Introduction

Lipoxygenase (Lox) belongs to a large family of enzymes known as non-heme iron-containing dioxygenases; Lox is widely found in animals, plants, and fungi and is particularly abundant in seeds (Shibata et al. 1994; Porta and Rocha-Sosa 2002; Liavonchanka and Feussner 2006). Most Lox-generated metabolites from different reactions are collectively called oxylipins, which play important roles in the regulation of plant growth and development (Berg et al. 2006), senescence (Seltmann et al. 2010), wounding and stress response (Veronico et al. 2006), and plant defense (Wang et al. 2008). The Lox content in wheat grain is very low, but the reactive oxygen species produced during enzymatic oxidation of fat not only can degrade the pigment, which influences the color of flour, noodles, steamed bread, and bread flour products (Manna et al. 1998; Loiseau et al. 2001), but also can effectively improve the nutritional and processing qualities of flour products (Shiiba et al. 1991; Trufanov et al. 2007; Permyakova et al. 2010; Permyakova and Trufanov 2011).

Flour color is a key quality trait for wheat and plays a critical role in determining end-use products and marketing (He et al. 2004; Fu et al. 2008). Flour contains abundant natural pigments (lutein and carotenoids), which significantly influence the color and nutrition of noodles and steamed bread flour products (Crawford et al. 2011). Previous studies showed that flour color is attributed to natural carotenoid pigments present in seeds and is affected by enzymes, such as Lox (Manna et al. 1998; Loiseau et al. 2001; Trufanov et al. 2007), phytoene synthase (Psy) (He et al. 2009; Crawford et al. 2011), and polyphenol oxidase (Baik et al. 1995; Chang et al. 2007) during the processing of end-use products.

Flour pigment is mainly degraded during milling and processing of flour products. Milling determines mechanical loss of pigments mainly located at the external layer of the kernel with a low quantity for pasta processing, in which the maximal pigment degradation by Lox activity occurs (Borrelli et al. 1999). Lox is one of the most important enzymes contributing to semolina bleaching (Manna et al. 1998; Borrelli et al. 1999). Wheat cultivars with low Lox activity may have high nutritional value (Žilić et al. 2010). The treated flour with Lox isozyme showed shorter dough development time and less resistance after peak time on a mixogram than the control flour without enzyme treatment (Shiiba et al. 1991). The negative correlation between specific Lox activity and dough extensibility explains the ambivalent role of this enzyme in gluten and dough quality (Permyakova et al. 2010). Lox activity is negatively correlated with flour strength (Trufanov et al. 2007). This finding proved that Lox activity not only affects the color of flour and flour products, but also is closely related with gluten strength and dough rheological properties. Lox is one of the most important factors affecting the storage characteristics of bread wheat. Leenhardt et al. (2006) found that low Lox activity or Lox gene deletions can effectively decrease lipid oxidation, thereby reducing grain oxidative deterioration and extending the storage period. Consequently, low Lox activity in bread wheat cultivars of grain or flour increases storage time, thereby enhancing the commercial value (Leenhardt et al. 2006; Borrelli et al. 2008).

Given the rapid progress of modern molecular and biological techniques, many Lox genes have been mapped and cloned in cereal crops, including barley (Hordeum vulgare L.) (van Mechelen et al. 1999), durum wheat (Triticum turgidum L.) (Carrera et al. 2007; Garbus et al. 2009; Verlotta et al. 2010), and bread wheat (Triticum aestivum L.) (Feng et al. 2010, 2012; Geng et al. 2012; Garbus et al. 2013). To date, partial or complete sequences of eight Lox genes, namely, Lpx-A3 (DQ474242 and DQ474244), Lpx-A1 - like (FJ518909), Lpx-B1.1 (DQ474240), Lpx-B1.2 (DQ474241), Lpx-B1.3 (HM126469), Lpx-B3 (DQ474243), Lpx-A2 (DQ448002), and Lpx-B2 (DQ448001), have been cloned from durum wheat and are located on chromosomes 4A, 4B, 5A, and 5B (Hessler et al. 2002; Carrera et al. 2007; Zhang et al. 2008 Garbus et al. 2009; Verlotta et al. 2010). Three alleles have also been identified for Lpx-B1.1, Lpx-B1.1a (HM126466), Lpx-B1.1b (HM126468), and Lpx-B1.1c (HM126470) (Verlotta et al. 2010). Four Lpx sequences have been identified and annotated in T. aestivum genome, including Lpx pseudogene in the A genome (Lpx-A1 - like; KC679303), LpxB1.1 a (KC679300) and LpxB1.2 (KC679301) genes in the B genome, and Lpx-D1 (KC679302) in the D genome (Garbus et al. 2009, 2013). Feng et al. (2010, 2012) cloned the gDNA or cDNA sequences of three Lox genes, i.e., TaLox1 (GU167920/GQ166692), TaLox2 (GU167921/GQ166691), and TaLox3 (HQ913602). Amplified gDNA sequences of TaLox-B1 from bread wheat cultivars with diverse grain Lox activities revealed the presence of two alleles, i.e., TaLox-B1a (HQ406779) and TaLox-B1b (HQ406780); one single-nucleotide polymorphism (SNP) has been detected in the third exon of TaLox-B1a and TaLox-B1b (Geng et al. 2012).

Although several Lox genes have been cloned, bread wheat contains a large Lox gene family and requires more Lox genes to precisely improve wheat quality in terms of Lox activity (Hart and Langston. 1977; Li et al. 1999). The Lox activity of bread wheat is also mainly influenced by Lox genes on chromosome 4 (Hart and Langston. 1977; Li et al. 1999; Verlotta et al. 2010; Garbus et al. 2013). Therefore, the current study focused on cloning Lox genes on chromosome 4B and identified the molecular characterization in bread wheat, as well as evaluated the association of these genes with Lox activity in Chinese bread wheat cultivars.

Materials and methods

Plant materials

Four Chinese winter wheat cultivars (Yunong 201, Zhoumai 18, Zhengmai 004, and Gaocheng 8901) were used for cloning TaLox genes in this study. The wheat plants were grown in a greenhouse under the condition of 16 h light at 25 °C (day) and 8 h dark at 22 °C (night). The root, stem, and leaf of each cultivar were collected at three-leaf seedlings and stored at −80 °C until analysis. The seedlings of Yunong 201 at the three-leaf stage was exposed at four different stress treatments of 4 °C low temperature, 42 °C high temperature, 100 mmol L−1 NaCl, and 20 % PEG 6000 treatments for 0, 1, 3, 6, 12, and 24 h, consecutively. A whole set of Chinese Spring nullisomic–tetrasomic lines and ditelosomic lines as well as a F 10 RIL population (UC 1110 × PI 610750) composed of 187 lines, kindly provided by Prof. Jorge Dubcovsky in University of California, Davis, were used to map the TaLox genes on chromosome location in bread wheat. A F 4:5 RIL population encompassing 110 lines, derived from Zhoumai 16 (with relatively high Lox activity) × Xianyang 83104 (with relatively low Lox activity), was used to examine the association of allelic variation of Lox genes on 4BS with Lox activity. A total of 136 bread wheat cultivars mainly from the wheat-producing area of China were used to evaluate the association of allelic variation of Lox genes on 4BS with Lox activity.

Field trials

During the 2011–2012 and 2012–2013 cropping seasons, the F 4:5 RIL population encompassing 110 lines and their parents were sown at the Zhengzhou Scientific Research and Education Center of Henan Agricultural University, China, and 136 Chinese winter wheat cultivars and advanced lines (Supplemental 1) were sown at Anyang (N36.1°, E114.5°), Zhengzhou (N34.9°, E113.6°), and Zhumadian (N32.9°, E114.1°) in randomized complete blocks with two replicates. Each plot comprised two 2 m rows spaced 25 cm apart with 75 plants in each row. Test plots were managed according to local practices. The field trials were kept free of weeds and diseases by broad-range herbicides and fungicides. After harvest, grain samples were cleaned. Falling number tests indicated that the plants were free of sprouting damage (data not shown).

Assays of grain Lox activity and colorimeter parameters

The total Lox activity in the mature seeds of the wheat cultivar was measured by the accumulation of hydrogen peroxide in the Lox reaction as described by Surry (1964). Extraction was performed as described by Geng et al. (2011) and Feng et al. (2012) with minor modifications. A single mature seed was crushed and suspended in 500 µL of cold extraction buffer (40 mM Na phosphate, pH 6.8). The homogenate was incubated on ice for 1 h with vortexing every 20 min. After centrifugation at 4 °C and 12,000 rpm for 10 min, the supernatant containing Lox was used for subsequent assay of Lox activity. The protein concentration of the enzyme solution was determined using the ELx808 model enzyme-labeled instrument (BioTek Instruments Inc., USA) with bovine serum albumin as standard. Negative controls of the samples were prepared by inactivating the crude enzymes by heat treatment at 100 °C for 10 min. The crude enzymes were incubated at 30 °C for 3 min in a cocktail of 2 mM linoleic acid (99 %), 0.05 % Tween 20, and 1 M NaOH. The reaction was stopped by adding an equal volume of 0.1 M NaOH. Lox activity was determined by measuring the conjugated diene absorption at 234 nm with an ultraviolet (UV)–visible spectrophotometer [UV-2600, Unico (Shanghai) Instruments Co., Ltd., Shanghai, China]. A 1-cm-thick quartz cuvette was used to measure the solution. One unit of Lox activity was defined as an increase in absorbance at 234 nm/min/g of whole wheat meal under assay conditions. For each data, Lox activity was determined in duplicate extracts of a single seed meal with parallel spectrophotometric measurements and mean values. If the coefficient of variation was >10 % for spectrophotometric assay of Lox activity in the duplicates, the test was repeated.

Wheat grains of each cultivar were milled into flour in a Chopin CD1 laboratory mill (Chopin Technologies, Paris, France) according to the method of Chen et al. (2013a). Flour color parameters of 136 Chinese wheat cultivars surveyed were measured by Colorimeter CR-410 (Konica Minolta Holdings, Inc.), i.e., L* (lightness), a* (redness) and b* (yellowness).

Cloning and sequence analyses of TaLox-B2 and TaLox-B3 genes

The cDNA sequences of bread wheat TaLox1 (GQ166692) and TaLox2 (GQ166691) genes were used for BLAST search against the wheat expressed sequence tag (EST) database in GenBank. All wheat ESTs sharing high similarity with the reference gene were subjected to overlapping sequence assembly (E value <10−50, score >500 bp, and identity >95 %; NCBI 2012). These ESTs were aligned, and the 5′ or 3′ end nucleotide sequences were compared by DNAMAN Version 6.0 software (http://www.softlandsl.com/free/dnaman+6+full.html). Subsequently, 20 primer sets were designed in the different conserved regions of the alignment sequences to obtain new sequences of TaLox genes in bread wheat. Finally, one primer set of Lox-P1 successfully amplified a full-length Lox cDNA (Table 1). Based on the Lox cDNA sequences cloned, three chromosome-specific primer sets of Lox-P2, Lox-P3, and Lox-P4 (Table 1) were redesigned to amplify the full-length gDNA sequence. All primers were designed by Premier Primer 5.0 software (http://www.primer-premier.findmysoft.com/) and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

Table 1 Primer sets used for identification of TaLox-B2 and TaLox-B3 genes in this study
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Genomic DNA was isolated from seedlings following a method modified from that of Lagudah et al. (1991) and Chen et al. (2012). PCR amplifications were performed in a BioRad-S1000 or ABI 9700 thermal cycler. Reactions were conducted in a 20 μL volume containing 50 ng of genomic DNA, 5 pmol of each primer, 200 μmol of each dNTP, 1× reaction buffer (50 mmol KCl, 10 mmol Tris–HCl, 1.5 mmol MgCl2, pH 8.4), and 1 unit of Taq DNA polymerase (TIANGEN Biotech Co., Ltd., Beijing). The high-fidelity Taq DNA polymerase (Takara Bio, Inc.) was used to generate amplification fragments for cloning TaLox genes. Reaction conditions were 94 °C for 5 min, followed by 35 cycles of 94 °C for 45 s, 60 °C to 66 °C (specific annealing temperature of each primer pair is presented in Table 1) for 45 s, 72 °C for 90 s, and a final extension of 10 min at 72 °C. PCR products were separated and analyzed on 1.0–1.5 % (w/v) agarose gels, stained with ethidium bromide, and visualized with UV light. Targeted fragments with expected sizes based on the primer-binding sites in the DNA sequence were purified from the gels using Quick DNA extraction kit [TaKaRa Biotechnology (Dalian) Co., Ltd.]. Purified products were ligated into pMD19-T vector [TaKaRa Biotechnology (Dalian) Co., Ltd.] and transformed into cells of the Escherichia coli DH-5α strain. Plasmids containing targeted fragments were extracted by Plasmid Rapid Isolation Kit (Beijing CoWin Biotech Co., Ltd.) and sequenced from 10 clones for each sample by Sangon Biotech (Shanghai) Co., Ltd.

Complete multiple alignments of sequences and translations of nucleotides into amino acid sequences were performed by DNAMAN Version 6.0 software (http://www.softlandsl.com/free/dnaman+6+full.html). Reliability of sequencing results was verified by examining the sequence chromatograms using Chromas Version 1.4.5 and FinchTV 1.5.0 (http://www.geospiza.com/Products/finchtv.shtml). Phylogenetic trees of Lox genes were obtained by MEGA 6.06 software (http://www.megasoftware.net/history.php). Neighbor-joining tree was bootstrapped using five distance methods: (1) observed divergence, (2) Kimura, (3) Jukes–Cantor, (4) maximum likelihood with Poisson, and (5) maximum likelihood with Hasegawa–Kishino–Ya.

Quantitative RT-PCR analysis of TaLox-B2 and TaLox-B3 genes

Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and first-strand cDNA was synthesized using the SuperScript™ First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Gene-specific primers Lox-P5 and Lox-P6 were designed to examine the expression levels of TaLox-B2 and TaLox-B3 genes. Wheat β-actin gene (Table 1) was selected as internal control with the primer Wac (Chen et al. 2013b).

The expression profiles of TaLox-B2 and TaLox-B3 genes were measured using the cDNA samples from different tissues (root, stem, and leaf) of the three wheat cultivars (Zhoumai 18, Zhengmai 004, Gaocheng 8901) as well as from seeds of different developmental stages of cultivar Aikang 58. Quantitative RT-PCR was performed using the Bio-Rad iQ5 Sequence Detection System (Applied Biosynthesis, CA, USA) with the SYBR Premix Ex Taq II [TaKaRa Biotechnology (Dalian) Co., Ltd.]. The PCR conditions consisted of an initial denaturation step for 2 min at 94 °C, followed by 40 cycles of 10 s at 95 °C, 10 s at 58 °C, 20 s at 72 °C, and a final extension of 10 min at 72 °C. Wheat β-actin gene was selected as the internal control. The 2−△△CT method was used to normalize and calibrate transcript values relative to the endogenous β-actin control. Six independent samples with triplicate repeats were analyzed.

Statistical analysis

For the 136 Chinese wheat cultivars and 110 F4:5 RIL population, the Lox activity of each genotype was measured in cropping seasons and averaged to examine the association between Lox activity and newly developed markers. The Lox activities of all materials were measured with two replications and averaged for statistical analyses. Analysis of variance was conducted by PROC MIXED in the Statistical Analysis System (SAS Institute 2000) with genotype classes as categorical variables to derive the mean Lox activity for each class and test the significance level for the two classes. The differences in Lox activities among genotypes with different PCR band profiles (660, 677, or 788 bp) were tested by least significant range multiple comparisons.

Results

Discovery of TaLox-B2 and TaLox-B3 genes in bread wheat

Based on a set of gDNA or cDNA sequences of Lox genes of durum wheat (DQ474240, HM126467, and HM126469), bread wheat (HQ913602, GU167920, and GU167921), and barley (L37358, L37359, and L35931), a new cDNA sequence of TaLox gene with 2586 bp was generated with the amplification of primer set Lox-P1 (Table 1) in Chinese cultivar Yunong 201. The full-length gDNA sequence of this new TaLox gene was assembled with the overlapped fragments by three successful amplifications of primer sets Lox-P2, Lox-P3, and Lox-P4 (Table 1). Physical mapping by a set of nullisomic–tetrasomic lines and ditelosomic lines of Chinese Spring indicated that this new TaLox gene was located on the short arm of chromosome 4B in Chinese Spring (Fig. 1). Therefore, this new TaLox gene was designated as TaLox-B2 because of the existence of TaLox1 (designated as TaLox-B1 gene in this study) on 4BS of bread wheat according to the nomenclature of McIntosh et al. (2007) and Geng et al. (2012). Sequence analysis of gDNA and cDNA of TaLox-B2 gene indicated that this gene was composed of seven exons and six introns (Fig. 2). TaLox-B2 gene with 4267 bp showed sequence similarities of 78.3 % with TaLox1 (GU167920), 60.4 % with TaLox2 (GU167921), and 60.9 % with TaLox3 (HQ913602) at the DNA levels. The deduced amino acid sequence showed that TaLox-B2 gene could encode an 861-aa protein and contained a Lox superfamily domain at the 170–838 interval as predicted using Pfam 27.0 software (http://www.pfam.xfam.org/).

Fig. 1
figure 1

Physical mapping of TaLox-B2 and TaLox-B3 genes by Lox-B23 marker in nullisomic–tetrasomic lines and ditelosomic lines in the Chinese Spring background. M: DL2000; 01: Chinese Spring; 02: N1A-T1D); 03: N1B-T1A; 04: N1D-T1B; 05: N2A-T2D; 06: N2B-T2A; 07: N2D-T2B; 08: N3A-T3D; 09: N3B-T3D; 10: N3D-T3A; 11: N4A-T4D; 12: N4B-T4A; 13: N4D-T4B; 14: N5A-T5B; 15: N5B-T5A; 16: N5D-T5B; 17: N6A-T6B; 18: Dt-4DL; 19: Dt-4DS; 20: Dt-4AL; 21: Dt-4AS; 22: Dt-4BS; 23: Dt-4BL

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Fig. 2
figure 2

Schematic representation of the structures of the Lox genes in durum and bread wheat. Filled boxes represent exons and lines represent introns

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However, when the primer set of Lox-P3 was used to amplify the TaLox-B2 gene, two fragments (approximately 1500 and 1400 bp) with different sizes could be obtained simultaneously in the Chinese cultivar Yunong 201. Sequences of both fragments after ligation with T-Easy vector indicated that the approximately 1500-bp fragment belonged to the TaLox-B2 gene, whereas the approximately 1400-bp fragment did not belong to any known TaLox genes. A new TaLox gene was also generated by sequencing several subclones encompassing overlapped sequences amplified with primer sets TaLox-P1, TaLox-P2, and TaLox-P3. The physical mapping by a set of nullisomic–tetrasomic lines and ditelosomic lines of Chinese Spring indicated that this new TaLox gene was also located on the short arm of chromosome 4B (Fig. 1). Therefore, this new TaLox gene was designated as TaLox-B3 because of the existence of TaLox-B1 and TaLox-B2 genes according to the nomenclature of McIntosh et al. (2007) and Geng et al. (2012). Linkage analysis further showed that TaLox-B3 gene was close to SSR marker Barc20 (GrainGenes breakpoint interval report: 4BS8-0.57-0.81) and was located on chromosome 4BS (Fig. 3). Sequence analysis of gDNA and cDNA of TaLox-B3 gene indicated that this gene consists of seven exons and six introns (Fig. 2). TaLox-B3 gene with 4246 bp showed sequence similarities of 79.3 % with TaLox1, 62.1 % with TaLox2, 62.5 % with TaLox3, and 84.8 % with TaLox-B2 at the DNA levels. However, the deduced amino acid sequence indicated that the TaLox-B3 gene showed sequence similarities of 97.8 % with the TaLox-B2 gene and encoded a protein of the same size (861 aa), which also contained a domain of lipoxygenase superfamily at the same interval with TaLox-B2 gene (170–838).

Fig. 3
figure 3

Linkage map constructed with TaLox-B3, SSR markers and DArT markers on the chromosome 4B in the RIL population derived from UC 1110/PI 610750

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Sequence blast of TaLox-B1, TaLox-B2 and TaLox-B3 genes in URGI database (http://www.wheat-urgi.versailles.inra.fr/) indicated that they were identical to three different contigs, i.e., IWGSC_chr4BS_ab_k71_contigs_longerthan_200_4957383 (TaLox-B1 gene), IWGSC_chr4BS_ab_k71_contigs_longerthan_200_4963368 (TaLox-B2 gene) and IWGSC_chr4BS_ab_k71_contigs_longerthan_200_4959255 (TaLox-B3 gene), respectively. It suggested that TaLox-B1, TaLox-B2 and TaLox-B3 genes were not allelic and belonged to different genes.

Molecular characterizations of TaLox-B2 and TaLox-B3 genes in Chinese bread wheat cultivars

Furthermore, when primer set Lox-P3 was used to amplify TaLox-B gene, three different types of fragments appeared in various Chinese wheat cultivars: type I with two fragments of approximately 1500 and 1400 bp in Yunong 201 and Chinese Spring, type II with only an approximately 1500 bp fragment in Gaocheng 8901, and type III with only an approximately 1400 bp fragment in Zhengmai 004. Sequencing results indicated that the approximately 1500-bp fragment of Gaocheng 8901 exhibited 100 % identity with approximately 1500-bp fragment of Yunong 201 and Chinese Spring. The approximately 1500-bp fragment of type II belonged to the TaLox-B2 gene. However, sequencing results of the approximately 1400-bp fragment of type III indicated that a new TaLox-B2 allele was discovered in Zhengmai 004 after the complete alignment of this sequence with the TaLox-B2 and TaLox-B3 genes. The full-length sequence of this new TaLox-B2 allele was obtained by assembling overlapped sequences of subcloned fragments amplified with primer sets Lox-P1 to Lox-P4. This new TaLox-B2 allele in Zhengmai 004 was designated as TaLox-B2b, and the TaLox-B2 allele in Yunong 201 was designated as TaLox-B2a according to the nomenclature of McIntosh et al. (2007) and Geng et al. (2012). Compared with the TaLox-B2a allele (Fig. 4), the TaLox-B2b allele exhibited two deletions (137 bp in the third intron and 3 bp in the fourth intron) and a 13-bp insertion in the third intron, as well as several SNPs. Complete alignment of TaLox-B2b allele with other TaLox genes of bread wheat indicated that TaLox-B2b allele showed sequence similarities of 99.4 % with TaLox-B2a allele, 85.1 % with TaLox-B3, 80.3 % with TaLox1, 62.0 % with TaLox2, and 61.6 % with TaLox3 at the DNA levels (Supplemental 2). A polygenetic tree of different lipoxygenase genes from tetraploid and hexaploid wheat were shown in Supplemental 3.

Fig. 4
figure 4

Part alignment of DNA sequences of TaLox-B2a, TaLox-B2b and TaLox-B3a alleles in bread wheat. The sequences of the forward primers and complementary sequences of the reverse primers for Lox-B23 are boxed and the introns are underlined

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Based on the sequences of TaLox-B2a, TaLox-B2b, and TaLox-B3 genes, a new marker Lox-B23 (Table 1) was developed to precisely distinguish the differences of TaLox-B2a, TaLox-B2b, and TaLox-B3 in Chinese bread wheat cultivars. The marker Lox-B23 could simultaneously generate two different expected fragments of 788 and 677 bp sizes in Yunong 201 or Zhoumai 18 with both TaLox-B2a and TaLox-B3 genes, including only one expected 788 bp fragment in Gaocheng 8901 with TaLox-B2a allele and one expected 660 bp fragment in Zhengmai 004 with TaLox-B2b allele (Fig. 5). Sequencing results confirmed the reliability of the marker Lox-B23. A total of 136 Chinese wheat cultivars were identified with allelic variations of the TaLox-B2 and TaLox-B3 genes. The results indicated that 122 out of 136 Chinese wheat cultivars contained TaLox-B2a allele, and the remaining 14 cultivars included TaLox-B2b allele at the TaLox-B2 locus (Supplemental 1). Up to 85 out of 136 Chinese wheat cultivars contained TaLox-B3 gene, and the remaining 51 cultivars lacked this gene at the TaLox-B3 locus. This finding suggested that the wheat cultivars with TaLox-B2a allele and TaLox-B3 gene at the TaLox-B2 and TaLox-B3 loci were predominant in the surveyed Chinese wheat cultivars. The TaLox-B3 gene was presented only in some Chinese wheat cultivars. The cultivars with TaLox-B3 were designated as TaLox-B3a allele, and the cultivars without TaLox-B3 were designated as TaLox-B3b allele. Notably, TaLox-B2 gene existed in all of the cultivars surveyed, whereas all wheat cultivars with TaLox-B3a gene contained TaLox-B2a allele (Supplemental 1).

Fig. 5
figure 5

Identification of TaLox-B2 and TaLox-B3 alleles by marker Lox-B23 in some Chinese wheat cultivars. M: DL2000; 01: Zhoumai 18; 02: Gaocheng 8901; 03: Zhoumai 16; 04: Yanzhan 4110; 05: Zhengmai 004; 06: Huaimai 19; 07: Zhengmai 9023; 08: Zhoumai 22; 09: Shaan 160; 10: Xianyang 83104; 11: Aikang 58; 12: Yunong 201; 13: Xinmai 18; 14: Yunong 211; 15: Xinong 979; 16: Yannong 15

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Associations of allelic variation of TaLox-B genes with Lox activity and colorimeter parameters in Chinese bread wheat

Analysis of variance indicated that the TaLox-B genes contributed significantly to variation of Lox activity in Chinese bread wheat cultivars and the influence of environment (Year, replicate and year × location, etc.) on Lox activity was also significant (P < 0.05) (Table 2). Analysis of associations of allelic variation of TaLox-B2 and TaLox-B3 genes with Lox activity and colorimeter parameters indicated that wheat cultivars with TaLox-B2a allele exhibited a significantly higher Lox activity than that of cultivars with TaLox-B2b allele. Wheat cultivars with TaLox-B3a allele showed significantly higher Lox activity than that of cultivars with TaLox-B3b allele (Table 3). Wheat cultivars with TaLox-B2b allele showed the significantly highest L*, the lowest a* (absolute value) and b* amongst cultivars with three different TaLox-B alleles. In the surveyed Chinese wheat cultivars, five allelic combinations were discovered at the TaLox-B1, TaLox-B2, and TaLox-B3 loci, namely, TaLox-B1a/TaLox-B2a/TaLox-B3a, TaLox-B1a/TaLox-B2a/TaLox-B3b, TaLox-B1a/TaLox-B2b/TaLox-B3b, TaLox-B1b/TaLox-B2a/TaLox-B3a, and, TaLox-B1b/TaLox-B2a/TaLox-B3b. Further analysis of association of TaLox-B allelic combination with Lox activity and colorimeter parameters indicated that wheat cultivars with the TaLox-B1a/TaLox-B2a/TaLox-B3a combination exhibited higher Lox activity than that of cultivars with the other four combinations (Table 4). By contrast, wheat cultivars with TaLox-B1a/TaLox-B2b/TaLox-B3b combination significantly showed the lowest Lox activity, a* (absolute value) and b* but the highest L* among cultivars with the five combinations (P < 0.05, Table 4).

Table 2 Mean-square values from ANOVA for Lox activity in Chinese bread wheat cultivars
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Table 3 Association of TaLox-B alleles with Lox activities and colorimeter parameters in Chinese wheat cultivars surveyed
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Table 4 Comparison of Lox activities and colorimeter parameters of bread wheat cultivars with different TaLox-B allelic combinations
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To determine the influence of TaLox-B3 gene on Lox activity in bread wheat, a F 4:5 RIL population containing 110 lines derived from Xianyang 83104 with TaLox-B1b/TaLox-B2a/TaLox-B3b (61.7 A 234 min−1g−1) and Zhoumai 16 with TaLox-B1b/TaLox-B2a/TaLox-B3a (80.0 A 234 min−1g−1) was examined by the markers Lox16, Lox 18, and Lox-B23. The average Lox activity of each line in F 4:5 generations was used to analyze the association of TaLox-B3 gene with Lox activity. The results indicated that out of the RIL population, 64 and 46 lines had TaLox-B1b/TaLox-B2a/TaLox-B3a and TaLox-B1b/TaLox-B2a/TaLox-B3b combinations, respectively. The average Lox activity of the wheat lines with TaLox-B1b/TaLox-B2a/TaLox-B3a (77.6 A 234 min−1g−1) was significantly higher than that of lines with TaLox-B1b/TaLox-B2a/TaLox-B3b (68.1 A 234 min−1g−1) (P < 0.05). This finding suggests that the existence of TaLox-B3 gene could significantly increase the Lox activity in bread wheat.

Expression profile analysis of TaLox-B2 and TaLox-B3 genes

Expression profiles of the TaLox-B2 and TaLox-B3 genes were examined by RT-PCR in the wheat cultivars Zhoumai 18 (TaLox-B2a/TaLox-B3a), Zhengmai 004 (TaLox-B2b/TaLox-B3b), and Gaocheng 8901 (TaLox-B2a/TaLox-B3b) using gene-specific primers Lox-P5 and Lox-P6 (Table 1). RT-PCR results indicated that both TaLox-B2 and TaLox-B3 genes could express in the root, stem, and leaf of Zhoumai 18 with TaLox-B2a/TaLox-B3a, whereas TaLox-B3 gene could not relatively express in those parts of Gaocheng 8901 and Zhengmai 004 with TaLox-B3b allele (Fig. 6a, b). Quantitative RT-PCR also showed that the relative expression level of TaLox-B2 gene was significantly higher in the leaf than in the root and stem of Zhoumai 18, Zhengmai 004, and Gaocheng 8901 (Fig. 6a). Notably, the relative expression levels of TaLox-B2a (Zhoumai 18 and Gaocheng 8901) gene in the leaf were significantly higher than those of TaLox-B2b (Zhengmai 004). However, the relative expression levels of TaLox-B2a and TaLox-B2b genes in the root and stem were insignificantly different (Fig. 6a). The relative expression level of TaLox-B3 gene was also significantly higher in the root than in the stem and leaf, with the lowest expression level in the stem of Zhoumai 18 with TaLox-B3a allele (Fig. 6b). Additionally, expression profiles of the TaLox-B2 and TaLox-B3 genes in seeds of the different developmental stages were shown in a Chinese current elite cultivar Aikang 58 (Fig. 7) with TaLox-B2a and TaLox-B3a genes.

Fig. 6
figure 6

Relative expression levels in the roots, stems, and leaves of three cultivars with different TaLox-B alleles. Different letters on the top of the bars indicated the significant difference at 0.05 probability level

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Fig. 7
figure 7

Relative expression level of TaLox-B2 and TaLox-B3 genes in seeds of different developmental stages of Aikang 58 (TaLox-B2a/TaLox-B3a)

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Discussion

The role of Lox in wheat grain quality formation is largely understood. Leenhardt et al. (2006) and Borrelli et al. (2008) found that the low Lox activity or Lox gene deletions can effectively reduce the oxidation of lipids, thereby extending the storage period. This report proved that Lox is one of the most important factors affecting the storage characteristics of bread wheat. Consequently, Lox activity significantly affects wheat quality improvement and seed storage; however, Lox activity is controlled by a large Lox gene family (Hart and Langston. 1977; Li et al. 1999). Although several Lox genes have been discovered and cloned, the current study focused on cloning new Lox genes and obtaining two from these that are closely associated with Lox activity in bread wheat to precisely illustrate the molecular and genetic bases of Lox activity.

In wheat, previous studies have revealed that Lpx1 and Lpx3 are found on group 4 chromosomes, and Lpx2 loci are located on group 5 chromosomes (Hart and Langston 1977; Li et al. 1999; Hessler et al. 2002; Carrera et al. 2007; Zhang et al. 2008; Garbus et al. 2009; Verlotta et al. 2010). LoxA, LoxB, and LoxC genes in the barley genome belong to a single copy of cloning, in which the gene sequence is relatively single (van Mechelen et al. 1995, 1999). In the A genome, a partially deleted copy of Lpx-1 (Lpx-A1-like) was found, colocalizing within a 42-kb region with Lpx-A3. This finding confirmed that these two genes in both genomes were close to each other. The Lpx-A1-like gene is also found in 4A chromosomes of bread wheat (Garbus et al. 2013). Lox genes are located on the same chromosome segments in durum wheat and bread wheat and contained ortholog with barley (Li et al. 1999; Nachit et al. 2001). In barley, the Lox gene sequence is used as probe, and homologous cloning technology indicated multiple Lox gene cloning in wheat (Carrera et al. 2007; Feng et al. 2010, 2012). Seven Loxs, namely, Lpx-A1_like, TaLox3, Lpx-B1.1 a, Lpx-B1.2, TaLox-B1, Lpx-D1, and TaLox1, have been isolated and found on chromosomes 4A, 4B, and 4D in bread wheat (Feng et al. 2010, 2012; Geng et al. 2012; Garbus et al. 2013). This finding showed that Lox activity of bread wheat is mainly influenced by Lox genes on chromosome 4, whereas the two new Lox genes were located on chromosome 4B in the current study.

Grain Lox activity showed high heritability in bread wheat. Genotype and environment have great influence on Lox activity, and the genotype effect is higher than the effects of environment and genotype–environment interaction (Borrelli et al. 1999). Introns are integral elements of eukaryotic genomes that perform various important functions and actively participate in gene evolution (Fedorova and Fedorov 2003). In previous studies on wheat grain phytoene synthase (Psy) genes, divergence in the introns of Psy genes showed high associations with grain yellow pigment (YP) content (He et al. 2009; Crawford et al. 2011). Compared with TaLox-B2a, TaLox-B2b contained two deletions (137 bp in the third intron and 3 bp in the fourth intron), a 13-bp insertion in the third intron, and several SNPs, suggesting a similar molecular mechanism for intron splicing. Particularly, TaLox-B2a and TaLox-B2b showed the same exon size and common exon–intron structure with 99.4 and 100 % of identities in gDNA and cDNA sequences. TaLox-B2a and TaLox-B2b are highly conserved in structure and located parallel to the same chromosome location. This finding strongly implied that alternative splicing occurred in post-transcriptional modification of TaLox-B2b, resulting in a nonsense mutation generating a premature translation termination before the third exon; as a consequence, this mutation influences the expression of TaLox-B2b, leading to reduced grain Lox activity.

The Loxs are expressed in different organelles with different functions and are encoded by multiple gene families in higher plants (Porta and Rocha-Sosa 2002; Liavonchanka and Feussner 2006). Feng et al. (2012) found that Lox genes expressed different quantities in various tissues and organs of wheat. In the current study, qRT-PCR results indicated that both TaLox-B2 (TaLox-B2a or TaLox-B2b) and TaLox-B3 genes could be expressed in the root, stem, and leaf. The TaLox-B2 gene was mainly expressed in the leaf, whereas the TaLox-B3 gene was mainly expressed in the root.

Functional markers developed from polymorphic sites within genes that accurately discriminate counterpart alleles at a locus should be used increasingly in wheat breeding (Bagge et al. 2007; Liu et al. 2012). To date, less functional markers for Lox genes have been reported in bread wheat. In the present study, a co-dominant functional marker, Lox-B23, for wheat grain Lox activity was developed and validated. The marker was highly relevant to Lox activity and can be used in wheat breeding programs aimed at improving the color of flour for various wheat-based end products.

Few cultivars with the 788-bp fragment still showed a low Lox activity, and the other cultivars with the 660-bp fragment exhibited a high Lox activity. These results may be attributed to multiple genes conditioning the Lox activity and environmental effects, particularly those on group 4 and 5 chromosomes (Hart and Langston 1977; Li et al. 1999; Feng et al. 2010, 2012; Garbus et al. 2013). Geng et al. (2012) developed dominant complementary functional markers Lox16 and Lox18, which can accurately discriminate the two alleles of TaLox-B1. Lox16 and Lox18 showed high associations with higher and lower Lox activities, respectively. Therefore, developing functional markers for the Lox gene on group 4 and 5 chromosomes and implementing them for wheat Lox evaluation in combination with Lox-B23 might be necessary for breeding wheat cultivars with low or high Lox activity.

In summary, we cloned two novel Lox genes on chromosome of 4BS, and identified two alleles of TaLox-B2 gene in Chinese bread wheat cultivars, and analyzed the association of allelic variation of TaLox genes with Lox activity. Furthermore, quantitative real-time PCR indicated that TaLox-B2 and TaLox-B3 genes could express in various organs of wheat. This study could provide useful information to further understand the molecular and genetic bases of Lox activity in bread wheat.

Author contribution statement

FC and DC designed this study. FZ and FC wrote the manuscript. FZ and PW performed experimental data for cloning of Lox genes and identification of Lox activity. FZ and NZ performed the qRT-PCR analyses. All authors reviewed the manuscript.