Article
QTLs for soybean seed isoflavones are linked to laccases, BANYULS and the MBW complex
Xueying Li 1, #, Jun Zhang1, 2, #, Liangyu Chen 1, Zhenhui Wang1. Cheng Yu1, Xiao Han 1, Yang Song1, Dan Yao 3, Sujie Fan1, Jeremy Murray4, Songnan Yang1, *, Jian Ma1, * and Jian Zhang1,5*
1 College of Agronomy, Jilin Agricultural University, 130118 Changchun, Jilin, China; xueyingl@jlau.edu.cn (X.L.); zhangjun@jlau.edu.cn (Jun.Z.); 20200045@mails.jlau.edu.cn (L.C.); wzhjlau@163.com(Z.H.W.); yucheng199811@163.com (C.Y.); hanxiaoyy@jlau.edu.cn (X.H.); songyangjlnd@163.com (Y.S.); fansujie@jlau.edu.cn (S.F.)
2 National Crop Variety Approval and Characteristic Identification Station, Jilin Agricultural University, 130118 Changchun, Jilin, China
3 College of Life Science, Jilin Agricultural University, 130118 Changchun, Jilin, China; dyao@jlau.edu.cn (D.Y.)
4 CAS Center for Excellence in Molecular Plant Science(CEMPS), Chinese Academy of Science, Shanghai, China; jmurray@sippe.ac.cn (JM)
5 Department Biology, University of British Columbia, Okanagan, Kelowna, BC, Canada
* Correspondence: soy@jlau.edu.cn (S.Y.); majian197916@jlau.edu.cn (J.M.); zhangjian@jlau.edu.cn (Jian.Z.)
# Xueying Li and Jun Zhang contributed equally to this work
Abstract: Isoflavones are secondary metabolites present in seeds of soybean [Glycine max (L.) Merr.] which have been recognized their benefit to human health. In this study, QTL mapping for soybean isoflavone gylcones including daidzin, glycitin and genistin and total isoflavones content was performed in population of 178 F2:6 recombinant inbred lines (RILs) which was generated from cross between varieties Jinong17 and Jinong18. A genetic linkage map covering 1248 cM was constructed using the simple sequence repeat (SSR) molecular markers. The results revealed 22 isoflavone- related QTLs, 5 for daidzin, 7 for genistin, 6 for glycitin, and 4 for total isoflavone content. Seven of these represent new QTLs. Twenty candidate genes were identified, including eight laccases with presumed role in lignin biosynthesis, and the transcriptional regulator BANYULS and all three components of the MYB-bHLH-WD40 (MBW) complex that regulate its expression. These findings suggest that alterations in lignin and proanthocyanidin metabolism influence isoflavone accumulation in seeds. These leads might be helpful in the efforts to breed new soybean varieties with improved isoflavone composition and content.
Keywords: soybean isoflavones; genetic mapping; quantitative trait loci; simple sequence repeat.
1. Introduction
Soybean is an important cash crop in many parts of the world. Soybean seeds are a good source of isoflavones, metabolites that are widely considered to be beneficial to human health as potential anticancer agents, and as therapy to reduce menopausal symptoms [1,2]. Soybean isoflavones are found in different parts of the plant, particularly in the hypocotyl, cotyledon, and seed coat [3]. The chemical structure of isoflavones is similar to that of female estrogens, and consequently they are often called phytoestrogens. Daidzin, glycitin and genistin are the main isoflavone compounds in soybean seeds[4]. Daidzin is a specific inhibitor of aldehyde dehydrogenase which may help to suppress ethanol consumption[5], and has also been shown to help prevent bone loss[6], provide neuroprotection and neuronutrition [7], and to have anti-oxidant and anti-inflammatory activities [8,9]. Glycitin may be useful for suppressing cartilage destruction in osteoarthritis [10] and protecting lung tissues from lipopolysaccharide-induced inflammation [11]. Genistin was also shown to be a potent anti-adipogenic and anti-lipogenic agent [12]. From the plant perspective, isoflavones function in plant disease resistance, plant-environment interactions [13,14], and act as important signals in interactions with beneficial N-fixing bacteria [15]. Soybean is also an important anti-bacterial plant protection element [16,17]. It plays an important role in the defensive response that protects plants from UV-induced damage [18-20]. In addition, soybean isoflavones are signaling substances between plants and microorganisms [21].It was found that the levels of genistein, daidzein and daidzein increased significantly when Sclerotinia sclerotioruminfected soybean [22].Other studies showed that the content of isoflavone metabolites in soybean leaf tissue changed significantly before and after aphid attack, indicating that the substances had a relationship with soybean aphid resistance [23,24].These research results indicated that increasing of soybean isoflavone content would be accompanied by the introduction of other good traits.
Since soybean products are rich source of isoflavones, increasing the total isoflavone content in seeds has become an important objective in soybean breeding.
The content and composition of soybean isoflavones are quantitative traits controlled by multiple genes and influenced by the environment [25]. Isoflavonoids are produced from the p-coumaryl CoA by the action of Chalcone Synthase (CHS) and/or Chalcone Reductase (CHR), followed by the sequential action of Chalcone Isomerase (CHI) and Isoflavone Synthase (IFS) [26].The availability of a soybean genetic linkage map has greatly boosted soybean genetic research on isoflavonoids. Liang (2010) identified six QTLs for isoflavone content localized in linkage groups J, N, D2, and G by using an RIL population [27].WANG et al. [28]detected 34 QTLs for isoflavone content in another RIL population, of which 23 were newly discovered loci, with one marker, qGTD2_1, explaining 3.4-11.0% of the phenotypic variation. Zhang et al. [29] detected 21 QTLs for isoflavones content, distributed on 8 chromosomes, which explained 4.48-8.83% of the phenotypic variation; of them, only one QTLs showed negative effects on isoflavones content. To date, in addition to known genes in the isoflavone synthesis pathway, researchers have identified 61 daidzein-related QTLs, 68 genistein-related QTLs, 71 glycitin -related QTLs and 62 total isoflavone content-related QTLs, which are recorded in the Soybase Genome Database (http://www.soybase.org). There have been some reports on transcription factors that regulate key enzymes of isoflavone synthesis. Chu et al. [30] reported one candidate gene,GmMYB29 , that is significantly correlated with isoflavone content and can activate IFS2 and CHS8 promoters. Vadivel et al. [31] studied transcription factor GmMYB176 , and showed it regulates isoflavones by activating the expression of CHS8 . However, the available markers density and population sizes used in previous studies were not sufficient to indentify the underlying genes [32]. Identification of QTLs affecting the isoflavone content will provide a theoretical basis for soybean quality improvement through molecular marker-assisted breeding and can provide leads on the key components of the pathway, and how they are regulated. Newly identified QTLs from our study would advance the understanding of the epistatic interactions associated with isoflavones content. SSR markers have multiple alleles in a single locus and are highly polymorphic, which is very beneficial to genetic research. In the field of soybean research, polymorphism of SSR molecular markers has been fully confirmed [33]. RIL population consists of F2 individuals who continuously self-cross, sib mate, or randomly cross within the population until the genotypes of the individuals in the family are completely homozygous.
In this study, a genetic linkage map was constructed using 58 SSR molecular markers and population of 178 F2:6 RILs, and QTLs were mapped for daidzin, glycitin, genistin and total isoflavone content. We present new QTLs and identify candidate genes and discuss their potential for metabolic engineering of soybean seed isoflavones.
2. Results
2.1. Variation of seed isoflavone content
The mean isoflavone content in dry seeds of the 178 RILs and the parental lines is shown in Table 1. Jinong17 had significantly higher values for all isoflavone contents compared to Jinong18, indicated that the two parents differed in the genes controlling individual and total isoflavone contents. The average total isoflavone content was 1235.6 µg/g, or 1.2% of the dry weight. The three main isoflavones, daidzin, glycitin and genistin, accounted for more than 90% of the total isoflavone content. The average content of the three components in the population was daidzin > genistin > glycitin.
The population exhibited positively skewed distribution for individual and total isoflavones with values ranging from 0.068 to 2.18, indicating their seed isoflavone composition and content was more like Jinong17 which has a higher isoflavones content. The peak shape of daidzin and glycitin was platykurtic because their kurtosis values were negative, while genistein and the total content of isoflavones had a sharp distribution.
Table 1. Isoflavone content of the RIL population and their parents Jinong17 and Jinong18.