Changes in anthocyanin and isoflavone concentrations in black seed-coated soybean at different planting locations

This study assessed the altitudinal variations in the anthocyanin and isoflavone contents of six black seed coated soybean [Glycine max (L.) Merrill] cultivars. The black soybean cultivars Heugcheong, Seonheuk, Geomjeong 1, Geomjeong 2, Cheongja 2, and Cheongja 3 were planted at Milyang (12 m above mean sea level — low altitude) and Muju (600 m — high altitude), Korea on 10 June 2005 and 2006. The total anthocyanin and isoflavone contents and individual components were investigated by reverse-phase high performance liquid chromatography (HPLC). All black soybean cultivars cultivated in high altitude possessed significantly higher total anthocyanin (p < 0.01) and isoflavone (p < 0.01) contents than those grown in low altitude. For anthocyanin composition, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, and peonidin-3-O-glucoside contents were significantly higher while delphinidin-3-O-glucoside contents was significantly lower at high altitude. The composition of individual isoflavones, 6″-O-malonyldaidzin, and 6″-O-malonylgenistin contents significantly increased at high altitude.     

A SNP genetic linkage map based on the ‘Hamilton’ by ‘Spencer’ recombinant inbred line population identified QTL for seed isoflavone contents in soybean

Soybean is one of the most important crops worldwide for its protein and oil as well as the health beneficial phytoestrogens or isoflavone. This study reports a relatively dense single nucleotide polymorphism (SNP)‐based genetic map based on ‘Hamilton’ by ‘Spencer’ recombinant inbred line population and quantitative trait loci (QTL) for seed isoflavone contents. The genetic map is composed of 1502 SNP markers and covers about 1423.72 cM of the soybean genome. Two QTL for seed isoflavone contents have been identified in this population. One major QTL that controlled both daidzein (qDZ1) and total isoflavone contents (qTI1) was found on LG C2 (Chr 6). And a second QTL for glycitein content (qGT1) was identified on the LG G (Chr 18). These two QTL in addition to others identified in soybean could be used in soybean breeding to optimize isoflavone content. This newly assembled soybean linkage map is a useful tool to identify and map QTL for important agronomic traits and enhance the identification of the genes involved in these traits.     

Identification of quantitative trait loci for seed isoflavone concentration in soybean (Glycine max) against soybean cyst nematode stress

Isoflavones are plant secondary metabolites produced in soybean (Glycine max), which provide plant defense against pathogens and are beneficial to human health. Soybean cyst nematode (SCN) is a major yield‐limiting pest in most soybean‐producing area across the world. Traits, seed isoflavones and SCN resistance are quantitative in nature, and their phenotypic evaluations are expensive. Quantitative trait loci (QTL) underlying the two traits will be helpful to develop SCN‐resistant lines with elevated isoflavones using marker‐assisted‐selection (MAS). This study aims to identify isoflavones and SCN‐related QTL in a soybean population consisting of 109 RILs, which was developed from a cross between two commercial soybean cultivars viz. ‘RCAT1004’ and ‘DH4202’ and grown in four non‐SCN and SCN‐infested fields during 2015 and 2016. While single marker ANOVA identified 10 QTL for isoflavones and five for SCN (p < 0.01), simple interval and multiple QTL mappings identified four QTL associated with isoflavones (LOD ≥ 2.2). These results contribute to a better understanding of the genetics of the two traits and provide molecular markers that can be used in MAS to facilitate developing SCN‐resistant soybeans with increased isoflavones.     

Unconditional and conditional QTL underlying the genetic interrelationships between soybean seed isoflavone,and protein or oil contents

Selection for soybean (Glycine max L. Merr.) rich in isoflavones, protein and oil has been difficult due to negative genetic interrelationships. In this study, genetic interrelationships among seed isoflavones and protein and oil contents were evaluated using both unconditional and conditional QTL mapping. Daidzein (DZ), genistein (GT), glycitein (GC) and total isoflavone (TI) contents were analysed in F_5:6, F_5:7 and F_5:8 recombinant inbred lines (RILs) derived from a cross between ‘Zhongdou 27’(TI 3791 μg/g; protein content 42.84%; oil content 18.73%) and ‘Jiunong 20’ (TI 2061 μg/g; protein content 34.05%; oil content 21.42%). When DZ, GT, GC and TI were analysed for their genetic relationships with protein or oil contents, eight conditional QTL were detected, which included DZ|pro, GC|pro, GT|pro, TI|pro, DZ|oil, GC|oil, GT|oil and TI|oil. Seventeen QTL that had significant genetic associations between seed isoflavone, and seed protein or oil contents were found, including two for DZ conditioned on protein (qDZ|proK‐1, qDZ|proF‐2); one for GC conditioned on protein (qGC|proM‐1); three for GT conditioned on protein (qGT|proM‐1, qGT|proA2‐1, qGT|proL‐1); three for TI conditioned on protein (qTI|proM‐1, qTI|proA2‐1, qTI|proF‐2); one for DZ conditioned on oil (qDZ∣oil K_1); one for GC conditioned on oil (qGC∣oilI_1); four for GT conditioned on oil (qGT∣oil A2_1, qGT∣oil F_1, qGTF_2, qGT∣oilD2_1); three for TI conditioned on oil (qTI∣oilA2‐1, qTI∣oilE‐1, qTI∣oilL‐1). Few epistatic interactions among loci were detected. These loci may be valuable for improving seed isoflavone, protein and oil contents.     

Dynamic quantitative trait loci underlies isoflavone accumulation in soybean seed

Most of quantitative trait loci (QTL) underlying soybean seed isoflavone contents were derived from the harvest stage of plant development, which uncover the genetic effects that were expressed in earlier seed developmental stages. The aim of this study was to detect conditional QTL associated with isoflavone accumulation during the entire seed development. A total of 112 recombinant inbred lines developed from the cross between ‘Zhongdou 27’ (higher seed isoflavone content) and ‘Jiunong 20’ (lower seed isoflavone content) were used for the conditional QTL analysis of daidzein (DZ), genistein (GT), glycitein (GC) and total isoflavone (TI) accumulations through composite interval mapping with mixed genetic model. The results indicated that the number and type of QTL and their additive effects for individual and total isoflavone accumulations were different among R3 to R8 developmental stages. Three unconditional QTL and six conditional QTL for DZ, four unconditional QTL and five conditional QTL for GT, six unconditional QTL and five conditional QTL for GC, six unconditional QTL and seven conditional QTL for TI were identified at different developmental stages, respectively. Unconditional and conditional QTL that affect individual and total isoflavone accumulations exhibited multiple expression patterns, implying that some QTL are active for long period and others are transient. Two genomic regions, Satt144‐Satt569 in linkage group F (LG F; chromosome 13, chr 13) for DZ, GC, GT and TI accumulations and Satt540‐Sat_240 in LG M (chr 07) for TI and GC accumulations, were found to significantly affect individual and total isoflavone accumulations in multiple developmental stages, suggesting that the accumulation of soybean seed isoflavones is governed by time‐dependent gene expression.     

Molecular loci associated with seed isoflavone content may underlie resistance to soybean pod borer (Leguminivora glycinivorella)

Soybean pod borer (SPB) (Leguminivora glycinivorella (Mats.) Obraztsov) causes severe loss of soybean (Glycine max L. Merr.) seed yield and quality in some regions of the world, especially in north‐eastern China, Japan and Russia. Isoflavones in soybean seed play a crucial role in plant resistance to diseases and pests. The aim of this study was to find whether SPB resistance QTL are associated with soybean seed isoflavone content. A cross was made between ‘Zhongdou 27’ (higher isoflavone content) and ‘Jiunong 20’ (lower isoflavone content). One hundred and twelve F_5:10 recombinant inbred lines were derived through single‐seed descent. A plastic‐net cabinet was used to cover the plants in early August, and thirty SPB moths per square metre were put in to infest the soybean green pods. The results indicated that the percentage of seeds damaged by SPB was positively correlated with glycitein content (GC), whereas it was negatively correlated with genistein (GT), daidzein (DZ) and total isoflavone content (TI). Four QTL underlying SPB damage to seeds were identified and the phenotypic variation for SPB resistance explained by the four QTL ranged from 2% to 14% on chromosomes Gm7, 10, 13 and 17. Moreover, eleven QTL underlying isoflavone content were identified, and ten of them were encompassed within the same four marker intervals as the SPB QTL (BARC‐Satt208‐Sat292, Satt144‐Sat074, Satt540‐Sat244 and Satt345‐Satt592). These QTL could be useful in marker‐assisted selection for breeding soybean cultivars with both SPB resistance and high seed isoflavone content.     

黑龙江省大豆籽粒异黄酮含量生态差异

大豆异黄酮由于其特有的生理保健功能而受到日益广泛的关注。为了明确黑龙江省大豆籽粒异黄酮含量的生态差异规律, 2005选定5个生态条件差异明显的试验点及4个大豆品种进行试验。结果表明, 年份、地点、基因型及基因型×环境互作对大豆籽粒异黄酮总含量及3种酸解处理后所得苷元的含量具有显著效应。2006年的大豆异黄酮总含量及3种苷元组分含量的平均值显著高于其他2年;大豆籽粒异黄酮含量不同基因型间及不同地点间均差异显著,大豆籽粒异黄酮总含量及其3种酸解后所得苷元含量与纬度呈极显著正相关。据此认为,大豆籽粒异黄酮含量在黑龙江省存在优势生产区, 对大豆籽粒异黄酮含量进行品质区划是可行的。     

Effects of Foliar Application of Elicitors on Red Clover Isoflavone Content

Red clover (Trifolium pratense L.) contains high concentrations of isoflavones, compounds that have received much interest lately due to their presumed benefits for human health. In this experiment we tested the possibility to induce isoflavone production in the foliage of two greenhouse‐grown red clover cultivars (‘Azur’ and ‘Start’) through the application of elicitor compounds. Foliar applications of different concentrations of acetic acid (50, 100, 250 and 500 mm ), yeast extract (1, 2, 3 and 4 g l^?1), and chitosan (125, 250, 500, and 1000 mg l^?1) were carried out on plants at the late vegetative stage, which were harvested 2 or 8 days after spraying. Concentrations of genistein, daidzein, formononetin and biochanin A were determined by high performance liquid chromatography. The two cultivars differed in isoflavone concentrations, ‘Azur’ having on average 36 % higher biochanin A, formononetin and total isoflavone concentrations than ‘Start’ (P < 0.05). A cultivar × sampling date interaction (P < 0.1) reflected a 20 % increase over time in total isoflavone concentration with ‘Azur’, which was not observed with ‘Start’. Effects of elicitors were limited, contrasts indicating overall, 12, 14 and 15 % greater total isoflavone concentration in yeast extract (P < 0.1), chitosan (P < 0.05) and acetic acid (P < 0.05)‐treated plants, respectively, than in untreated control plants. There were few differences between the various elicitors and none between concentrations of each elicitor.     

Effect of cold storage on vitamin C,phenolics and antioxidant activity of five orange genotypes [Citrus sinensis (L.) Osbeck]

The effect of cold storage on antioxidant profile and the antioxidant activity of five sweet orange genotypes [Citrus sinensis (L.) Osbeck], three blood (pigmented) varieties with different anthocyanin contents (‘Tarocco Messina’, ‘Tarocco Meli’ and ‘Moro’) and two blond varieties (‘Ovale’ and ‘Valencia late’), stored at 6 ± 1 °C for 65 d was investigated. During fruit storage, anthocyanins, hydroxycinnamic acids, vitamin C, flavanones and total phenolics were determined, and juice antioxidant capacity was measured by two different in vitro tests (DPPH scavenging activity and inhibition of induced linoleic acid peroxidation). The results showed an increase in anthocyanins, flavanones and hydroxycinnamic acids and a slight decrease in vitamin C in the blood oranges. Cold storage negatively affected flavanone concentration, while positively influenced vitamin C in blond orange varieties. Both antioxidant activity tests showed an increase in antioxidant capacity during storage caused mainly by phenolic accumulation (blood oranges) and vitamin C increase (blond oranges). Finally, correlations between antioxidant activity and total or individual phenolic components were examined.     

二月兰花瓣花色苷组成分析

为了研究二月兰花色苷成分组成特点,本试验以3份二月兰为试材,利用特征颜色反应确定二月兰花瓣的色素类型,并通过高效液相色谱-质谱联用技术(HPLC-Q-TOF-MS)分析花色苷种类及其含量。研究结果表明,3份二月兰花瓣中均不含类胡萝卜素,除白色花瓣以外,其余均含花色苷。蓝紫色花瓣的花色苷含量最高,粉色次之。在二月兰花瓣中共检测到了7种花色苷成分,通过与已有文献比对,推定其成分为矢车菊素3-芸香糖苷、锦葵素3,5-二葡萄糖苷、矢车菊素3-香豆酰葡萄糖苷-5-葡萄糖苷、矢车菊素3-p-香豆酰二葡萄糖苷-5-葡萄糖苷、矢车菊素3-阿魏酰槐糖苷-5-丙二酰葡萄糖苷、矢车菊素3-香豆酰槐糖苷-5-丙二酰葡萄糖苷、矢车菊素3-(6′-丙二酰葡糖苷-2′-(6′′-香豆酰-2′′-羟基苯甲酰葡糖苷))-5-丙二酰葡糖苷。粉色花瓣中仅含矢车菊素的花色苷衍生物,而蓝紫色花瓣中则含矢车菊素和锦葵素的花色苷衍生物。本研究为二月兰花色苷成分的进一步分离和鉴定工作提供了参考,同时也为二月兰花色的分子育种提供理论依据。     

Changes in hydrophilic and lipophilic antioxidant activity and related bioactive compounds during postharvest storage of yellow and purple plum cultivars

Eight plum cultivars (four dark-purple and four yellow) were harvested at the commercial ripening stage, and changes of fruit quality properties were evaluated during cold storage and subsequent shelf-life, with special emphasis on bioactive compounds (phenolics, anthocyanins and carotenoids) and antioxidant activity (TAA). From the eight plum cultivars, four showed the typical climacteric ripening pattern (‘Blackamber’, ‘Larry Ann’, ‘Golden Globe’ and ‘Songold’) while four behaved as suppressed-climacteric types (‘Golden Japan’ ‘Angeleno’, Black Diamond’ and ‘TC Sun’), the latter being described for the first time. At harvest, large variations in phytochemicals and antioxidant activity were found among cultivars in peel and pulp tissues, although phytochemical concentration and antioxidant activity were higher in the peel than in the flesh (2–40-fold depending on the bioactive compound). During storage, increases in total phenolics for all cultivars (peel and pulp), in total anthocyanin content in the peel of the dark-purple plums, and total carotenoids in the peel and pulp of the yellow cultivars were observed. This behaviour of the bioactive compounds was reflected in TAA changes, since hydrophilic-TAA (H-TAA) was correlated with both phenolics and anthocyanins, while lipophilic-TAA (L-TAA) was correlated with carotenoids. L-TAA comprised about 30–50% of the TAA in plum tissues. Carotenoids and phenolics (and among them the anthocyanins) could be the main lipophilic and hydrophilic compounds contributing to L-TAA and H-TAA, respectively. No significant loss of bioactive compounds and TAA occurred during prolonged plum storage. Moreover, for a better evaluation of the antioxidant potential of plums, the contribution to carotenoids should not be overlooked.     

Identification and validation of an over‐dominant QTL controlling soybean seed weight using populations derived from Glycine max × Glycine soja

Heterosis, or hybrid vigour, has been used to improve seed yield in several important crops for decades and it has potential applications in soybean. The discovery of over‐dominant quantitative trait loci (QTL) underlying yield‐related traits, such as seed weight, will facilitate hybrid soybean breeding via marker‐assisted selection. In this study, F₂ and F_2 : 3 populations derived from the crosses of ‘Jidou 12’ (Glycine max) × ‘ZYD2738’ (Glycine soja) and ‘Jidou 9’ (G. max) × ‘ZYD2738’ were used to identify over‐dominant QTL associated with seed weight. A total of seven QTL were identified. Among them, qSWT_13_1, mapped on chromosome 13 and linked with Satt114, showed an over‐dominant effect in two populations for two successive generations. This over‐dominant effect was further examined by six subpopulations derived from ‘Jidou12’ × ‘ZYD2738’. The seed weight for heterozygous individuals was 1.1‐ to 1.6‐fold higher than that of homozygous individuals among the six validation populations examined in different locations and years. Therefore, qSWT_13_1 may be a useful locus to improve the yield of hybrid soybean and to understand the molecular mechanism of heterosis in soybean.     

The transition time from the juvenile phase to the adult phase differs in soybean cultivars ‘Enrei’ and ‘Peking’

Plants develop juvenile phase to adult phase in vegetative stage. Although soybean is a very important crop worldwide, there has been only one study of the juvenile–adult phase change. In this study, we determined that the juvenile–adult phase change occurred at different stages in two soybean cultivars that differ in their photosensitivity. Cultivar ‘Enrei’ (E1e2e3E4) is weakly photosensitive and cultivar ‘Peking’ (E1E2E3E4) is strongly photosensitive. In ‘Enrei’, the leaf size gradually increased at a constant leaf position regardless of the difference in day length. In ‘Peking’ plants transferred to short‐day conditions at several leaf development stages, leaf size gradually increased at different leaf positions. Expression of miR156 by ‘Enrei’ transferred to short‐day conditions had nearly the same pattern as that of ‘Enrei’ grown under long‐day conditions. In ‘Peking’, the expression of miR156 had different patterns in younger leaves of plants subjected to either a short‐day treatment or long‐day conditions. These results indicate that the E2 and E3 loci that regulate photosensitivity also regulate the expression of miR156 and the juvenile–adult phase change in soybean.     

Mapping quantitative trait loci (QTLs) underlying seed vitamin E content in soybean with main,epistatic and QTL × environment effects

Soybean (Glycine max (L.) Merr.) seed contains small amounts of tocopherol, a non‐enzymatic antioxidant known as lipid‐soluble vitamin E (VE). Dietary VE contributes to a decreased risk of chronic diseases in humans and has several beneficial effects on resistance to stress in plants, and increasing VE content is an important breeding goal for increasing the nutritional value of soybean. In this study, quantitative trait loci (QTLs) underlying VE content with main, epistatic and QTL × environment effects were identified in a population of F_5 : 6 recombinant inbred lines from a cross between ‘Hefeng 25’ (a low‐VE cultivar) and ‘OAC Bayfield’ (a high‐VE cultivar). A total of 18 QTLs were detected that showed additive main effects (a) and/or additive × environment interaction effects (ae) in different environments. Moreover, 19 epistatic pairs of QTLs were found to be associated with α‐tocopherol (α‐Toc), γ‐tocopherol (γ‐Toc), δ‐tocopherol (δ‐Toc) and total VE (TE) contents. The QTLs identified in multienvironments could provide more information about QTL by environment interactions and could be useful for the marker‐assistant selection of soybean cultivars with high seed VE contents.     

High-performance liquid chromatographic analysis of anthocyanins in induced amphidiploids of Iris laevigata Fisch. × I. ensata Thunb.

Summary Anthocyanins of colchicine-induced amphidiploids of I. laevigata × I. ensata were analyzed by the HPLC procedure and compared with those of parental species and F₁ hybrids of I. laevigata × I. ensata. The amphidiploids were characterized by seventeen anthocyanins, and exhibited all anthocyanins of the parental species and the F₁ hybrids. Malvidin 3RGac5G and petunidin 3RGac5G in anthocyanins detected were major pigments in the amphidiploids, which exhibited strongly the malvidin 3RGac5G-petunidin 3RGac5G type of I. ensata rather than the petunidin 3RGac5G-malvidin 3RGac5G type of I. laevigata. Moreover, a higher degree of anthocyanin content was observed in the amphidiploids and the F₁ hybrids. Namely, the anthocyanin content of the amphidiploids and the F₁ hybrids were 2.81 and 2.45 times as much as that of the mid-parent. This implies that there is a complementary effect on anthocyanin synthesis of parental genes in them. Finally, the utility of the amphidiploids was discussed.Contribution from the Laboratory of Plant Breeding, Faculty of Agriculture, Miyazaki Univ., No. 64.     

An HPLC investigation of flower colour and breeding of anthocyanins in species and hybrids of Alstroemeria

The tepals of 28 Chilean species of Alstroemeria and 183 interspecific hybrids were analysed for anthocyanin content by high-performance liquid chromatography (HPLC). The anthocyanins were identified as 3-rutinosides of 6-hydroxydelphinidin, 6-hydroxyeyanidin, cyanidin, and delphinidin and 3-glyeosides of cyanidin and delphinidin, some of which were acylated with malonic acid. Comparisons of the anthocyanin contents in parents and offspring showed that no anthocyanidin or acylation pattern was dominant, and that offspring values were close to mid-parent values for the percentage of malonated anthocyanins, whereas the inheritance of cyanidin, 6-hydroxycyanidin, and delphinidin seems more complicated. Flower colour, hue, and intensity were measured by CIELab in fresh tepals and compared with their anthocyanin content and the estimated flavonoid concentrations. Colour intensity was positively correlated with anthocyanin concentration. Compared with flowers containing exclusively cyanidin 3-glycosides, the hues of flowers with delphinidin 3-glycosides were bluer and with 6-hydroxycyanidin 3-glycosides redder, respectively. Both malonation of anthocyanin and co-pigmentation with flavonoids caused a shift to bluish hues, irrespective of the anthocyanidins. By quantifying both chemical and colorimetric characteristics a model for the effect of anthocyanin on Alstroemeria flower colour was established. Breeding of new cultivars of Alstroemeria is discussed.     

Increased anthocyanin content in purple pericarp × blue aleurone wheat crosses

Due to their antioxidant activity, anthocyanins are of increasing interest for nutritionists, food scientists and plant breeders. Anthocyanins in wheat grains are expressed in either the pericarp or aleurone layer. Previous studies revealed that different anthocyanins are present in wheat varieties carrying genes for either the purple pericarp or the blue aleurone trait. Progeny from crosses between red‐, purple‐ and blue‐grained wheat varieties were selected over several cycles for grain colour by visual scoring. Bulked F₅ grains were evaluated for their total anthocyanin content by UV‐VIS spectrophotometry and HPLC‐MS. The results demonstrate that it is possible to increase the anthocyanin content by the combination of the different genetic backgrounds for purple pericarp and blue aleurone, even though the majority of progeny were within the range of the purple‐ and blue‐grained check varieties. Visual scoring for grain colour is efficient, reliable and fast for selection in early breeding generations. Advanced breeding lines with high anthocyanin content can be identified by simple extraction methods and spectrophotometric measurements.     

Genetic analysis and identification of two soybean mosaic virus resistance genes in soybean [Glycine max (L.) Merr]

Soybean mosaic virus (SMV) commonly affects soybean production worldwide, and the SC18 strain has been widespread in China. This study aimed to characterize and map the SC18 resistance genes present in soybean cultivars ‘Kefeng No. 1’ and ‘Qihuang 22’. Inheritance analysis revealed that two independent single dominant genes in Kefeng No. 1 and Qihuang 22 confer resistance to SC18. Using simple sequence repeat (SSR) markers and bulked segregant analysis, the Kefeng No. 1 and Qihuang 22 resistance genes were located on soybean chromosomes 2 and 13, respectively. We further screened two populations of recombinant inbred lines with 32 SSR markers in the target region, where the resistance gene in Kefeng No. 1 was fine mapped to an 80‐kb region containing six putative genes. Sequence and expression analyses of these genes revealed that SMV resistance in Kefeng No. 1 was probably attributable to three of the candidate genes (i.e. Glyma.02G127800, Glyma.02G128200 and Glyma.02G128300). Collectively, the results of this study will greatly facilitate the cloning of SC18 resistance genes and marker‐assisted breeding of SMV‐resistant soybean cultivars.