Barley (Hordeum vulgare L.) is the fourth largest cereal crop planted worldwide with the extensive utilization in various fields. Barley grain has been widely used as livestock feed and raw material for malting and brewing, while it is also used as the major food source in some regions around the world and appreciated as a component of a healthy diet (Baik and Ullrich, 2008; Newton et al., 2011; Ullrich, 2011). Starch, of which amylose and amylopectin are the two components, is the main constituent in barley grain like other cereal grains (James et al., 2003; Zeeman et al., 2010; Asare et al., 2011). The proportion of amylose to amylopectin in barley endosperm affects certain properties of the starch which may further influence the grain malting, food and feed quality as well as the product application (James et al., 2003; Jaiswal et al., 2010; Jane et al., 2010). As compared to normal starches, high-amylose starches with the high amylose/amylopectin ratio are usually used for producing resistant starches and the products with high gelling strength in related industries, while amylose-free starches with the low amylose/amylopectin ratio generally show the suitability for frozen foods industrial applications because of the excellent freeze-thaw stability (Bird et al., 2000; Jobling, 2004; Jaiswal et al., 2010; Jane et al., 2010).

ADP-glucose Pyrophosphorylase (AGPase), granule-binding starch synthase (GBSS), soluble starch synthase (SSS), starch branching enzyme (SBE), and debranching enzyme (DBE) are known as the 5 classes of enzymes involving in the starch biosynthesis. Of these, GBSS as the key enzyme participates in amylose synthesis, while SSS, SBE, and DBE are believed to have unique functions and concerted actions in amylopectin synthesis (James et al., 2003; Fan et al., 2016). Each class of these enzymes has several isoforms encoded by the related genes, furthermore, each relevant gene has multiple alleles with different genetic effects. Starch synthase IIa (SSIIa) encoded by SSIIa (SSII-3) gene is the one of a family enzymes that work progressively in the conversion of ADP-glucose to starch polymers by elongating short amylopectin chains which has the degree of polymerization (DP) ≤ 10 to intermediate chains of amylopectin (DP = 12~24) in the cereal endosperm (Fontaine et al., 1993; Luo et al., 2015). It has been reported in the barley, wheat, rice and maize that the composition and content as well as properties of grain starch can be changed by the altered SSIIa (Yamamori et al., 2000; Umemoto et al., 2002; Morell et al., 2003; Zhang et al., 2004; Konik-Rose et al., 2007).

Understanding the molecular and genetic mechanism of amylopectin is important in barley starch quality improvement. However, nearly all the studies in barley about amylopectin synthesis enzymes/genes were conducted with mutant materials. Up to now, there are no reports on the study about detecting QTL for barley grain amylopectin concentration via linkage mapping analysis. Furthermore, there is only one report on GWAS for grain amylopectin content in barley, which identified 17 associated single nucleotide polymorphisms (SNPs) using a collection of 254 European spring barley varieties (Shu and Rasmussen, 2014).

In this study, we mapped QTLs responsible for amylopectin content in barley by using a bi-parental population together with an association mapping panel both already genotyped with SNPs developed by genotyping-by-sequencing (GBS). Identification of the relevant QTL by the combination of linkage analysis and GWAS has not been reported before. Furthermore, the candidate genes in the mapping region were predicted and analyzed by the annotation according to syntenous and comparative genomics with rice (Oryza sativa L.). The SSII-3 (SSIIa) gene was subsequently characterized and markers related to the gene were designed and validated. The results of this study reveal new genetic insights into grain amylopectin content in barley and provide the sequence-based marker for use in marker-assisted selection (MAS) for barley breeding.

The main constituent of barley grain is starch which comprises the two components of amylose and amylopectin. Variations in amylose/amylopectin content and amylopectin structure can affect starch properties which may further influence grain qualities for different utilizations (James et al., 2003; Jaiswal et al., 2010; Jane et al., 2010). As to measuring the amylopectin content, the colorimetric method is used relatively easily and inexpensively, however, the problem experienced in the use of this method has been detailed by Gibson et al. (1997) and the method extensively used in the amylopectin content measurement is through the difference calculation between total starch content and amylose content (Shu and Rasmussen, 2014). In this study, we use the Megazyme total starch assay kit together with the Megazyme amylose/amylopectin assay kit (a modification of a Con A method) to measure the amylopectin content phenotype.

Almost all the quality-related phenotypes in the grain crop are the quantitative trait which was generally studied via QTL or GWAS mapping to identify specific gene/locus. The complementarity of classical linkage analyses and genome-wide association study has been well demonstrated by Brachi et al. (2010). Furthermore, the developing sequencing technologies has greatly increased the SNPs discovery in many species (Davey et al., 2011). GBS is a relatively low cost, simple and efficient approach for discovering and genotyping the sample with genome-wide SNP markers (Elshire et al., 2011; Poland and Rife, 2012). High-density genetic maps constructed by SNP markers through GBS have great value for applications in crop breeding and genetics research. In the present study, the grain amylopectin content trait of 178 DH lines and 185 barley varieties, which were all genotyped with SNPs developed via GBS, was measured in diverse environments.

As easily influenced by multiple factors, most of QTLs relevant to quantitative traits were detected only under a few environmental conditions or with the small effect. Hence, the QTL identified stably in various environments and genetic backgrounds as well as with the large effect could be effectively used in MAS for breeding. In our study, a total of four QTLs on chromosome 3, 4, 5, and 7H were detected in two locations from the DH population (Table 3). Among them, qAPC-7-1 was identified in both environments and determined 12.98% of phenotypic variation (Table 3; Figure 2A). In addition, qAPC-7-1 was co-localized with an associated SNPs (SNP3120) detected by GWAS from the collection of 185 barley varieties. According to Comadran et al. (2012), and considering a LD of 4-6 cM in barley, the associated SNP3210 on chromosome 7H located at 70.68 cM (in the reference genome of cv. Morex) (Table S5), which was co-localized with qAPC-7-1, was located just near the starch synthase IIa gene (MLOC_69670, SSIIa), also designated as SSII-3. 17 SNPs associated with grain amylopectin content were identified by GWAS in the report of Shu and Rasmussen (2014). Among them, one significant SNP region on 7H (5 SNPs from 12.75 to 15.37 cM) was overlapping with SNP4298 (15.93 cM on 7H) detected in our study. The region was very close to the waxy locus which was known to be essential in amylose synthesis and metabolism. Although the accumulated knowledge indicated that the waxy gene is the key gene mainly responsible for amylose content, it also has influence on amylopectin structure and content (Denyer et al., 1996; Hori et al., 2007).

The starch biosynthesis pathway has been extensively and deeply researched in recent decades. SSIIa (SSII-3), which is known as an important gene in amylopectin biosynthesis, has the significant effect on starch content and quality (James et al., 2003; Nakamura et al., 2005; Fan et al., 2016). In our study, candidate genes relevant to amylopectin synthesis were predicted in the co-localized mapping region based on the gene annotation. The gene (Os06g0229800) encoding starch synthase II that played an important role in amylopectin biosynthesis was barley SSIIa (SSII-3) gene. According to the isolation and characterization results of the two bi-parental SSIIa alleles of the DH population, a 33-bp polymorphic difference in exon 2 was found (Figure 6). qRT-PCR analysis result indicated that the expression levels in each stage after flowering of SSIIa allele in Naso Nijo were higher than those in TX9425 (Figure 7B). Furthermore, the expression pattern of SSIIa alleles in our study was consistent to Radchuk et al. (2009). According to those results, we can conclude that the deletion was likely responsible for reducing SSIIa transcript, thus resulting in a reduced amylopectin concentration in barley endosperm.

The conventional breeding method combining with MAS could significantly raise the breeding efficiency (Tian et al., 2010). In this study, a sequence-based marker (SSIIa-DNA) was designed according to the 33-bp difference between the SSIIa alleles (Table S4). Amplified by primer SSIIa-DNA, two types (58 type-TX9425 and 124 type-Naso nijo) were separated within 182 barley varieties of the association mapping panel, in addition, there was a significant difference between the two types (Figure 7A). The result further indicated that the 33-bp nucleotide sequence was a functional polymorphic difference associated with grain amylopectin content and the developed molecular marker was suitable for MAS in barley breeding.

In summary, a QTL responsible for barley grain amylopectin content was detected in both bi-parental and association mapping panel respectively by linkage analysis and GWAS. A strong candidate for the SSIIa gene and a functional polymorphism (a insertion/deletion of 33bp in exon 2) associated with different levels of grain amylopectin content were identified. Furthermore, a sequence-based molecular marker had been developed with potential application for MAS in breeding for desirable grain amylopectin content in barley.

XF and JZ measured the grain amylopectin content, performed QTL, GWAS and candidate gene analysis, and wrote the manuscript. WD and YS prepared the grain flour for amylopectin content measurement and prepared the DNA and RNA of materials for relevant experiment. CL and BG managed the field experiments. RX supervised the project. All authors read and approved of the final manuscript.