Cloning and functional analysis of soluble acid invertase 2 gene (SbSAI-2) in sorghum.

MAIN CONCLUSION: The sorghum soluble acid invertase gene SbSAI-2 was cloned and the function verified in Pichia pastoris and rice, showing the SbSAI-2 affects composition and content of sugar in stem juice. Sugar metabolism is one of the most important metabolic processes in plants, in which soluble acid invertase plays a key role. However, the structure and function of the soluble acid transferase gene in sorghum are still fully unclear. In this study, SbSAI-2 was cloned from the sorghum variety BTx623, and two transcripts were found through sequence analysis, with only one transcript translated into an active protein. There is 72% homology between SbSAI-2 and OsVIN2. The construction of Osvin2 mutant lines and SbSAI-2-1 overexpression lines in Oryza sativa L. japonica. cv. Nipponbare were produced to clarify the invertase functionality. While the invertase activity in the stem of the Osvin2 mutant line was reduced, with no significant difference (P > 0.05), and the contents of fructose and glucose in stem tissue did not change significantly (P > 0.05), and the content of sucrose increased by 38.89% (P < 0.01). In SbSAI-2-1 overexpression lines, the invertase activity in stem was increased by more than 20 times (P < 0.01). The contents of glucose and fructose in stem tissues were increased by two and three times, respectively (P < 0.01), while the content of sucrose was significantly decreased, which was below the detection limit (P < 0.01). This study indicated that SbSAI-2 is a key enzyme related to sucrose metabolism and affects the composition and content of sugar in stems. The result provided further the gene function verification and laid a foundation for the development of molecular markers.

Change of soluble acid invertase gene (SAI-1) haplotype in hybrid sorghum breeding program in China.

Sugar metabolism is the most important and core one which drives plant growth and development. Invertases are key enzymes that regulate sugar metabolism. A still-growing number of studies have revealed that invertases play a crucial role in various aspects of plant growth and development. Crop yield is the product of sugar metabolism; it could be deduced that invertase also regulated the yield formation. So we have done a series of research on soluble acid invertase in sweet sorghum from enzyme activity to gene cloning and functional marker development. In this paper, we sequenced full length of SAI-1 gene in 69 grain sorghum parent lines, trying to see how it differs in their gene sequences and their distribution in related hybrid varieties released in the past. To our surprise, the result showed that B-lines and restore lines (R-line) have almost different SAI-1 haplotype distribution. The change of haplotype of SAI-1 gene is associated with yield gain as with grain sorghum breeding progress, which proved that SAI-1 may take a very important role in yield formation. And we also found the SAI-1 gene tends to become shorter as with the breeding advance, which means short sequence in introns, while exon remains unchanged leading to higher gene efficiency. The best SAI-1 haplotype combination of sorghum hybrid was also found for different planting regions. These findings are of great significance for improving breeding efficiency, understanding heterosis, and germplasm enhancement. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01231-2.

High-resolution mapping of QTL for fatty acid composition in soybean using specific-locus amplified fragment sequencing.

KEY MESSAGE: We constructed a high-density linkage map comprising 3541 markers developed by specific-locus amplified fragment sequencing, and identified 26 stable QTL including nine novel loci, for fatty acid composition in soybean. Soybean oil quality and stability are mainly determined by the fatty acid composition of the seed. In the present study, we constructed a high-density genetic linkage map using 200 recombinant inbred lines derived from a cross between cultivated soybean varieties Luheidou2 and Nanhuizao, and SNP markers developed by specific-locus amplified fragment sequencing (SLAF-seq). This map comprises 3541 markers on 20 linkage groups and spans a genetic distance of 2534.42 cM, with an average distance of 0.72 cM between adjacent markers. Inclusive composite interval mapping revealed 26 stable QTL for five fatty acids, explaining 0.4-37.0% of the phenotypic variance for individual fatty acids across environments. Of these QTL, nine are novel loci (qLA1, qLNA2_1, qPA4_1, qLA4_1, qPA6_1, qSA12_1, qPA16_1, qOA18_1, and qFA19_1). These stable QTL harbor three fatty acid biosynthesis genes (GmFabG, GmACP, and GmFAD8), and 66 genes encoding lipid-related transcription factors. These stable QTL and tightly linked SNP markers can be used for marker-assisted selection in soybean breeding programs.

Construction of a high-density genetic map based on large-scale markers developed by specific length amplified fragment sequencing (SLAF-seq) and its application to QTL analysis for isoflavone content in Glycine max.

BACKGROUND: Quantitative trait locus (QTL) mapping is an efficient approach to discover the genetic architecture underlying complex quantitative traits. However, the low density of molecular markers in genetic maps has limited the efficiency and accuracy of QTL mapping. In this study, specific length amplified fragment sequencing (SLAF-seq), a new high-throughput strategy for large-scale SNP discovery and genotyping based on next generation sequencing (NGS), was employed to construct a high-density soybean genetic map using recombinant inbred lines (RILs, Luheidou2×Nanhuizao, F5:8). With this map, the consistent QTLs for isoflavone content across various environments were identified. RESULTS: In total, 23 Gb of data containing 87,604,858 pair-end reads were obtained. The average coverage for each SLAF marker was 11.20-fold for the female parent, 12.51-fold for the male parent, and an average of 3.98-fold for individual RILs. Among the 116,216 high-quality SLAFs obtained, 9,948 were polymorphic. The final map consisted of 5,785 SLAFs on 20 linkage groups (LGs) and spanned 2,255.18 cM in genome size with an average distance of 0.43 cM between adjacent markers. Comparative genomic analysis revealed a relatively high collinearity of 20 LGs with the soybean reference genome. Based on this map, 41 QTLs were identified that contributed to the isoflavone content. The high efficiency and accuracy of this map were evidenced by the discovery of genes encoding isoflavone biosynthetic enzymes within these loci. Moreover, 11 of these 41 QTLs (including six novel loci) were associated with isoflavone content across multiple environments. One of them, qIF20-2, contributed to a majority of isoflavone components across various environments and explained a high amount of phenotypic variance (8.7%-35.3%). This represents a novel major QTL underlying isoflavone content across various environments in soybean. CONCLUSIONS: Herein, we reported a high-density genetic map for soybean. This map exhibited high resolution and accuracy. It will facilitate the identification of genes and QTLs underlying essential agronomic traits in soybean. The novel major QTL for isoflavone content is useful not only for further study on the genetic basis of isoflavone accumulation, but also for marker-assisted selection (MAS) in soybean breeding in the future.

Isoflavone Content of Soybean Cultivars from Maturity Group 0 to VI Grown in Northern and Southern China.

Soybean isoflavone content has long been considered to be a desirable trait to target in selection programs for their contribution to human health and plant defense systems. The objective of this study was to determine isoflavone concentrations of various soybean cultivars from maturity groups 0 to VI grown in various environments and to analyze their relationship to other important seed characters. Forty soybean cultivars were grown in replicated trials at Wuhan and Beijing of China in 2009/2010 and their individual and total isoflavone concentrations were determined by HPLC. Their yield and quality traits were also concurrently analyzed. The isoflavone components had abundant genetic variation in soybean seed, with a range of coefficient variation from 45.01% to 69.61%. Moreover, individual and total isoflavone concentrations were significantly affected by cultivar, maturity group, site and year. Total isoflavone concentration ranged from 551.15 to 7584.07 µg g-1, and averaged 2972.64 µg g-1 across environments and cultivars. There was a similar trend regarding the isoflavone contents, in which a lower isoflavone concentration was generally presented in early rather than late maturing soybean cultivars. In spite of significant cultivar × year × site interactions, cultivars with consistently high or low isoflavone concentrations across environments were identified, indicating that a genetic factor plays the most important role for isoflavone accumulation. The total isoflavone concentration had significant positive correlations with plant height, effective branches, pods per plant, seeds per plant, linoleic acid and linolenic acid, while significant negative correlations with oleic acid and oil content, indicating that isoflavone concentration can be predicted as being associated with other desirable seed characteristics.

The physicochemical properties and Pickering emulsifying capacity of acorn starch.

In this work, the granule characteristics, functional properties, in-vitro digestibility, antioxidant capacity, and phenolic composition of acorn starch were investigated and compared to those of potato starch and corn starch, and its Pickering emulsifying ability was also evaluated. The results showed that the acorn starch granules were spherical and oval in shape, with a smaller particle size, and the amylose content and crystallinity degree were similar to those of corn starch. However, the acorn starch was difficult to swell, with poor aqueous solubility, though it had a strong gel strength and setback viscosity. Because acorn starch contained more free and bound polyphenols, its resistant starch content after cooking and ABTS and DPPH radical scavenging activities were significantly higher than those of potato starch and corn starch. Acorn starch also exhibited outstanding particle wettability and could stabilize Pickering emulsions. The assessed emulsion showed an outstanding effect for protecting ß-carotene against ultraviolet irradiation and was positively correlated with the acorn starch addition amount. The obtained results may serve as a reference for the further development of acorn starch.

Formation mechanism of Pickering emulsion gels stabilized by proanthocyanidin particles: Experimental and molecular dynamics studies.

The feasibility of constructing a Pickering emulsion gel with proanthocyanidin particles (PAP) was evaluated in this study, and the related mechanism was revealed by combining instrumental characterization with molecular dynamics simulation. The results showed that PAP was composed of nano/micron spherical particles or their fragments, which had excellent wettability. Suitable PAP addition amount (w, ≥1%) and oil volume fraction (φ, 40-90 %) were beneficial to the formation of stable Pickering emulsion gel. The oil droplet size of gel was inversely proportional to w and φ. The mechanical parameters (gel strength, loss modulus, and storage modulus) were positively correlated with w and φ. Molecular dynamics simulation indicated that the proanthocyanidin molecules in the oil-water system could spontaneously reside and aggregate at the interface, and their interactions with water and oil reduced interfacial tension, which was consistent with the experimental results. This study provides a reference for other polyphenol-based Pickering emulsions.

Interaction of genotype-ecological type-plant spacing configuration in sorghum [Sorghum bicolor (L.) Moench] in China.

Grain sorghum has been a significant contributor to global food security since the prehistoric period and may contribute even more to the security of both food and energy in the future. Globally, precise management techniques are crucial for increasing grain sorghum productivity. In China, with diverse ecological types, variety introduction occasionally occurs across ecological zones. However, few information is available on the effect of ecological type on genotype performance and how plant spacing configuration influences grain yield in various ecological zones. Hence, a series of two-year field experiments were conducted in 2020 and 2021 in four ecological zones of China, from the northeast to the southwest. The experiments included six widely adapted sorghum varieties under six plant spacing configurations (two row spacing modes: equidistant row spacing (60 cm) mode and wide (80 cm)-narrow (40 cm) row spacing mode; three in-row plant spacings: 10 cm, 15 cm, and 20 cm). Our results indicated that ecological type, variety, and plant spacing configuration had a significant effect on sorghum yield. Ecological type contributed the highest proportion to the yield variance (49.8%), followed by variety (8.3%), while plant spacing configuration contributed 1.8%. Sorghum growth duration was highly influenced by the ecological type, accounting for 87.2% of its total variance, whereas plant height was mainly affected by genotype, which contributed 81.6% of the total variance. All test varieties, developed in the south or north, can reach maturity within 94-108 d, just before fall sowing in central China. Generally, sorghum growth duration becomes longer when a variety is introduced from south to north. A late-maturing variety, developed in the spring sowing and late-maturing regions, possibly could not reach maturity in the early-maturing region. The row spacing modes had no significant affect on sorghum yield, but the equal-row spacing mode consistently caused higher yields with only one exception; this might imply that equal-row spacing mode was more advantageous for boosting sorghum yield potential. In contrast, decreasing in-row plant spacing showed significant positive linear associations with sorghum grain yield in most cases. In addition, these results demonstrated that sorghum is a widely adapted crop and enables success in variety introduction across ecological zones.

Interpretation of genotype-environment-sowing date/plant density interaction in sorghum [Sorghum bicolor (L.) Moench] in early mature regions of China.

Sorghum [Sorghum bicolor (L.) Moench] is an important crop for food security in semiarid and arid regions due to its high tolerance to abiotic and biotic stresses and its good performance in marginal lands with relatively low fertility. To deeply understand the interrelationship among sorghum genotype, environment, sowing dates, and densities in the spring sowing early maturing (SSEM) areas of China, and to provide a basis for specifying scientific and reasonable cultural practices, a two-year field experiment was conducted with six popular varieties at six locations. Combined ANOVA showed that the yield difference between years was significant (P<0.05); the yield differences among locations, varieties, sowing dates, and densities were all highly significant (P<0.01). The variety effect was mainly influenced by location, year, sowing dates and their interactions. The sowing effect was mainly influenced by the location, year, variety and their interactions The plant density effect was significantly influenced by location and location-year interaction. Of the contributions of various test factors to yield variance, the location was the largest one (38.18%), followed by variety (12.31%), sowing date (1.53%), density (0.54%), and year (0.09%), with all these single factors accounting for 52.65%. The total contribution of all two-factor interactions accounted for 14.24%, among which the greatest contributor was location-hybrid interaction (8.07%). The total contribution of all three-factor interactions accounted for 14.58%, of which year-location-hybrid interaction was the largest contributor (9.02%). Sowing dates significantly affected model of sorghum growth and development, especially during the late period. The key climatic factors affecting yield were different among the six locations. Weather factors during the grain filling stages contributed much more than those during the early stage to grain yield. Mid-maturing varieties are recommended other than early maturing varieties for the SSEM areas even when late sowing occurs. Sowing as early as possible is recommended for areas with very short frost-free period (Harbin, Tongliao, and Datong). Proper delayed sowing is recommended for areas with a relative long frost-free period (Gongzhuling, Baicheng and Zhangjiakou). This research will provide a conducive reference for sorghum production in similar areas.

[Rapid determination of fatty acids in soybeans [Glycine max (L.) Merr.] by FT-near-infrared reflectance spectroscopy].

Current breeding programs dealing with fatty acid (FA) concentrations in soybean [Glycine max (L. ) Merr.] require large numbers for gas chromatographic analyses, thus it is important to develop a method for rapid determination of fatty acid by Near-Infrared Reflectance spectroscopy (NIRS) in soybeans. The objective of this work was to study the potential of fourier-transform near-infrared reflectance spectroscopy (FT-NIRS) to estimate the fatty acid concentrations in Chinese soybean varieties. One hundred and eight of soybean cultivars or lines (the calibration set: 64; the external validation set: 44) were scanned within 4000-12500 cm(-1) of wavenumbers using a standard sample cup by NIRS machinery, and analyzed the fatty acids by gas chromatograph (GC) methods. Equations were developed using partial least squares (PLS) regression and cross validation for multivariate calibration in this study. The optimal spectral region was selected from 6101.9 to 5446.5 cm(-1) based on the OPUS 5.0 software. Cross validation results showed that major FA components such as oleic acid (R2(CV) = 0.94), linoleic acid (R2(CV) = 0.87), linolenic acid (R2(CV) = 0.85), and total saturates (R2(CV) = 0.88) were accurately determined by the proposed equations as compared with the reference data obtained by the GC method. External validation results also demonstrated that equation for oleic acid had the highest predictive ability R(2)val = 0.91), root mean square error of predication (RMSEP) value was 2.47 g x kg(-1) dry weight, the ratios of RMSEP to the standard deviation (SD) was 0.29, which was usable for quality assurance application. Moreover, equations for palmitic acid, stearic acid, linoleic acid, linolenic acid, and total saturates were predicted with the determination coefficients ranging from 0.66 to 0.76, RMSEP values from 0.37 to 2.74 g x kg(-1) dry weight, and RMSEP/SD values from 0.47 to 0.53, which could be used for sample screening. Therefore, we confirmed that a reliable estimation of the major fatty acid components is possible by using NIRS technique in soybeans.

[Development of new soybean germplasm with null lipoxygenase and Kunitz trypsin inhibitor genes].

Soybean is one of the most important sources of plant protein for human. Soybean protein is a kind of high-quality protein composed of balanced amino acids, which contains all kinds of amino acids, especially 8 amino acids necessary for human. But it also contains some components that are not good for human and affect food quality, such as lipoxygenase (Lox) and trypsin inhibitor (Ti). Those are important anti-nutritious factors. Nutritional value and processing quality of soybean can be improved by means of development of new variety with null Lox and Ti. In this paper, new soybean germplasms that pyramided multiple genes of high quality, null lipoxygenase and trypsin inhibitor genes (Ix1, Ix2, Ix3 and ti) were developed by means of cross and biochemical marker-assisted selection of progenies for null lipoxygenase and trypsin inhibitor genes using known Lox and Ti markers (protein markers). Female parents were soybean varieties Ludou 4, Zhongpin 661, Yudou 8,91D15, wei8640 popularized in Huanghuaihai Plain. Male parents were varieties introduced from US, trypsin-inhibitor (Ti)-deficient varieties P. I. L83-4387 and near isogenic lines of varieties Century for lipoxygenase (Lox)-deficient genes, Century-2 (Ix2), Century-2.3 (Ix2Ix3) and Century-1.3 (Ix1 Ix3). These new germplasms will promote soybean breeding for improved quality production,and utilization.

[Development of a new type soybean germplasm with null lipoxygenase isozymes].

Soybean protein is a kind of high-quality protein composed of balanced amino acids. It contains all kinds of amino acids, especially eight amino acids necessary for human, but also contains some components that are not good for human and affect food quality, such as Lipoxygenase (Lox) and Trypsin inhibitor (Ti). Nutritional value and processing quality of soybean can be improved by means of development of new variety with null Lox and Ti. In this paper, a new type soybean germplasm with null lipoxygenase isozymes was developed by Institute of Crop Sciences, Chinese Academy of Agricultural Sciences through years of biochemical marker assistant selection for null lipoxygenase by means of isoelectric focusing-polyacrylamid gel electrophoresis (IEF-PAGE) in the hybrid progenies of "96P17" (Female parent, a null lipoxygenase 2.3 line) and "93704" (Male parent, a null lipoxygenase 1.3 line). It is the first new soybean germplasm with null Lox1.2.3 genes in our country, which will contribute to soybean breeding for high quality, soybean production and utilization. In this paper, the development process of new type soybean germplasm is described.

Evaluation of the chemical quality traits of soybean seeds, as related to sensory attributes of soymilk.

The soybean seed chemical quality traits (including protein content, oil content, fatty acid composition, isoflavone content, and protein subunits), soymilk chemical character (soluble solid), and soymilk sensory attributes were evaluated among 70 genotypes to determine the correlation between seed chemical quality traits and soymilk sensory attributes. Six sensory parameters (i.e., soymilk aroma, smoothness in the mouth, thickness in the mouth, sweetness, colour and appearance, and overall acceptability) and a seven-point hedonic scale for each parameter were developed. Significant positive correlations were observed between overall acceptability and the other five evaluation parameters, suggesting that overall acceptability is an ideal parameter for evaluating soymilk flavour. The soymilk sensory attributes were significantly positively correlated with the characteristics of the glycinin (11S)/beta-conglycinin (7S) protein ratio, soluble solid, and oil content but negatively correlated with glycitein and protein content. Our results indicated that soymilk sensory attributes could be improved by selecting the desirable seed chemical quality traits in practical soybean breeding programs.

[Development of a new soybean variety with null trypsin inhibitor and lipoxygenase 2.3 genes--zhonghuang 16 and its cultivation practices].

Soybean protein is a kind of high-quality protein composed of balanced amino acids, which contains all kinds of amino acid, especially 8 amino acids necessary for human, but also contains some components that are not good for human and affect food quality, such as Trypsin inhibitor and Lipoxygenase. Nutritional value and processing quality of soybean can be improved by means of development of new variety with null Lox and Ti. In this paper, a new soybean variety Zhonghuang 16 (originally name as Zhongzuo 96-952) was developed by Institute of Crop Breeding and Cultivation Chinese Academy of Agricultural Sciences through years of biochemical marker assistant selection for null trypsin inhibitor by Native-polyacrylamid gel electrophoresis (Native-PAGE) and null lipoxygenase by means of isoelectric focusing-polyacrylamid gel electrophoresis (IEF-PAGE) in the hybrid progenies of "ti15176" (Female parent)--a high-yielding, mosaic virus resistant and null trypsin inhibitor line and "Century-2.3" (Male parent)--a null lipoxygenase near isogene line of an elite American variety "Century". This variety was subjected to Beijing regional trial for summer-sowing soybean during 1999-2000, and to Beijing demonstration test in 2001. In 2002, it was passed the examination and approval by the Beijing Committee of Crop Variety Examination and Approval because of its outstanding characteristics such as high and stable yielding, good quality (high protein and fat content, high protein content and good protein quality-null Ti and Lox2.3), disease resistant and good general character. It is the first new soybean variety with null Ti and Lox2.3 genes in our country. In this paper, the development process and cultivation of Zhonghuang 16 were described.

Simultaneous determination of geniposide and its metabolites genipin and genipinine in culture of Aspergillus niger by HPLC.

When cultivated with Aspergillus niger, geniposide, an important drug, is transformed into genipin and genipinine. A simple and rapid HPLC method for simultaneous determination of geniposide and its two metabolites in broth of A. niger is described. The chromatographic separation was achieved on a C18 ODS column (250 x 4.6 mm) by gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the gradient mixtures. The flow rate was 1 mL/min, the detection wavelength was 238 nm and the column temperature was kept at 28 degrees C. The retention times of geniposide, genipin and genipinine were 10.9, 13.8 and 21.5 min, respectively. The mean absolute recoveries of three analysts were over 98%. Quantification limits were 0.01 microg/mL for geniposide and 0.02 microg/mL for the two metabolites. The method was applied for the quantification of geniposide, genipin and genipinine during fermentation and the evaluation of the bioavailabilities of these three compounds in Caco-2 monolayer.