Analysis and mapping quantitative trait loci for histidine content in barley (Hordeum vulgare L.) using microsatellite markers

Mining the gene of histidine content in barley grain helps with the breeding of functional barley varieties. The study constructed a recombinant inbred lines (RILs) containing 193 families derived from the cross between Ziguangmangluoerling (ZGMLEL) (♀) and Schooner No.3 (♂). The histidine (HIS) content in the grain of the mapping population and its parents were determined by an automatic amino acid analyzer. The HIS content of ZGMLEL was 0.53 mg/g. The grain HIS content of Schooner No. 3 was 0.21 mg/g, and the grain HIS content of population ranged from 0.23 to 0.54. Genetic linkage maps, including those of seven chromosomes of barley, were constructed by using 180 pairs of simple sequence repeat (SSR) markers, with a total genetic distance of 2671.03 cM and average marker spacing of 14.84 cM. Quantitative trait locus (QTL) IciMappingV3.3 was used to analyze QTL of HIS content in barley grains, and three QTLs were detected. Mapping results showed that the three loci were located on chromosomes 2H, 4H, and 7H, respectively. The major QTL with a contribution rate of 10.11% was located on barley chromosome 4H (HVBAMMGB84-BMAG0808). The additive effect is positive (0.025). Thus, it comes from the high-value parent ZGMLEL. Another major QTL with a contribution rate of 13.75% was located on barley chromosome 7H (GBM1303-GMS056). The minor QTL with a contribution rate of 6.01% was located on chromosome 2H (Scssr03381-Scssr07759). The additive effects of 4H and 7H QTLs were negative (− 0.02 and − 0.033). So, they came from the male parent Schooner. The results provided a reference for further fine mapping, cloning, and transformation of HIS genes in barley grains.


Introduction
Barley (Hordeum vulgare L.) is one of the earliest domesticated ancient cereal crops in the word (Khahani et al. 2019). Barley is a food and economic crop, as well as a medicine and food homologous crop. It is rich in minerals, lipids, and amino acids. Its grains are also rich in protein, physiologically active substances, total flavonoids, c-aminobutyric acid (GABA), b-glucan and other nutritional functional components (Arndt et al. 2006;Xu et al. 2019). It has special efficacy and great medicinal value for the prevention and treatment of diabetes, hypertension, cardiovascular disease, chronic uremia (such as HIS), and other chronic diseases (Chan et al. 2009;Yang et al. 2020). Functional barley is one of the popular topics in barley research.
Amino acids are among the structures that make up proteins and play an irreplaceable role in most cell functions, regulation, and metabolism (Shen et al. 2019). HIS is a common amino acid that is not essential to adults, but essential to young children. So, it is called a semi-essential amino acid. HIS mainly exists in the liver and skin stratum corneum. It has a strong vasodilating effect; it can participate in the regulation of the nervous system and stimulation of the gastric acid and pepsin secretion (Gu et al. 2018;Hu et al. 2018). It has an important effect on the treatment of anemia, gastrointestinal disease, asthma, angina pectoris, cardiac insufficiency, and uremia (Lin et al. 2008;Kang et al. 2016). HIS is now used in the pharmaceutical and food industry but also with alanine co-synthesis of carnosine, which has antioxidant and anti-aging effects. Increasing the content of HIS in food is one of the important ways of histidine supplementation in humans. Thus, the production of functional meat products has become a popular research topic. As the role of HIS in the field of medicine was gradually recognized, it became one of the most commonly used drugs in medical treatment in China; so, the demand for HIS is also increasing (Sun et al. 2010).
Mining QTLs by constructing a mapping population is the main method of barley QTL research at present. Proteins and flavonoids are functional components of barley, and their QTLs have been reported (Yang et al. 2017). Agronomic trait QTLs include flowering (Ibrahim et al. 2018), disease resistance (Read et al. 2003), grain length (Watt et al. 2018), grain yield (Long et al. 2018;Falcon et al. 2019;Hu et al. 2019), spike height, spike length, spike grain number, plant height, flag leaf length, leaf area, and leaf width (Islamovic et al. 2013;Liu 2015;You 2016).
The grain amino acid content of barley has been confirmed to be a quantitative character that is affected by both gene and environment (Xu et al. 1988). Agronomic measures, meteorological factors and regional conditions under environmental conditions will affect barley's amino acids content Nie et al. 2010;Hu et al. 2017;Li 2017;Xu et al. 2017;Bai et al. 2018). Significant progress has been made in the composition and determination of amino acid content in barley and the changes of amino acid content under different malt processing conditions (MacLeod 1951;Pomeranz et al. 1976;Williams et al. 1984;Han et al. 2009;Luo et al. 2014). For example, Nie et al. (2010) analyzed and detected the changes in the amino acid content in barley malt during germination and drying. They concluded that HIS belongs to class III (the most important) amino acid in the process of malting. It is the most important yeast metabolism.
Genetic studies have shown that barley grain amino acids were affected by endosperm triploid and maternal plant diploid genes (Yan et al. 1997). Through further research, Xu et al. (1996) found that the inheritance of HIS content in two-rowed barley grains was controlled by seed direct gene and maternal effect, and a strong interaction existed between direct dominance and the environment. However, the research on barley amino acid QTL is weak. At present, only QTLs of gammaaminobutyric acid (GABA) content in barley grains are distributed on the chromosomes 4H, 5H, 6H and 7H (Luo et al. 2014;Yang et al. 2017). However, the QTL of HIS in barley has not been reported yet.
In the previous study, we found a variety with high HIS content in the grains (ZGMLEL), and established RILs with this variety. In this study, we used the constructed RIL mapping population as materials to analyze the correlation between HIS content in the mapping population grains and 12 morphological characters. The QTL of HIS content in barley grains were excavated. The results lay the foundation for fine mapping and transformation of histidine content genes in barley grains.

Plant materials
In the winter of 2007 in Kunming, the hybrid F 1 was prepared by using ZGMLEL as the female parent, and Schooner No.3 as the male parent. The F 2 population was obtained by self-crossing. P 1 , P 2 and F 8 populations were planted in the experimental fields of Kunming Academy of Agricultural Sciences (altitude 1916 m) and Songming experimental fields (altitude 1902 m) from 2008 to 2011, according to the cultivation rules of RILs. The characters of F 7 and F 8 were stable and were considered as RILs.

Cultivation of the materials
On 28 October 2016, 193 F 9 RILs lines of ZGMLEL 9 Schooner No.3 were planted in Yanhe Town, Yuxi City (altitude 1646 m). The previous stubble of the experimental site was rice, the soil was clay loam, and soil fertility was medium. The previous study of this project showed that the yield of regenerated wheat grain and straw was the highest at the stage of 3-leaf stage and 1 heart. In this experiment, the seedlings were cut 60 days after sowing and repeated for 2 days. The sowing rate of each plant was the same as that of the base fertilizer, that is, the sowing rate was 100 grains/m, the compound fertilizer (N: P 2 O 5 : K 2 O = 13 : 5 : 7) was the base fertilizer, and the fertilizer application rate was pure N 120 kg/hm 2 , which emerged on November 6. Topdressing with pure N 30 kg/hm 2 was performed on 2 January 2017. the other management was the same as the local conventional high-yield wheat field.

Determination of HIS content
The grain HIS content of RILs mapping population and parents was determined by Hitachi-L8900 automatic amino acid analyzer.

Investigation and determination of morphological traits
After the population plant matured, five plants were randomly selected for each repeat to measure 12 morphological traits, including available spike, invalid spike, main spike length, plant height, total length of internode and others. The mean value of each trait was calculated. Taking the OD260/OD280 ratio between 1.8 and 2.0 as the evaluation index, the purity of DNA was detected by ultraviolet spectrophotometer (DU-7400), and the DNA concentration was calculated by using the OD260 value.

Screening of SSR markers
The 500 pairs of SSR primers used in this experiment were uniformly selected from the barley genome. The primer sequences were from http://www.wheat.pw. usda.gov/cgi-bin/graingenes, and the primers were synthesized by Beijing Dingguo Changsheng Biotechnology Co., Ltd. The synthetic primers were used to screen for polymorphism between ZGMLEL and Schooner No. 3. The selected polymorphic primers were amplified by PCR in the population.

PCR amplification and detection of reaction product
The PCR reaction system was 10 ll; 0.5 ll for upstream primer and downstream primer, 2 ll for DNA, 1.5 ll for PCR Buffer, 0.4 ll for dNTPs, 4.9 ll for ddH 2 O, and 0.2 ll for Taq polymerase.
PCR amplification procedure: 94°C for 5 min, 94°C for 40 s, 55°C annealing for 40 s, 72°C extension 1 min for 35 cycles, and a final extension at 72°C for 5 min.
Detection of PCR amplification products: the amplification products were separated by denaturing 8% polyacrylamide gel electrophoresis and then detected by silver staining. The specific reagents are mentioned in the study of Sun et al. (2009).

SSR marking data record
We placed the silver-stained gel under the film observation lamp to read the tape with reference to Yang et al. (2017). We recorded it with QTL IciMapping software. If the band type was the same as the female ZGMLEL recorded ''0,'' and if it is same as the father's choice recorded ''2,'' the hybrid band is recorded as ''1'' and the missing band is recorded as ''-1.'' QTL mapping of amino acids in grains of RILs We used QTL IciMappingV3.3 to construct the molecular genetic linkage map of barley RIL population and used the Inclusive Composite Interval Mapping (ICIM) method to map QTL. Using LOD C 2.5 as the threshold of QTL existence, We analyzed the linkage relationship between histidine content and marker genotypes in the mapping population, determined the relative position of histidine QTLs on chromosomes, and estimated their genetic effects (Wang 2009). The nomenclature of QTL followed the method of McIntosh (2013).

Results and analysis
Analysis of the variation of the grain HIS content in RILs The grain HIS frequency map and variation table of ZGMLEL RILs are shown in Fig. 1; Table 1. Combined with Fig. 1; Table 1, the population kurtosis was -0.73, and the population deviation value was  -0.59, indicating that the HIS content distribution in the population grain was the slope peak (left-deviated normal distribution). The strains with lower HIS content accounted for a larger proportion in the population, and the intermediate HIS content lines were relatively less in the population. According to Table 1, the average HIS content of the population was between the two parents. The HIS content of the male parent Schooner No.3 was 0.21 mg/g, which was a low-value parent, and the HIS content of the female parent ZGMLEL was 0.53 mg/g, which was a highvalue parent. The HIS content in population grains ranged from 0.23 mg/g to 0.54 mg/g, with the average value of was 0.38 ± 0.01 mg/g, and the coefficient of variation was 1.90%. The average value of the population was higher than that of the parents, and the content of HIS was higher than that of the strains with high parent content, indicating that the HIS content showed a trend of high parental segregation in the RIL mapping population.

Correlation analysis between grain HIS content and 12 morphological traits in RILs
The correlation between HIS content in population grain and 12 morphological characters is presented in Table 2. The HIS content was significantly or very significantly correlated with six traits. Significant negative correlation was found with available spike and panicle weight per plant. Extremely significant positive correlation was found with invalid spike and blighted grains. Extremely significant negative correlation was found with number of solid grains and setting rate. Further analysis showed that the HIS content in the grain was most closely related to setting rate, and the correlation coefficient was -0.34. The correlation between histidine and 12 morphological traits showed that the morphological traits of histidine and barley yield were significant or negative. Thus, increasing barley grain yield might reduce histidine content.

SSR marker analysis of ZGMLEL RILs population
(1) SSR marker analysis Among the 500 pairs of SSR primers, 180 could obtain clear and polymorphic bands, and the segregation of the above markers in the population was basically consistent with the Mendel segregation ratio of 1:1, which was a homozygous locus.
(2) Construction of genetic map We used 180 pairs of primers with good polymorphism between parents to conduct population amplification and constructed seven linkage groups. These seven linkage groups were constructed (Fig. 2). The related information of the linkage groups was sorted out in Table 3. According to the Table 3, the total length of the genetic linkage map was 2671.03 cM, and the average genetic distance was 14.84 cM. The longest covering distance was found in the 7H chromosome, which was 542.89 cM, and the number of markers was the highest (39). The average genetic distance between markers was 13.92 cM. The shortest distance was 233.72 cM on chromosome 6H, and number of markers was the lowest (15). The average distance between markers was 15.59 cM.

QTL analysis of grain HIS content in the ZGMLEL RILs
The QTL information of grain HIS content in ZGMLEL RILs is shown in Fig. 3 and Table 4. According to the Table 4, three QTLs related to the HIS content in barley grains were excavated; these were located on 2H, 4H and 7H chromosomes, which b Fig. 2 Positions of QTLs location of barley histidine (HIS) content

Discussions
Barley is listed as a health food by the United States Department of Agriculture (Newman and Newman 2008). Whole-grain food made from barley is considered to be ''healthy food'' for humans and plays an important role in preventing chronic diseases such as cardiovascular disease and cancer (Gangopadhyay et al. 2015;. The study on the correlation between grain HIS content and 12 morphological characters in barley showed the significant negative correlation between grain HIS content and barley yield. Thus, increasing barley yield may decrease grain HIS content. Using the RIL population to make a genetic mapping and mining QTL can analyze the additive effect of QTLs, This population is suitable for plant QTL mapping. Many barley QTLs were obtained by using this population. For example, Lu (2015) used the RIL population constructed by barley (GP 9 H602) as a material for genetic linkage map and QTL analysis b Fig. 3 The LOD value and additive effect of HIS on chromosomes of 2H, 4H, and 7H; (1) In this study, we used ZGMLEL 9 Schooner No.3 RIL mapping population to map three QTLs of HIS content in barley grains, which were located on chromosomes 2H, 4H, and 7H, respectively. In previous studies, QTLs of other traits were found in barley 2H, 4H, and 7H Emebiri et al. 2005;Li 2007;Nie et al. 2010;Guo 2012;Yu 2014;Liu 2015;Wang 2016;Yang et al. 2017;Fei 2018). However, no reports on histidine QTL in barley grains are available. So, the three QTLs found in this study were all new QTLs controlling histidine content in barley grains. Further analysis revealed that the locus found in this study on the 4H chromosome had common markers with the protein sites reported by Yang et al. (2017) on 4H (BMAG0808). This result may be due to the fact that the mapping populations used in this study are consistent with those used by Yang. Amino acids are the basic units of proteins. Therefore, a certain correlation may exist between QTLs controlling protein content and histidine content in barley grains.. We speculated that there might be a certain correlation might exist between the QTL controlling the protein content in barley grains and the QTL controlling the histidine content in barley grains.

Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.  The negative value of additive effect indicated that the gene came from the male parent Schooner a The positive value of additive effect indicated that the gene came from the female parent ZGMLEL