- Research article
- Open Access
Construction of the first high-density genetic linkage map and identification of seed yield-related QTLs and candidate genes in Elymus sibiricus, an important forage grass in Qinghai-Tibet Plateau
BMC Genomics volume 20, Article number: 861 (2019)
Elymus sibiricus is an ecologically and economically important perennial, self-pollinated, and allotetraploid (StStHH) grass, widely used for forage production and animal husbandry in Western and Northern China. However, it has low seed yield mainly caused by seed shattering, which makes seed production difficult for this species. The goals of this study were to construct the high-density genetic linkage map, and to identify QTLs and candidate genes for seed-yield related traits.
An F2 mapping population of 200 individuals was developed from a cross between single genotype from “Y1005” and “ZhN06”. Specific-locus amplified fragment sequencing (SLAF-seq) was applied to construct the first genetic linkage map. The final genetic map included 1971 markers on the 14 linkage groups (LGs) and was 1866.35 cM in total. The length of each linkage group varied from 87.67 cM (LG7) to 183.45 cM (LG1), with an average distance of 1.66 cM between adjacent markers. The marker sequences of E. sibiricus were compared to two grass genomes and showed 1556 (79%) markers mapped to wheat, 1380 (70%) to barley. Phenotypic data of eight seed-related traits (2016–2018) were used for QTL identification. A total of 29 QTLs were detected for eight seed-related traits on 14 linkage groups, of which 16 QTLs could be consistently detected for two or three years. A total of 6 QTLs were associated with seed shattering. Based on annotation with wheat and barley genome and transcriptome data of abscission zone in E. sibiricus, we identified 30 candidate genes for seed shattering, of which 15, 7, 6 and 2 genes were involved in plant hormone signal transcription, transcription factor, hydrolase activity and lignin biosynthetic pathway, respectively.
This study constructed the first high-density genetic linkage map and identified QTLs and candidate genes for seed-related traits in E. sibiricus. Results of this study will not only serve as genome-wide resources for gene/QTL fine mapping, but also provide a genetic framework for anchoring sequence scaffolds on chromosomes in future genome sequence assembly of E. sibiricus.
The tribe Triticeae (Poaceae) includes several major cereal crops (wheat, barley, and rye) and many ecologically and economically important forage grasses . Elymus L. is the largest genus in the Triticeae, which comprises about 150 polyploid perennial grass species widely distributed worldwide . Asia is the most important center of origin where approximately 80 Elymus species were found . Many Elymus species are closely related to wheat and barley, and may thus serve as potential gene pool for the improvement of stress tolerance (cold, drought and disease) and other important agronomic traits . Elymus sibiricus (Siberian wild rye), which is indigenous to northern Asia, is an important perennial, cold-season and self-pollinated forage grass of the genus Elymus . Based on the cytogenetic analysis, E. sibiricus is allotetraploid species, containing St and H genomes. The St genome is derived from Pseudoroegneria spicata (Pursh) A. Löve, and the H genome is derived from the genus Hordeum . Elymus sibiricus is widely grown and used for forage production and grassland eco-engineering in the Qinghai-Tibet Plateau region of China, owing to its good forage quality, drought and cold tolerance, and excellent adaptability to local special environments [7, 8]. Despite E. sibiricus has various agricultural uses and economically importance, its serious seed shattering makes seed production difficult for this species. For cereal crops and forage grasses, seed yield is affected by many seed yield-related traits, such as spike length, seed width, floret number per spike, 1000-seed weight, and seed shattering, among which seed shattering is a major cause of yield loss . Previous study showed that serious seed shattering may result in up to 80% seed yield losses if harvesting is delayed . As a result, selection for high seed retention and genetic improvement of seed shattering are important breeding objectives for this species. Several major quantitative trait loci (QTLs) and genes for seed shattering have been reported in cereal crops like rice, wheat, barley, maize and sorghum, and a few forage grasses. For example, in rice, SH4 , qSH1 , OsCPL1 , SHAT1 , and SH5  were identified as major genes for seed shattering, their functions and interactions in regulating abscission layers formation and development were also revealed. In addition, in hybrid Leymus (Triticeae) Wildryes, a major-effect QTL for seed retention was identified on linkage group (LG) 6a, which aligns to other seed shattering QTLs in American wildrice, Zea and Triticum . Together, these studies indicate the presence of QTLs and genes with large effects on seed shattering, and the potential to understand which QTLs or genes play a role in regulating seed shattering.
The availability of genetic map makes feasible the identification of genes for monogenic traits or major loci for quantitative traits, it also provides an important basis for the study of genome structure and evolution . It is particularly important for future positional gene cloning, marker-assisted selection, and comparative genome analysis . The utility of genetic linkage map depends on the types and number of markers used . High-density linkage map lays a foundation for genome assembly and fine mapping of quantitative trait loci (QTL) . To date, several molecular marker systems have been used for the construction of genetic linkage map, including amplified fragment length polymorphism (AFLP) , restriction fragment length polymorphisms (RFLP) , random amplified polymorphic DNA (RAPD) , simple sequence repeat (SSR) , sequence-related amplified polymorphism (SRAP) , and single-nucleotide polymorphism (SNP) . Among these markers, SNP marker is considered as the most promising molecular marker for high-density genetic map construction due to their abundant and wide distribution in genome. The advent of massive parallel next-generation sequencing (NGS) technologies could identify and obtain thousands of SNPs at the whole genome level, thus making it possible to construct high-density SNP genetic maps. However, whole-genome sequencing and genotyping large populations are still cost-prohibitive . Reduced representation library sequencing is considered to be one efficient strategy to bring down the cost through genome reduction [28, 29]. For example, restriction site-associated sequencing (RAD-seq) sequences only the DNA fragment with restriction sites, and has been used for large-scale SNP discovery and genetic mapping in many species [30, 31]. As a modified reduced representation sequencing technique, specific-locus amplified fragment sequencing (SLAF-seq) has several distinguishing advantages such as reduced sequencing costs, deep sequencing, marker efficiency optimization through pre-designed reduced representation scheme, and double-barcode method for large populations. It is an efficient method for large-scale De Novo SNP discovery and genotyping of large population . Recently, SLAF-seq has been increasingly used for high-density genetic linkage map construction in several crops , forage grasses , and animal species .
Toward improving the understanding of E. sibiricus genome arrangement and the genetic control of seed yield-related traits, we constructed a genetic linkage map and identified QTLs related to seed shattering as well as other seed traits. Two E. sibiricus genotypes were selected based on their variation for seed yield-related traits. We applied SLAF-seq to develop thousands of SLAF markers (SLAFs) and construct the first high-density genetic linkage map in E. sibiricus, then identified QTLs and candidate genes for eight seed yield-related traits. These results could lay a foundation for future functional genetic dissection of key genes related to seed shattering and other seed traits.
Analysis of SLAF-seq and SLAF markers
After SLAF library construction and high-throughput sequencing, 253.25 Gb of raw data containing 1267.20 M reads were generated. The average percentage of Q30 (quality scores of at least 30) bases was 93.03%. The average guanine-cytosine (GC) content was 46.69%. To estimate the validity of library construction, we used Oryza sativa ssp. japonica (genome size = 382 M) as control. A total of 901,095 reads with 92.17% Q30 bases and 45.32% GC content were generated (Table 1). The number of reads for male and female parents was 29,809,327 and 65,542,805, respectively. The average number of reads for offspring was 5,859,224.46 with 93.03% Q30 bases and 46.69% GC content. The number of SLAF markers generated for male and female were 232,429 and 326,923, respectively. The average number of SLAF marker in the progeny was 202,120 (Table 2). The average sequencing depth was 31.95-fold and 7.51-fold for parents and each progeny, respectively.
We detected 370,470 SLAF markers, among which 97,387 were polymorphic, 269,579 and 3504 were non-polymorphic (72.77%) and repetitive (0.94%), respectively. Polymorphic markers included mapped biallelic markers and unmapped biallelic markers, monomorphic markers with only one tag in parents were recognized as non-polymorphic markers, mutiallelic markers with tag number larger than 4 in parents were recognized as repetitive markers. Mutiallelic SLAFs which could not be used for recombination rate calculating were removed from further analysis. After filtering the SLAF markers lacking the parent information, 46,135 polymorphic SLAFs were successfully genotyped and further classified into eight segregation patterns (ab×cd, ef × eg, lm × ll, nn × np, aa×bb, hk × hk, cc × ab, ab×cc) (Fig. 1). The mapping population was obtained from the F1 hybrid plant of two homozygous parents, therefore, the 18,343 SLAF markers with aa×bb segregation pattern in the F2 population were used for genetic map construction.
Basic characteristics of the genetic maps
We further filtered the SLAF markers using four criteria . These SLAF markers that belonging to following four types were removed from mapping construction: SLAF markers from parents with sequencing depth less than 10X; SLAF markers with more than five SNPs; SLAF markers with missing in more than 10% of offspring and segregation-distorted markers (Chi-square, p < 0.01). Only the SLAF markers that passed the four-step filtering process were used for constructing a high-quality genetic map. The final map included 1971 markers with 2610 SNP on the 14 linkage groups (LGs) and was 1866.35 cM in length (Fig. 2). The length of each linkage group ranged from 87.67 cM (LG7) to 183.45 cM (LG1), with an average marker density of 1.66 cM between adjacent markers (Table 3). The maximum number of markers (565) were found on LG11, whereas LG8 possessed the minimum number of markers (29) (Additional file 4: Figure S1, Additional file 1: Table S1). The “Gap ≤ 5” value was used to reflect the degree of linkage between each marker, ranging from 73.08 to 100%, with an average of 92.09%. The largest gap on this map was 11.03 cM located in LG14. The number of SNP on each linkage group varied from 35 (LG7) to 712 (LG 11), with an average of 186.
In total, only 26 markers showed a significant (p < 0.05) segregation distortion and were mapped on the final map, accounting for 1.32% of mapped markers (Table 4). Most of the linkage groups (LGs) had segregation distortion markers with the exceptions of LG1, LG3, LG4, LG13, and LG14. The frequencies of distorted markers on LG6 (19.23%) and LG12 (19.23%) were higher than those of the other linkage groups. LG11, which possessed the maximum mapped markers (565 SLAF markers), had the lowest frequency of distorted marker (3.85%).
Quality evaluation of the genetic map
To evaluate the quality of the genetic map, haplotype mapping and heat mapping were carried out. The haplotype map reflected the double exchange of the population, which is caused by genotyping error, suggesting a possible recombination hotspot. The haplotype maps of each linkage group were developed for the parental controls and 200 offspring using 1971 SLAF markers. The results showed that most of the recombination blocks were distinctly defined. The LGs 9, 10, and 13 had no missing data, while LG 8 had the largest missing data (3.53%), with an average of 0.73%. Most of the LGs were uniformly distributed (Additional file 5: Figure S2). The heat maps were constructed based on the pair-wise recombination value from the 1971 mapped markers to reflect the recombination relationship between mapped markers on each single linkage group (Additional file 6: Figure S3). The results confirmed the order of mapped SLAF markers on each linkage group.
Phenotypic analysis of the parents and F2 population revealed significant variations in all eight seed yield-related traits (Table 5, Additional file 2: Table S2). The coefficient of variation (CV) among all traits ranged from 7.24% (WS in 2018) to 58.08% (FN in 2016). We analyzed the correlation between years and traits (Table 6). Our results showed a correlation between phenotypic data detected in different years with exception of WS between 2016 and 2017, and SW1 between 2016 and 2017. For example the correlation for seed shattering (SSc) between 2016 and 2018, 2017 and 2018, 2016 and 2017 were 0.841, 0.783, and 0.360, respectively. Floret number per spike (FN) was significant correlated between 2016 and 2018, 2017 and 2018. Spike length (SL) was significant correlated during 3 years. We calculated the heritability of these traits, all traits had relatively high heritability. The highest heritability (0. 6718) was found for seed shattering (SSc), the lowest heritability (0.4638) was found for floret number per spike (FN). These results were consistent with the correlation analysis between different years. The correlation were found between most traits, for example, awn length (AL) was positively correlated with width of seed (WS), 1000-seed weight (SW1) and spike length (SL). Seed shattering (SS) was positively correlated with floret number per spike (FN). The absolute values of Skewness and Kurtosis for most traits with exception of FN (2017), WS (2017 and 2018), and SW1 (2017) were less than 1 (Table 5). Besides, the normal frequency distributions of eight traits were analyzed and the P-value was more than 0.05 except for SL (2017), FN, SS, WS (2017 and 2018) and SW1 (2017) (Fig. 3).
QTL mapping and comparative genome analysis
A total of 29 QTLs were detected for eight seed-related traits on 14 linkage groups, of which 3 for spike length (SL), 2 for floret number per spike (FN), 6 for seed shattering (SS, SSD and SSc), 7 for awn length (AL), 3 for width of seed (WS), and 8 for 1000 seed weight (SW1). The LOD and PVE (the percentage of phenotypic variation explained) for all QTLs ranged from 3 to 10.62, 2.17 to 10.85%, respectively (Fig. 4, Table 7). Six QTLs detected for seed shattering explained 2.17 to 9.48% of the phenotypic variation. Among the six QTLs, 1 QTLs were detected on LGs 6 using breaking tensile strength (BTS) data, 2 QTLs were detected on LGs 3 and 11 using seed shattering degree (SSD) data, 3 QTLs were detected on LGs 2, 3 and 11 using seed shattering rate (SSc) data. Especially, seed shattering QTLs on LG3 and LG11 could be detected using two methods and at two years (2016 and 2017), respectively. Seven QTLs for awn length (AL) were detected on five linkage groups (LG1, LG5, LG6, LG11 and LG13), among which the QTL on LG1 explained the maximum phenotypic variation of 10.37%. On LG12, a QTL for seed width (WS) was detected and explained the largest phenotypic variation of 10.85% among all QTLs. Moreover, QTLs for awn length (AL) and 1000 seed weight (SW1) were detected on more than five LGs, suggesting a complex genetic mechanism of these traits. A total of 16 QTLs could be consistently detected for two or three years, for example, two QTLs for spike length (SL) on LG14 were detected in 2017 and 2018, two QTLs for seed shattering on LG11 were detected in 2016 and 2017, three QTLs for 1000-seed weight (SW1) on LG9 and three QTLs for awn length (AL) on LG1 were detected for three years.
The 1971 mapped SLAF markers generated from E. sibiricus were compared with the genome sequences of wheat and barley. The Circos plot and Colinear graph was constructed to show the linear relationships between E. sibiricus and wheat and barley, illustrating a corresponding relationship between the mapped markers and their genomic locations (Fig. 5). The numbers of matching markers between E. sibiricus and each species were 1556 (79%) for wheat, 1380 (70%) for barley (Fig. 5a). We further broken down alignments to each subgenome of wheat (A, B and D), the number of matching markers on ChrA, ChrB and ChrD was 311, 523 and 521, respectively (Fig. 5b and c). The largest number of matching markers was found on Chr2A (79), CHr3B (129) and Chr7D (98) for each subgenome, respectively. For barely, the number of matching marker on each chromosome ranged from 139 (Chr1) to 237(Chr2), with an average of 184.
A total of 43 markers within seed yield-related QTL regions could be identified on barley and wheat chromosomes, of which 3 markers for spike length (SL), 2 markers for floret number per spike (FN), 8 markers for seed shattering (SS, SSD and SSC), 7 markers for awn length (AL), 6 markers for seed width (WS), and 17 markers for 1000-seed weight (SW1) (Table 7). These markers were distributed across different chromosomes of each species. For example, 8 markers linked with seed shattering distributed on wheat chromosomes 1A, 3B, 6A, 6B, and 7B, and barley chromosomes 1H, 2H, 3H and 5H. We further identified 30 candidate genes for seed shattering within six QTL regions based on the functional annotation of barley and wheat genomes, of which 15, 7, 6 and 2 genes were involved in plant hormone signal transcription, transcription factor, hydrolase activity and lignin biosynthetic process, respectively (Fig. 6, Additional file 3: Table S3). In particular, among candidate genes for plant hormone, 3 genes were involved in regulation of abscisic acid-activated signaling pathway, 6 genes were involved in ethylene response pathway, 3 genes were involved in auxin-activated signaling pathway, 1 genes for gibberellin, and 1 gene for jasmonic acid. Based on our abscission zone transcriptome data of two E. sibiricus genotypes (XH, high seed shattering, ZN, low seed shattering). A total of 20 unigenes involved in plant hormone, transcription factor, and hydrolase activity were predicted from “XH-WS vs ZN-WS”, of which 14 genes were up regulated in high seed shattering genotype XH, 6 genes (2 for ethylene activity, 1 for gibberellin activity, 1 for MYB transcription factor activity, 1 for xylanase activity and 1 for glycosyl hydrolase activity) were up regulated in low seed shattering genotype (Fig. 7). Together, these results suggested these candidate genes might be associated with the regulation of seed shattering in E. sibiricus.
The first linkage map for E. sibiricus by next-generation sequencing
E. sibiricus breeders have been working to improve economically important traits including yield potential, stress tolerance and persistence. To understand the molecular and genetic mechanisms underlying these important traits, we need some modern methods and tools to effectively identify QTLs and candidate genes involved in these traits. Molecular marker development and genetic linkage map construction are important preliminary basic works for undertaking molecular breeding activities in any crops . Especially, markers strongly related to preferred traits could be used in marker-assisted selection (MAS) to speed up the genetic improvement of desired agronomic traits. To date, a variety of molecular markers including RAPD, SRAP, AFLP and SSRs have been wildly used for linkage map construction in a number of forage grasses such as tall fescue , meadow fescue , ryegrass , orchardgrass [38, 39], alfalfa , white clover , red clover , and Elymus wheatgrass . However, the number of mapped markers are limited in many previously reported maps. SLAF sequencing, as a next-generation sequencing technology for genome-wide SNP discovery, has been successfully used for high-density genetic map construction in many plant species including rice , cucumber , kiwifruit (Actinidia chinensis) , mei (Prunus mume) , sesame , even in animals such as chicken , and white shrimp . However, large scale SNP mining in E. sibiricus lagged behind other species due to its large, complex, and nature polyploidy. Here, we used the SLAF-seq to develop and identify 370,470 SLAF markers, of which 97,387 were polymorphic with a polymorphism rate of 26.29%. The polymorphism rate of SLAF markers between the two parents was 26.29%, higher than previous reports in cucumber (9.57%)  and sesame (5.12%) , suggesting a considerable difference between the two parental genotypes. The linkage map contained 14 linkage groups and spanned 1866.35 cM. Based on SLAF-seq, the map quality of the present genetic maps was similar to previously reported genetic maps for other several species, although, they were the first ones reported for this species. For example, Zhang et al.  reported the first high-density genetic map for sesame. A total of 1233 markers were mapped on the 15 linkage groups, with an average marker density of 1.20 cM. In general, these results proved that SLAF-seq is a powerful high-throughput technology for the whole-genome wide SNP discovery and is effective for E. sibiricus linkage map construction.
According to our results, the number of SLAF markers on each linkage group varied from 27 (LG7) to 565 (LG 11). Especially, some markers tended to highly cluster in some regions on LG9, LG10 and LG 11. The similar results were found in other plants such as grape , sunflower , and tree peony . This phenomenon may result from the non-random distribution of mapped markers on linkage group and the uneven recombination rates and marker polymorphism between mapping parents on some chromosomes . Moreover, four gaps larger than 10 cM were located on LG1, LG7, LG8, and LG14. The lack of maker polymorphism and a shortage of marker detection in these regions may have contributed to this finding [51, 52].
Application of this map for the QTL detection of seed yield-related traits
Like most native grasses, E. sibiricus has serious seed shattering which cause large seed yield losses during harvest, making commercial seed production difficult. In this study, Y1005 and ZhN06 were selected and used as parents because they are genetically divergent and have several contrasting seed traits. In the F2 mapping population, eight seed yield-related traits showed considerable phenotypic variation. For example, seed shattering in mapping population ranged from 5.14 gf to 18.80 gf during 2016, from 5.66 gf to 20.68 gf during 2017, and from 6.53 gf to 19.62 gf during 2018. Some progenies had lower seed shattering than their parents. Thus, the use of low seed shattering genotype provides QTLs for low seed shattering that could prove robust for breeding lower seed shattering genotypes.
Genetic linkage map allows for comparative genetic studies with other species, provides important information about the genome structure and evolution of a species, and lays a foundation for studying complex and important agronomic traits . The major objective of this study was to reveal the genetic mechanisms controlling seed yield-related traits in E. sibiricus. Our comparative genome analysis indicated that E. sibiricus was more closely related to wheat and barley. In this study, a total of 8 markers within seed shattering QTL (SS, SSD and SSC) regions could be identified on wheat chromosomes 1A, 3B, 6A, 6B, and 7B, and barley chromosomes 1H, 2H, 3H and 5H. In wheat, the Q gene on chromosome 5A was identified as a major domestication gene, which encodes an AP2 transcription factor largely responsible for free seed shattering . TaqSH1, encoding a BEL1-like protein, was located on the homoeologous group 3 chromosome in wheat. Overexpression of TaqSH1 gene in transgenic Arabidopsis plants could down-regulate several well-known abscission-related genes such as HAESA and KNAT1/6, suggesting this gene might play as a key upstream regulator for abscission zone development . In barley, the non-brittle rachis trait was controlled by btr1 and btr2 genes on chromosome 3H. Further, Pourkheirandish et al.  cloned and identified the Btr1 and Btr2 genes and elucidated the mechanism underlying the disarticulation of the wild type barley spike.
Based on our comparative genome analysis, marker 5164 and marker 144,585 were on wheat Chr3B, marker 124,682 was on barely Chr 3H. Together, these results highlight the possibility for seed shattering candidate gene identification in E. sibiricus based on these genetic maps and the synteny with model grasses. QTLs on other genomic regions may provide new candidate genes for seed shattering in E. sibiricus.
Candidate genes for seed shattering
Plant hormone play an important role in regulating plant growth and development processes. In this study, we identified 15 potential seed shattering candidate genes involved plant hormone including abscisic acid (ABA), ethylene, auxin, gibberellin and jasmonic acid. Abscisic acid regulates many agronomically important development processes and numerous adaptive stress responses in plants . It plays a direct role in abscission of many plant organs such as seed, leaf, flower and fruit [5, 58]. Our previous studies found some ABA-related genes were up-regulated in the abscission zone and suggested these ABA-responsive genes may affect seed shattering [5, 8]. Ethylene is an important regulator of abscission of many plant organ such as seeds, fruit and leaf . Ethylene receptor genes (ETR1), the ethylene insensitive mutant of Arabidopsis, plays a role in delaying the shedding of floral parts . In our previous study, we reported that 5 ETR1 gene were up-regulated in abscission zone in Elymus nutans, suggesting the roles of ETR genes in regulating abscission . Ethylene-responsive factors (ERF) also regulate abscission of plant organs, including floral organs, leaf and seed. In tomato (Solanum lycopersicum) Ethylene-responsive factor 52 (SIERF52) is specifically expressed in pedicel abscssion zones. When the expression of SIERF52 was suppressed in transgenic plants flower abscission will be significantly delayed compared with wild type . Our transcriptome analysis showed 4 ethylene response factor genes were up-regulated in the abscission zone of high seed shattering genotype, suggesting their roles in promoting seed shattering. Jasmonic acid (JA) is an important regulator of plant growth, development and defense. It had good abscission-promoting effect and positively promoted the abscission of bean petiole expiants in the dark and light without enhancing ethylene production in bean petiole expiants . Similar results were reported in our study, we found three genes involved in JA-mediated signaling pathway were up-regulated in abscission zone of high seed shattering genotype XH-WS. However, it is difficult to identify which hormone or gene is key factor for regulating abscission process, as plant organ abscission is a complex and highly coordinated process involving multiple gene expressions in plant hormone signing pathway. A balance and interaction of these genes may have contributed to seed shattering in E. sibiricus.
Transcription factors play an important role in the signal transduction pathways. In this study we identified 7 transcription factor genes, such as MYB, MADS-box and WRKY genes. According to previous study in major crops. Many well-known genes for seed shattering are transcription factors genes like SH4  and qSH1 , STK . In rice, SH4 is a major QTL for seed shattering, which encodes a MBY3 transcription factor . And many MYB proteins are critical components of multiple hormone-mediated transcriptional regulatory, including ethylene, abscission acid and auxin, which act as important regulators of plant organ abscission . In our previous study, we identified 14 MYB genes up-regulated in the abscission zone in E. nutans, indicating their potential roles in regulating the development of abscission zone . In Arabidopsis, STK is a MADS-box transcription factor gene, which regulates the formation of seed abscission zone . WRKY transcription factors are key components in abscisic acid signaling, and play roles in regulating many plant processes, including seed development, the response to stress, and seed shattering . Therefore, we inferred these candidate transcription factors identified in this study may have the similar functions in the regulation of seed shattering.
Seed shattering is generally caused by the development and degradation of abscission layers that is located in the rachilla just below each seed. Our previous study showed that increased hydrolytic enzymes activity like cellulase and polygalacturonase in abscission zone are highly related to high seed shattering degree in E. sibiricus . Cellulase is a key hydrolytic enzyme, which plays role in plant cell wall loosening during plant organ abscission . In rice, an endo-1,4,-β- glucanase gene named as OsCel9D, is an important regulator in modifying cell wall structure and component during abscission, and mutations of this gene will reduce cell elongation and affect cellulose biosynthesis and increase the pectin content, which finally hamper the abscission process in seed shattering . In addition, xyloglucan endotransglucosylase (XTHs) were suggested to loosen plant cell wall through cutting and rejoining the xyloglucans that tether adjacent cellulose miscrofibrils . In this study we identified 2 cellulase genes and 2 XTHs genes, and transcriptome analysis showed that these candidate genes were differently expressed in the abscission zone of two E. sibiricus genotypes, indicating these genes may have contributed to seed shattering.
In general, seed shattering is an important agronomic trait that need improvement during wild grass domestication. Seed shattering is a complex biological process affected by environment factors, cultivation management and multiple changes in the metabolism process, abscission layer cell structure, and functional gene expression level. Previous studies have reported many genes associated with seed shattering, they are involved in hydrolytic enzymes activity, lignin biosynthesis and degradation, plant hormone signaling and response, transcription factors and protein kinase activity [5, 8, 15, 58]. In this study, we constructed the first genetic linkage map and identified seed-related QTLs and candidate genes for seed shattering. Results from this study mostly confirmed previous findings and also reported some new potential candidate genes for seed shattering. More studies are needed to explore their potential roles and functions in seed shattering in the future. In addition, a combination of multiple methods, including genetic mapping and whole-genome association analysis and transcriptome analysis could help us to identify more major loci underlying seed shattering in E. sibiricus.
Materials and methods
Plant materials and DNA extraction
The mapping population of 200 F2 individuals was obtained from self-pollinating a single F1 plant, which was developed from a cross between “Y1005” (male parent) and “ZhN06” (female parent). Y1005 and ZhN06 were collected from Sichuan and Gansu provinces, China, respectively. The parents were selected based on a previous evaluation for agronomic traits and genetic diversity . The two parental genotypes are genetically divergent and have several contrasting seed traits, including seed shattering, 1000-seed weight, seed width, spike length, awn length, and floret number per spike.
DNA extraction was carried out with young healthy leaves using the Qiagen DNeasy 96-well procedure (QIAGEN, Valencia, Calif). The quantity and quality of genomic DNA samples were evaluated using the NanoDrop ND1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and by 0.8% agarose gel electrophoresis.
The F2 population of 200 individuals and parents were grown and evaluated in the field of Yuzhong research farm, Lanzhou University, Gansu, China (elevation 1720 m, longitude 103°34′ E, latitude 35°34′ N) for mapping. Plants were spaced 0.5 m within rows and 0.5 m between rows. Phenotypic data for seed yield-related traits were evaluated, including spike length (SL), floret number per spike (FN), seed shattering (SS), awn length (AL), width of seed (WS), and 1000-seed weight (SW1) for three consecutive years (2016–2018). E. sibiricus plant has a spike inflorescence containing 15–30 spikelets, each spikelet consists of approximately 5 florets . Three previously reported methods were used to accurately evaluate seed shattering in this study. Breaking tensile strength (BTS) method was firstly used to determine seed shattering. The BTS value was measured upon detachment of seed from the pedicels by pulling, which is negatively related with seed shattering degree. Totally, thirty randomly chosen spikelets from middle part of the spike of each plant were examined at maturity (4 weeks after heading), and their average BTS values were calculated . The second method is that seed shattering degree (SSD) was measured following the described procedure by Yao  with a minor modification. Three spikes from each plant were released at a height of 1 m and freely fell down onto a hard surface. The seed shattering degree was expressed by a percentage (%) of the number of shattered seeds to the total number of seeds. The third method is that seed shattering rate (SSc) was evaluated at maturity. Based on the number of naturally shattered seeds, seed shattering was rated as follows: 1 (> 80% shattering), 2 (60–80% shattering), 3 (40–60% shattering), 4(20–40% shattering), 5 (< 20% shattering). Other seed traits: SL, FN, AL, WS, and SW1 were measured according to the methods described by Zhang et al. . The descriptive statistics of phenotypic data and the correlation analysis between years and traits were calculated by using SPSS software (SPSS, version 19 for Windows, SPSS Inc., Chicago, IL, USA). Heritability (h2) for each trait was estimated based on previously reported method: h2 = σg2/ (σg2 + σgy2/n + σe2/nr), where σg2 is the genotypic variance, σgy2 is the variance caused by the interaction between genotype and year, σe2 is the error variance, n is the number of years, and r is the number of replications .
SLAF library construction and high-throughput sequencing
SLAF library construction was performed according to the methods described by Sun et al. . For maximum SLAF-seq efficiency, a pilot experiment was carried out to establish and optimize the conditions required to avoid repetitive SLAFs, achieve an uniform distribution of SLAFs, and obtain optimal SLAF yield. Briefly, HaeIII restriction enzymes (New England Biolabs, NEB, USA) was used to digest the genomic DNA of the two parents and F2 population. A poly-A was added to the 3′ ends of digested fragments. These digested fragments were then ligated with Dual-index sequencing adaptors, and amplified by PCR. The PCR was carried out in reaction solutions containing the diluted restriction-ligation DNA samples, dNTPs, Q5® High-Fidelity DNA polymerase (NEB) and PCR forward primers 5′- AATGATACGGCGACCACCGA-3′ and reverse primer 5′-CAAGCAGAAGACGGCATACG-3′. The PCR products were then purified using Agencourt AMPure XP beads (Beckman Coulter, High Wycombe, UK) and pooled.
The pooled samples were separated with 2% agarose gel. Fragments of 464–494 bp (with barcodes and adaptors) were excised and purified using a QIAquick gel extraction kit (Qiagen, Hilden, Germany). The obtained SLAFs in the quality-tested library were used for paired-end sequencing on an Illumina HiSeq 2500 sequencing platform (Illumina, San Diego, CA, USA). To check the reliability of testing processes, we used the genome of Oryza sativa as a quality control to undergo the same procedures of library construction and sequencing as the E. sibiricus mapping population.
Sequence data analysis and genotyping
SLAF marker identification and genotyping were performed according to the procedures described by Sun et al. . Low-quality reads (with quality score < Q30) were deleted and then the left reads were assigned to the two parents and F2 individuals according to the duplex barcodes. The clean reads were obtained after filtering the barcodes and the terminal 5-bp positions from each read. All paired-end reads (200 bp per read) generated from SLAF-seq raw reads were clustered according to their sequence similarity. Sequences with over 90% identity were grouped into one SLAF locus. As E. sibiricus is self-pollinated species, an F2 population was obtained by self-pollinating the F1 plant of a cross between two fully homozygous parents with genotype aa or bb. Therefore, we only used the SLAF markers with aa×bb segregation pattern for genetic map construction.
Map construction and QTL analysis
HighMap software was used for linkage map construction with four steps: SLAF marker grouping, SLAF marker ordering, genotyping error correction, and genetic map evaluation . The single-linkage clustering algorithm was applied to assign the markers into linkage groups. The modified logarithm of odds (MLOD) score > 5 was set up to partition marker loci into linkage groups (LGs). For comparative genome analysis, we carried out the BLAST search between the mapped SLAF makers and the whole genome sequences of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) by using an E-value cutoff of 1e-10 and 90% identity cutoff . For QTL identification, the genotypic data of the mapped markers on the linkage map was integrated with the field phenotypic data of eight seed traits. Logarithm of odds (LOD) scores larger than the 5% cutoff value was used to identify significant loci associated with seed traits. The threshold value was determined through 1000 permutation test according to the composite interval mapping (CIM) method from “qtl” package of R. An interval mapping model with LOD scores of 3.0 for potential QTL was used for QTL detection. MapQTL 6.0  was used to estimate the percentage of phenotypic variation and additive effect explained by a QTL for a trait.
Candidate gene identification
In order to gain an in-depth understanding of the potential functions of these seed yield-related QTLs and identify candidate genes for seed-related traits, the SLAFs within QTL regions were subsequently searched against the Hordeum vulgare and Chinese spring wheat reference genome (ftp://ftp.ensemblgenomes.org/pub/plants/release-39/fasta/hordeum_vulgare/dna/; http://www.wheatgenome.org/News/Latest-news/IWGSC-Reference-Sequence-v1.0-browser-now-available-at-URGI) by using the basic local alignment search tool (BLAST). For annotation, the assembled sequences were queried using BLASTX (E-value ≤1e-5) against 7 public databases like the NCBI non-redundant protein sequence (Nr), Gene Ontology (GO), Protein family (Pfam), Cluster of Orthologous Groups (COG), Annotated protein sequence database (Swiss-Prot), and Kyoto Encyclopedia of Genes and Genomes (KEGG), euKaryotic Orthologous Groups (KOG). The expression profiles of candidate gene were obtained from E. sibiricus abscission zone transcriptome data (https://www.ncbi.nlm.nih.gov/biosample/6545378). The formula log2 (FC) was used to calculate the transcript fold-change, and the false discovery rate (FDR) control method was applied for the correction for multiple tests . Significant differentially expressed transcripts (DETs) between two samples were identified only when an absolute value of the log2 (FC) ≥ 2 and FDR significance score ≤ 0.01 were set as the thresholds. A heatmap was constructed for candidate genes using the Heatmap Illustrator (HemI 1.0) program (Beijing Institude of Genomics, CAS, Beijing, China) .
Availability of data and materials
Raw Illumina sequencing data are available in NCBI SRA: SRX6857199-SRX6857400 (https://www.ncbi.nlm.nih.gov/sra/?term=SLAF-seq+of+Elymus+sibiricus), other datasets generated or analysed during this study are included in this published article and its supplementary files.
Basic local alignment search tool
Breaking tensile strength
Composite interval mapping
Cluster of orthologous groups
Coefficient of variation
Differentially expressed transcripts
False discovery rate
Floret number per spike
Kyoto encyclopedia of genes and genomes
Clusters of orthologous groups for eukaryotic complete genomes
Logarithm of odds
The modified logarithm of odds
RefSeq non-redundant protein
Polymerase chain reaction
The percentage of phenotypic variation explained
Quantitative trait loci
Specific-locus amplified fragment sequencing
- SSC :
Seed shattering rate evaluated by five level of classifications
- SSD :
Seed shattering degree assessed by dropping from a height
1000 seed weight
SNP type, Trv means transversion, Tri means transition
Width of seed
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This work was supported by Chinese National Natural Science Foundation (No. 31971751), Chinese National Basic Research Program (2014CB138704), the Fundamental Research Funds for the Central Universities (LZUJBKY-2019-37), Major Science and Technology Projects of Gansu Province and 111 program (B12002). We also thank Mingshu Cao from AgResearch New Zealand for language editing.
This study was financially supported by grants from Chinese National Natural Science Foundation (No. 31971751), Chinese National Basic Research Program (2014CB138704), and the Fundamental Research Funds for the Central Universities (LZUJBKY-2019-37).
Ethics approval and consent to participate
These plant materials used in this study were obtained from Lanzhou University. E. sibiricus are not endangered or protected species, thus, no permissions or licences were required for collecting these samples and conducting this experiment.
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SLAF marker and their locations on each linkage.
Phenotypic data of parents and F2 population in E. sibiricus.
Thirty identified candidate genes for seed shattering within six QTL regions based on the functional annotation of barley and wheat genomes.
High-density genetic linkage maps of E. sibiricus.
Haplotype map of linkage map.
Heat map of linkage map.
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Zhang, Z., Xie, W., Zhang, J. et al. Construction of the first high-density genetic linkage map and identification of seed yield-related QTLs and candidate genes in Elymus sibiricus, an important forage grass in Qinghai-Tibet Plateau. BMC Genomics 20, 861 (2019). https://doi.org/10.1186/s12864-019-6254-4
- Elymus sibiricus
- Seed yield-related traits
- High density genetic linkage map
- Comparative genome analysis