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.