglycerols (TAG), i.e. the storage lipids. Phospholipase D (PLD) catalyses the hydrolysis of phospholipids to phosphatidic acid, and PLDα is an abundant PLD isoform. Although wild-type (WT) seeds stored for 33 months were non-viable, 30%-50% of PLDα-knockdown (PLD-KD) soybean seeds stored for 33 months germinated. WT and PLD-KD seeds increased in lysophospholipid levels and in TAG fatty acid unsaturation during ageing, but the levels of lysophospholipids increased more in WT than in PLD-KD seeds. The loss of viability of WT seeds was correlated with alterations in ultrastructure, including detachment of the plasma membrane from the cell wall complex and disorganization of oil bodies. The data demonstrate that, during natural ageing, PLDα affects the soybean phospholipid profile and the TAG profile. Suppression of PLD activity in soybean seed has potential for improving seed quality during long-term storage.

Phospholipid and triacylglycerol profiles modified by PLD suppression in soybean seed.

Phospholipase D (PLD) is capable of hydrolyzing membrane phospholipids, producing phosphatidic acid. To alter phospholipid profiles in soybean seed, we attenuated PLD enzyme activity by an RNA interference construct using the partial sequence from a soybean PLDα gene. Two transgenic soybean lines were established by particle inflow gun (PIG) bombardment by co-bombarding with pSPLDi and pHG1 vectors. The lines were evaluated for the presence and expression of transgenes thoroughly through the T(4) generation. PLD-suppressed soybean lines were characterized by decreased PLDα enzyme activity and decreased PLDα protein both during seed development and in mature seeds. There was no change in total phospholipid amount; however, the PLD-attenuated transgenic soybean seed had higher levels of di18:2 (dilinoleoyl)-phosphatidylcholine (PC) and -phosphatidylethanolamine (PE) in seeds than the non-transgenic lines. The increased polyunsaturation was at the expense of PC and PE species containing monounsaturated or saturated fatty acids. In addition to increased unsaturation in the phospholipids, there was a decrease in unsaturation of the triacylglycerol (TAG) fraction of the soybean seeds. Considering recent evidence for the notion that desaturation of fatty acids occurs in the PC fraction and that the PC→DAG (diacylglycerol)→TAG pathway is the major route of TAG biosynthesis in developing soybean seed, the current data suggest that PLDα suppression slows the conversion of PC to TAG. This would be consistent with PLD playing a positive role in that conversion. The data indicate that soybean PLD attenuation is a potentially useful approach to altering properties of edible and industrial soybean lecithin.

Chlorophyll loss caused by soybean aphid (Hemiptera: Aphididae) feeding on soybean.

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a worldwide pest of soybean, Glycine max (L.) Merr. Studies to find control methods were initiated in 2000 when it was first detected in North America. A. glycines can reduce yields by as much as 50%, and it is the vector of several viral diseases. A. glycines removes phloem sap, which can result in a reduction of chlorophyll content. Quantification of chlorophyll loss caused by A. glycines feeding on soybean is of vital importance. The SPAD-502 chlorophyll meter is a device that has been used to measure chlorophyll loss caused by nonchewing insects. Chlorophyll loss was studied in no-choice tests on the infested and uninfested leaves of a susceptible check (KS4202). The minimum combined number of days and aphids needed to detect significant chlorophyll loss was 30 aphids confined for 10 d. In a similar experiment, seven resistant entries and two susceptible checks were evaluated. There was no significant chlorophyll reduction between infested and uninfested leaves of five of the resistant entries (K1621, K1639, Pioneer 95B97, Dowling, and Jackson). Percentage of loss of chlorophyll in the susceptible checks was approximately 40%; Jackson and Dowling had a significantly lower percentage loss (13 and 16%, respectively) compared with the susceptible checks. The percentages of chlorophyll loss of K1621, K1639, and Pioneer 95B97 were not statistically different from the percentage of loss of Jackson.

Feeding behavior by the soybean aphid (Hemiptera: Aphididae) on resistant and susceptible soybean genotypes.

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a major pest of soybean, Glycine max (L.) Merr. Since 2000, when A. glycines was detected in the United States, several studies on this insect have been done in different areas, but there is no report of any studies of stylet penetration behavior by A. glycines on resistant and susceptible soybean. Assessment of feeding behavior of this aphid species was compared on four resistant entries (K1639, Pioneer 95B97, Dowling, and Jackson) and a susceptible check (KS4202) by using the electrical penetration graph (EPG) technique. Feeding behavior of A. glycines adults was recorded during a 9-h period. The average time needed to reach the first sieve element phase by A. glycines was 3.5 h in KS4202, whereas it was 7.5 h in the resistant entries. The total duration in the sieve element phase was longer than an hour in KS4202, and only 2 to 7 min in the resistant entries. These results suggest that morphological or chemical factors in the phloem tissue of resistant plants affect stylet penetration activities of A. glycines. In the majority of the recordings, however, the aphid stylet reached the xylem phase before penetrating the sieve element, and the time that aphids spent ingesting xylem sap was not different among all entries. Therefore, it is possible that xylem sap in the resistant entries may contain toxic substances that change aphid behavior and that affect further activities in the sieve element phase.

Characterization of antibiosis and antixenosis to the soybean aphid (Hemiptera: Aphididae) in several soybean genotypes.

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is an introduced pest of soybean, Glycine max (L.) Merr., in North America, and it can reduce yields by 50%. Since 2000, when A. glycines was first detected in the United States, studies of this insect and possible control methods have been initiated. Plant resistance to this aphid species is one important component of integrated control. Reproduction of A. glycines was compared on 240 soybean entries in a pesticide-free greenhouse. Eleven entries had fewer nymphs produced, compared with the susceptible checks, and these entries were used in follow-up experiments to assess antibiosis and antixenosis. Antibiosis was estimated in true no-choice tests, in which adults were confined individually in double-sided sticky cages stuck to the upper side of leaves. Antixenosis was assessed in choice tests, in which all entries were planted in a single pot. Adult aphids were placed in the center of the pot, and 24 h later the number of adults on each plant was counted. Of the 11 entries evaluated, nine showed a moderate antibiotic effect to A. glycines, and the other two entries (K1639 and Pioneer 95B97) showed not only a strong antibiotic effect but also exhibited antixenosis as a category of resistance to A. glycines. The resistant soybean entries found in this work are potential sources for A. glycines control.