Genome-Wide Association Study Discovered Favorable Single Nucleotide Poly Morphisms and Candidate Genes Associated with Ramie (Boehmeria Nivea L.) Colloidal Matters

ABSTRACT

Ramie (Boehmeria nivea L.) bast fiber is one of the most ancient natural fibers, which is used for textile only after degumming. Hemicellulose, pectin, and hydrotrope are the main colloidal matters in ramie fiber, which are removed when degumming. However, the genetic variation and control loci are poorly understood. Here, we analyzed the genetic variation of the colloidal matters and discovered the favorable single nucleotide poly morphisms (SNP) by genome-wide association study using 319 ramie core germplasms. In total, 21, 5, and 10 SNPs were found to be the significantly key control loci associated with hemicellulose, pectin, and hydrotrope, respectively, which changed the amino acid sequences of the reference genes. Fourteen key genes involved in various sugars biosynthesis and metabolism pathways were identified to regulate the hemicellulose content in ramie. Meanwhile, 7 calcium transporting or binding related genes were identified to involve in the biosynthesis of calcium magnesium pectinate-the main component of pectin. Moreover, 306 genes related to hydrotrope were identified and their function were also discussed. Our study first reveals the genetic variation and candidate genes related to the colloidal matters of ramie bast fiber, and provides tools for marker-assisted selection in improvement of agricultural traits.

摘要

苎麻（Boehmeria nivea L.）韧皮纤维是最古老的天然纤维之一，脱胶后才用于纺织. 半纤维素、果胶和水溶助长剂是苎麻纤维中的主要胶体物质，在脱胶时被除去. 然而，人们对遗传变异和控制位点知之甚少. 在这里，我们分析了胶体物质的遗传变异，并通过全基因组关联研究使用 319 种苎麻核心种质发现了有利的单核苷酸多态性 (SNP). 总共发现 21、5 和 10 个 SNP 分别是与半纤维素、果胶和水溶助长剂相关的显着关键控制位点，它们改变了参考基因的氨基酸序列. 鉴定了参与各种糖类生物合成和代谢途径的 14 个关键基因，以调节苎麻中的半纤维素含量. 同时，鉴定出7个钙转运或结合相关基因参与果胶的主要成分果胶钙镁的生物合成. 此外，还鉴定了 306 个与水溶助长剂相关的基因，并讨论了它们的功能. 我们的研究首次揭示了与苎麻韧皮纤维胶体物质相关的遗传变异和候选基因，为标记辅助选择改良农业性状提供了工具.

KEYWORDS:

• Ramie (Boehmeria nivea L.)
• bast fiber
• SNP
• content of colloidal matters
• candidate genes
• GWAS

关键词:

• 苎麻
• 韧皮纤维
• 胶体物质的含量
• 候选基因

Introduction

Bast fiber extracted from the stem bark of plants is one of the most important fiber types for human use. Ramie (Boehmeria nivea L.), which is native to China and one of the oldest fiber crops, has been utilized for producing textiles for more than 4700 years (Li 1970). The superior fiber obtained from its woody stem is long and highly durable, pure white in color and silky in texture, with a high degree of hygroscopicity and superior heat dissipation. These superior characteristics make ramie a highly versatile and useful natural raw textile material, and its high-end textile products have become increasingly popular in the worldwide.

Natural ramie fibers are composites made up of hollow cellulose fibrils fused together by hemicellulose and lignin matrix (John and Anandjiwala 2008). The main component of ramie phloem is cellulose (65–75%), and the second component is colloidal matters (25–30%), also known as bast non-cellulosic substances, which includes hemicellulose (14–16%), hydrotrope (6–8%), pectin (4–5%), lignin (0.8–1.5%), circumstances wax (0.2–2.0%), and others (Biswas et al. 2013). 30–100 cellulose molecules constitute microfibrils, then the microfibrils form the main framework of the fiber cell wall (Mokhothu and John 2015), which are the principle components of raw textile materials. Before using as raw textile materials, ramie bast fiber are performed degumming to remove the colloidal matters, which have extremely complicated composition and structure.

Hemicellulose are polysaccharides that comprise of several sugar moieties, which contain glucose, arabinose, xylose, mannose, galactose, and also other monomers. In fiber cell wall, hemicellulose molecules are held to cellulose by hydrogen bonds to form a hemicellulose-cellulose network, which is seemingly the main structural constituent of the fiber. The sugar group of hemicellulose side chain crosslinks lignin polymer through covalent bonds such as aryl ether bond, phenyl glycoside bond, acetal bond, and ester bond (Sella Kapu and Trajano 2014; Zhou et al. 2016). Hemicellulose forms a network structure through its side chain interaction. Because of its network structure with cellulose and other biological molecules, hemicellulose is the most difficult colloidal matter to remove in the degumming process.

Pectin, one class of high molecules polymerized by galacturonic acid, is a major component of primary cell wall of natural fiber, and plays the role of closely bonding adjacent fibrous cell together (Willats et al. 2001). Pectin can be divided into soluble pectin and insoluble pectin. The soluble pectin is mainly methyl pectinate, which is characterized by high solubility in water. The insoluble pectin, also known as raw pectin, mainly exists in the form of calcium and magnesium salts of pectinic acid, which has a network structure and is insoluble in water but can be hydrolyzed quickly in dilute acid and dilute alkali solutions (Evans, Akin, and Foulk 2002). Thus, pectin is easy to remove in degumming process. In natural ramie bast fiber, there are some micromolecules, pigments, sugars and other substance that are easily soluble in water, called hydrotrope. Although hydrotrope accounts for 6–8% of the colloidal matter in ramie bast fiber, it is not the difficulty of ramie fiber degumming because it is easy to remove in degumming process.

Presently, the method of ramie fiber degumming includes chemical degumming, physical degumming and biological degumming. However, it can induce various adverse effects on the environment or ramie fiber using these methods. For example, chemical degumming is time-consuming, multiprocess, high energy consumption, large consumption of water resources and serious environmental pollution (Pan, Day, and Mahalanabis 1999), which seriously restricts the development and application of ramie industrial degumming. The long degumming cycle, large fiber damage and high difficulty in strain screening of bacterial degumming are important factors limiting its large-scale development and application. Breeding varieties with low content of colloidal matter is an effective way to solve the problem caused in fiber degumming. Analyzing the genetic basis of glial components, mining key candidate genes, and then improving crops by means of molecular biology is a fast and effective way to breed new crop varieties. However, little attention has been paid to the genetic basis and the related regulatory genes in colloidal matters of ramie bast fiber.

Previously, we found that the chemical compositions in different parts of ramie bast fiber showed significant differences (Ou et al. 2020), and also found that (Ou et al. 2019) different stripping methods significantly affect the yield and quality of ramie fiber, suggesting the complex components of ramie bast fiber. Genetic improvement of ramie varieties by means of molecular biology is an effective strategy to cultivate high-quality fiber varieties. In this study, we focus on excavating the significantly associated single nucleotide polymorphism (SNP) of ramie colloidal matters (hemicellulose, hydrotrope and pectin) by whole-genome scanning with 319 core germplasms of ramie. The related regulatory genes are screening by aligning the ramie genome, and the function of the predicted candidate genes are also discussed. This research will provide the genetic basis of the components of ramie gum and reveal the key genetic regulatory sites. Our study will provide significant reference for ramie molecular breeding.

Material and methods

Plant material and phenotypic analysis

A total of 319 core germplasms of ramie collected from China, India, Indonesia, Japan and Cuba (Table S1), were used in this study. These germplasms were planted in the Yuanjiang Experimental Station, Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences (E112°36“, N28°83”). All germplasms were propagated by asexual propagation, and each germplasm was planted in a row with six plots (one plant per plot), with a distance of 50 cm between plots and 70 cm between rows. The bast fiber of the six plants for each germplasm were mixed harvested in June (season 1) and August (season 2), 2018, respectively, and dried under natural conditions for the determination of hemicellulose, hydrotrope, and pectin.

Before determination, the bast fiber for each germplasm was divided into two equal parts: one was discarded and the other was retained, then the retained one was divided into two equal parts via the same method. The retained sample was separated repeatedly by this method, until its weight was about 40–50 g. The middle part of the final retained sample was used for phenotypic analysis, and three replicates were set for each germplasm, with ~3 g for each replicate. Sample was dried to constant weight in an oven in 105°C, immediately put in a dryer for 30 ± 5 min, and weighed (the weight of hydrotrope, hemicellulose and pectin was recorded as W_hy1, W_hc1 and W_p1, respectively). Next, the sample was put in a conical bottle filled with 150 ml of extractant. Distilled water, 20 g·L⁻¹ of sodium hydroxide and 5 g·L⁻¹ of ammonium oxalate was the corresponding extractant for hydrotrope, hemicellulose, and pectin determination, respectively. After 20 min for extractant soaking, the sample was heated for 30 min in a microwave oven, then washed with running tap water. The clean sample was dried in 105°C, cooled in a dryer, and weighed (the weight of hydrotrope, hemicellulose and pectin was recorded as W_hy2, W_hc2, and W_p2, respectively.). The content of hydrotrope, hemicellulose and pectin was calculated with the corresponding following formula: W_hy = 100 × (W_hy1 - W_hy2)/W_hy1, W_hc = 100 × (W_hc1 - W_hc2)/W_hc1, W_p = 100 × (W_p1 – W_p2)/W_p1

GWAS

Resequencing was performed using the 319 core ramie germplasms and 3,494,975 high-quality SNPs was received (Huang 2020). Using these high-quality SNPs and the phenotypic data, phenotype-genotype association analysis and allele effect calculations were performed with the TASSEL 5.0 software. The general linear model adjusted using the Q-matrix (GLM-Qmatrix model) was used in the association analysis. Those with P < .1 and P < .05 were defined as significant trait-associated SNPs.

Identification and annotation of candidate genes

Sequences of favorable SNPs were used to blast the ramie genome (data unpublished), and the genes located in the region 100 kb upstream or downstream of the trait-associated SNPs were identified as candidate genes. Functional annotations of the candidate genes were performed by sequence comparison against NCBI non-redundant (NR, http://ftp.ncbi.nih.gov/blast/db/), Swiss-Prot (http://www.uniprot.org/), Gene Ontology (GO, http://www.geneontology.org/), Clusters of Orthologous Groups (COG, http://www.ncbi.nlm.nih.gov/COG/), Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/), EuKaryotic Orthologous Groups (KOG, http://ftp.ncbi.nih.gov/pub/COG/KOG/), and Pfam (http://pfam.xfam.org/) databases.

Results

Changes of hemicellulose, hydrotrope, and pectin in the 319 ramie germplasms

The content of hemicellulose, hydrotrope, and pectin of the ramie bast fiber were detected at June, 2018 (season 1), and August, 2018 (season 2), using 319 ramie core germplasms. The content of hemicellulose ranged from 0.114 to 0.229, with an average of 0.142 at season 1; from 0.055 to 0.155, with an average of 0.097 at season 2 (Table 1). The coefficient of variation (CV) at season 2 for hemicellulose was more than twofolds compared with that of season 1. The changes of hydrotrope content ranged from 0.038 to 0.161 for the two seasons, and the CV was about 19 at the two detected seasons, which showed a large variation among the 319 ramie germplasms (Table 1). The content of pectin ranged from 0.033 to 0.104, with a CV value of 21.11 at season 1; from 0.091 to 0.202, with a CV value of 11.09 at season 2 (Table 1). The changes of hemicellulose, hydrotrope, and pectin content at season 1 and season 2 showed a normal distribution with good continuity (Figure 1). These data illustrated the large variation amplitude of the detected traits of the core germplasm population, indicating that it could be an excellent population for marker-trait GWAS.

Figure 1. Normal distribution curves of hemicellulose, hydrotrope and pectin detected at season 1 (June, 2018) (a) and season 2 (August, 2018) (b).

Table 1. Phenotypic variation of different gum components of ramie bast fiber.

	Detected stage	Mean	SD	Max	Min	CV(%)
Hemicellulose	June, 2018	0.142	0.0134	0.229	0.114	9.39
	August, 2018	0.097	0.0185	0.155	0.055	19.11
Hydrotrope	June, 2018	0.092	0.0178	0.161	0.062	19.40
	August, 2018	0.065	0.0125	0.110	0.038	19.22
Pectin	June, 2018	0.064	0.0136	0.104	0.033	21.11
	August, 2018	0.149	0.0165	0.202	0.091	11.09

Identification of favorable SNPs and candidate genes associated with hemicellulose

In total, 80 significant SNPs associated with hemicellulose were identified, with 40 of them distributing on chromosome 1, 19 on chromosome 5, 5 on chromosome 11, and 7 on chromosome 13 (Figure 2, Table S2). There were 47 SNPs located on the intron of the reference genes, 21 located on exon, and 12 on UTR (Table 2, Table S2). The 21 SNPs located on the exon region involved 14 genes, whose amino acid sequences were changed due to the existence of SNPs. Two genes (Maker00076265 and Maker00086604) were early terminated when translated into amino acid sequence, which was induced by the existence of the SNPs located on the exon of Maker00076265 and Maker00086604. Maker00076265 encodes a cytochrome P450 protein of 486 amino acid residues (named BnP450-1), and the corresponding mutant gene encodes a protein of 334 amino acid residues (named BnP450-M1), whose predicted structure was different with BnP450–1 (Figure 3(a,b,e,f). Maker00086604 encodes an auxin response factor of 844 amino acid residues (named BnARF-1), and the corresponding mutant gene encodes a protein of 514 amino acid residues (named BnARF-M1), whose predicted structure was different with BnARF-1 (Figure 3(c,d,g,h).

Figure 2. Significant SNPs and candidate genes associated with hemicellulose identified at season 1 (June, 2018) (a) and season 2 (August, 2018) (b).

Figure 3. The predicted protein structure of BnP450–1 (a), BnP450-M1 (b), BnARF-1 (c) and BnARF-M1 (d). The ramachandran plots of BnP450–1 (e), BnP450-M1 (f), BnARF-1 (g) and BnARF-M1 (h).

Table 2. Hemicellulose significantly associated SNPs located on exon of the related genes.

ChrName	GeneID	Position	SNP ID	Allels	Mutation Effect	Gene Annotation
chr1	Maker00075072	4825385	Maker75072-385	C/T	Amino acid sequence change	NAC domain-containing protein
chr1	Maker00075196	2024018	Maker75196-018	C/G	Amino acid sequence change	hypothetical protein
chr1	Maker00076265	2888070	Maker76265-070	A/G	Early termination	cytochrome P450 90A1
chr1	Maker00075620	2891071	Maker75620-071	A/T	Amino acid sequence change	nuclear pore complex protein
chr1	Maker00075124	3436304	Maker75124-304	A/G	Amino acid sequence change	Influenza virus NS1A-binding protein-A-like protein
chr1	Maker00075124	3436358	Maker75124-358	A/G	Amino acid sequence change	Influenza virus NS1A-binding protein-A-like protein
chr1	Maker00075124	3437714	Maker75124-714	C/T	Amino acid sequence change	Influenza virus NS1A-binding protein-A-like protein
chr1	Maker00075124	3437729	Maker75124-729	A/T	Amino acid sequence change	Influenza virus NS1A-binding protein-A-like protein
chr1	Maker00075027	4418661	Maker75027-661	G/T	Amino acid sequence change	Ethylene-responsive transcription factor
chr1	Maker00075461	4467280	Maker75461-280	C/T	Amino acid sequence change	hypothetical protein L484
chr1	Maker00075786	5431369	Maker75786-369	A/G	Amino acid sequence change	hypothetical protein L484
chr4	Maker00000232	26020988	Maker00232-988	A/G	Amino acid sequence change	Alpha-mannosidase
chr4	Maker00000232	26020994	Maker00232-994	C/G	Amino acid sequence change	Alpha-mannosidase
chr5	Maker00066873	11646277	Maker66873-277	C/T	Amino acid sequence change	mitochondrial uncoupling protein 1 isoform X2
chr8	Maker00086604	12912278	Maker86604-278	A/T	Early termination	Auxin response factor
chr11	Maker00052155	13768879	Maker52155-879	A/G	Amino acid sequence change	Disease resistance protein RPM1
chr11	Maker00052155	13768906	Maker52155-906	A/G	Amino acid sequence change	Disease resistance protein RPM1
chr11	Maker00052155	13769035	Maker52155-035	A/C	Amino acid sequence change	Disease resistance protein RPM1
chr11	Maker00052155	13769081	Maker52155-081	G/T	Amino acid sequence change	Disease resistance protein RPM1
chr13	Maker00082564	10648380	Maker82564-380	C/T	Amino acid sequence change	Cytoplasmic 60S subunit biogenesis factor REI1 homolog1
chr13	Maker00082795	10655963	Maker82795-963	A/G	Amino acid sequence change	hypothetical protein L484

To receive candidate genes related to hemicellulose synthesis or metabolism, the genes located in the region 100 kb upstream or downstream of the trait-associated SNPs were identified, and their function were also analyzed. Particularly, two glucose biosynthesis related genes (BnG6PT3 and BnUGPA3), one trehalose (BnTPPA), one fructose (BnABF1), one glucan (BnGE134) biosynthesis related genes were identified nearly the SNPs of Maker75072–385, Maker75196–018, Maker75707–243, which were mapped onto the chromosome 1 (Figure 2, Table 3). On chromosome 13, one glucose (BnG6PI), one glucan (BnGUN7), and one xylan (BnIRX9 H) biosynthesis-related genes were detected, which was nearby the SNP Maker03162–140, Maker96309–904 and Maker24425–396, respectively. One galactose metabolism-related gene (BnPT225), one glucose biosynthesis-related gene (BnHXK1), one mannose biosynthesis-related gene (BnMANA1), and one glucan biosynthesis related gene (BnGBG6) were identified on chromosome 11, 2, 4 and 5, respectively. Glucose, mannose, glucan, trehalose, fructose, and xylan are main components of hemicellulose. The above genes identified nearby the significant SNPs related to hemicellulose may show a key role in controlling the hemicellulose content of ramie bast fiber.

Table 3. Candidate genes related with hemicellulose biosynthesis and metabolism.

	Name	GeneID	ChrName	Position	SNP ID	Allels	Annotation
1	BnTPPA	Maker00075985	chr1	4825385	Maker75072-385	C/T	trehalose-phosphate phosphatase A isoform X2
2	BnABF1	Maker00076017	chr1	4825385	Maker75072-385	C/T	acid beta-fructofuranosidase 1, vacuolar
3	BnG6PT3	Maker00074903	chr1	4825385	Maker75072-385	C/T	Glucose-6-phosphate exchanger SLC37A2
4	BnGE134	Maker00075678	chr1	2024018	Maker75196-018	C/G	Glucan endo-1,3-beta-glucosidase 4
5	BnUGPA3	Maker00075707	chr1	3254243	Maker75707-243	A/G	UTP-glucose-1-phosphate uridylyltransferase 3
6	BnPT225	Maker00051236	chr11	13768879	Maker52155-879	A/G	Probable sugar phosphate/phosphate translocator
7	BnG6PI	Maker00003162	chr13	1178140	Maker03162-140	A/G	glucose-6-phosphate isomerase
8	BnIRX9 H	Maker00024425	chr13	7585396	Maker24425-396	C/T	beta-1,4-xylosyltransferase
9	BnGUN7	Maker00096309	chr13	15148904	Maker96309-904	C/T	endoglucanase 9-like
10	BnHXK1	Maker00091684	chr2	18607028	Maker91684-028	C/T	Hexokinase-1
11	BnMANA1	Maker00000232	chr4	26020988	Maker00232-994	A/G	Alpha-mannosidase
12	BnGBG6	Maker00067238	chr5	11646277	Maker66873-277	C/T	glucan endo-1,3-beta-glucosidase 6-like

Identification of favorable SNPs and candidate genes associated with pectin

Twenty-nine significant SNPs associated with pectin were identified, which were mainly distributed on the chromosome 11 (10), chromosome 13 (7) and chromosome 8 (5) (Figure 4(a,b), Table S3). Eighteen of the SNPs located on the intron of the reference genes, 5 on UTR, and 6 on exon. The 6 SNPs located on the exon involved 5 genes, and the amino acid sequences encoded by 4 of the genes were changed due to the existence of SNPs (Table 4). Genes located in the region 100 kb upstream or downstream of the significant SNPs were scanned to identified the candidate genes related to pectin biosynthesis. In total, 189 genes were identified, which were mapped on the chromosome 13 (59), chromosome 9 (31), chromosome 3 (29), chromosome 8 (18), chromosome 11 (16), and chromosome 12 (11) of ramie (Table S4). KEGG analysis showed that 74.0% of the identified genes from the top 20 significant enriched differential pathways involved in metabolism, which included amino sugar and nucleotide sugar metabolism, phenylalanine metabolism, cyanoamino metabolism, and others (Figure 5(a), Figure S1). Calcium magnesium pectinate is the main component of pectin, and particularly, 7 calcium transporting or binding related genes were identified nearby the SNPs of Maker36972–744, Maker86695–027, Maker58329–659, Maker09298–307, and Maker63012–296 (Table 5), hinting the key genes functioning in the controlling of the pectin component.

Figure 4. Significant SNPs associated with pectin identified at season 1 (June, 2018) (a) and season 2 (August, 2018) (b). Significant SNPs associated with hydrotrope identified at season 1 (June, 2018) (c) and season 2 (August, 2018) (d).

Figure 5. Bar graph of the top 20 significantly enriched differential pathways of pectin (a) and hydrotrope (b) synthesis and metabolism related candidate genes generated by KEGG analysis.

Table 4. Pectin and hydrotrope significantly associated SNPs located on exon of the related genes.

ChrName	GeneID	Position	SNP ID	Allels	Mutation Effect	Gene Annotation
Pectin
chr3	Maker00030691	7008695	Maker30691-695	A/G	Amino acid sequence change	hypothetical protein L484
chr9	Maker00009488	5462307	Maker09488-307	A/G	Amino acid sequence change	probable E3 ubiquitin-protein ligase
chr11	Maker00037509	3410538	Maker37509-538	A/G	Nonsense mutation	hypothetical protein L484
chr13	Maker00083008	9507111	Maker83008-111	A/C	Amino acid sequence change	protein NRT1/PTR FAMILY 5.10-like
chr13	Maker00083008	9507114	Maker83008-114	C/G	Amino acid sequence change	protein NRT1/PTR FAMILY 5.10-like
chr13	Maker00082756	10875906	Maker82756-906	G/T	Amino acid sequence change	hypothetical protein L484
Hydrotrope
chr1	Maker00075094	3409136	Maker75094-136	A/C	Amino acid sequence change	Sorting nexin 2A
chr4	Maker00057207	21725134	Maker57207-134	C/T	Amino acid sequence change	hypothetical protein L484
chr4	Maker00000105	22097629	Maker00105-629	C/T	Nonsense mutation	hypothetical protein L484
chr4	Maker00000105	22097632	Maker00105-632	A/G	Amino acid sequence change	hypothetical protein L484
chr4	Maker00000423	22100622	Maker00423-622	C/G	Amino acid sequence change	6-phosphogluconate dehydrogenase
chr5	Maker00053489	8283061	Maker53489-061	A/C	Amino acid sequence change	Subtilisin-like protease
chr9	Maker00060587	1794541	Maker00060587	C/T	Amino acid sequence change	Probable E3 ubiquitin ligase
chr9	Maker00009325	3017383	Maker09325-383	C/T	Nonsense mutation	PRA1 family protein D
chr10	Maker00063152	3302097	Maker63152-097	A/G	Amino acid sequence change	Squamosa promoter-binding-like protein 12
chr11	Maker00037923	1394213	Maker37923-213	A/G	Nonsense mutation	putative LRR receptor-like serine/threonine-protein kinase
chr11	Maker00008667	16829064	Maker08667-064	A/T	Amino acid sequence change	Trigger factor-like protein TIG
chr11	Maker00008667	16829065	Maker08667-065	C/T	Amino acid sequence change	Trigger factor-like protein TIG
chr11	Maker00008667	16829101	Maker08667-101	G/T	Amino acid sequence change	Trigger factor-like protein TIG
chr14	Maker00019022	4812412	Maker19022-412	A/G	Nonsense mutation	Histone-lysine N-methyltransferase
chr14	Maker00019022	4813303	Maker19022-303	A/T	Nonsense mutation	Histone-lysine N-methyltransferase

Table 5. Calcium ion transporting and binding-related genes identified nearby the pectin associated SNPs.

ChrName	GeneID	Position	SNP ID	Allels	Gene Annotation
chr3	Maker00036972	14510744	Maker36972-744	A/G	Mitochondrial Rho GTPase 1, involving in calcium ion binding process
chr3	Maker00037313	14510744	Maker37313-744	A/G	Cation/calcium exchanger 5
chr8	Maker00086695	11753027	Maker86695-027	C/T	calcium-dependent protein kinase
chr8	Maker00087357	11753027	Maker87357-027	C/T	Calcium-transporting ATPase
chr12	Maker00058329	10932659	Maker58329-659	A/G	Proline-rich receptor-like protein kinase PERK4, calcium-mediated signaling
chr9	Maker00009298	5462307	Maker09298-307	A/G	putative calcium-binding protein CML22
chr10	Maker00063012	2852296	Maker63012-296	C/T	Calcium-binding protein CML37

Identification of favorable SNPs and candidate genes associated with hydrotrope

The hydrotrope of ramie bast fiber includes a variety of substances, such as micromolecules, pigments, sugars and other substance. In total, fifty-two significant SNPs related to hydrotrope ramie bast fiber were identified, which were distributed on 11 chromosomes of ramie (Figure 4(c,d), Table S5). Thirty-three of the SNPs located on the intron of the reference genes, 15 on exon and 4 on UTR (Table S5). The 15 SNPs located on exon involved 11 genes, and the amino acid sequences encoded by 10 of the genes were changed due to the existence of SNPs (Table 4). Genes located in the region 100 kb upstream or downstream of the significant SNPs were scanned to identified the candidate genes related to hydrotrope biosynthesis and metabolism. Totally, 306 genes were identified which were mainly distributed on the chromosome 14 (43), chromosome 13 (40), chromosome 3 (35), chromosome 6 (29), chromosome 11 (28) and chromosome 1 (27) (Table S6). The top 20 significantly enriched differential pathways showed that the candidate genes relatively evenly involved in fructose and mannose metabolism, nitrogen metabolism, ABC transporters, biosynthesis of amino acids and others processes (Figure 5(b), Figure S2), which is consistent with the multiple components of hydrotrope.

Discussion

The components of colloidal matter of ramie bast fiber are complex, and the genetic basis and molecular regulation mechanism of colloidal matter are still unclear up to now. Here, we performed a strategy of GWAS with 319 ramie core germplasms to identified significantly SNPs and key candidate genes associated with the regulatory of hemicellulose, pectin, and hydrotrope. In total, 153 significantly SNPs (80, 21 and 52 associated with hemicellulose, pectin, and hydrotrope, respectively) were identified, and 88 of them were located on the intron of the reference genes, 21 on UTR, and 42 on exon, suggesting most of the colloidal matter regulatory related SNPs being the nonsense mutation. The SNPs significantly associated with hemicellulose mainly distributed on the chromosome 1, 5, 11 and 13 of ramie, while the SNPs associated with pectin mainly located on the chromosome 1, 3, 6, 10 and 11. The significantly SNPs related to hydrotrope were distributed on the first, eleventh and thirteenth chromosome of ramie. These results denoted that the genetic regulation sites of the ramie colloidal matter were mainly concentrated on chromosome 1, 11 and 13.

Because of comprising about half of the total gummy components, hemicelluloses are the main removal objective in the degumming process of ramie bast fiber. Hemicelluloses are polysaccharides that comprise of sugar moieties, such as glucose, arabinose, xylose, mannose, galactose and also other monomers (Scheller and Ulvskov 2010). Due to its intricate network structure with cellulose and other biological molecules (Sella Kapu and Trajano 2014; Zhou et al. 2016), as well as its complex components, little of the genetic basis and the regulatory molecular mechanism of hemicellulose is known in ramie. Several researches have been conducted on other plant species for detecting the regulatory controlled sites of hemicellulose. For example, three QTLs for hemicellulose were detected in Brassica napus L. (Liu et al. 2013). Seven and 9 significant SNPs associated with hemicellulose were identified in sorghum (Laavanya et al. 2021) and Populus trichocarpa (Porth et al. 2013), respectively. In this study, 21 SNPs associated with hemicellulose were identified in ramie, which induced the changes of amino acid sequences of the reference genes. Especially, the two genes BnP450-1 and BnARF-1 were early terminated when translated into amino acid sequence, as a result of the mutation on their exons caused by the existence of the SNPs. Thus, the 21 SNPs could be considered as the key genetic controlled sites in regulating the hemicellulose in ramie. The homologous gene of BnP450-1 in Arabidopsis shows a regulating role in cell division of leaf and pollen germination via brassinosteroids signaling pathway (Vogler et al. 2014; Zhiponova et al. 2013). The P450 family genes play various roles in plant growth and development and stress resistance, and our study may show a novel function of P450 family genes in biosynthesis of hemicellulose. Meanwhile, we identified 12 key genes (Table 3) related to the synthesis and metabolism of a variety of sugars, such as glucose, glucan, xylose and others, which are the main components of hemicellulose. These genes could be considered as candidate genes regulating the hemicellulose contents in ramie.

Conclusion

In conclusion, we performed a GWAS strategy with 319 ramie core germplasms to identified the significantly associated SNP of ramie gum as well as the associated genes for the first time. Key SNPs associated with hemicellulose (21), pectin (5), and hydrotrope (10) were mainly located on the chromosome 1,11 and 13 of ramie genome. The key regulatory candidate genes of hemicellulose (14) and pectin (7) were identified, while 306 genes regulating multiple pathways were found to be related to hydrotrope synthesis, which was consistent with its multicomponent composition. Our study provides the genetic basis of the components of ramie gum and reveal the key genetic regulatory sites, which provides significant reference for ramie molecular breeding.

Summary

• The contents of hemicellulose, pectin, and hydrotrope in ramie bast fibers were systematically studied with 319 ramie core germplasms for the first time.

• The genetic variations of hemicellulose, pectin, and hydrotrope in ramie were first revealed in this study.

• The key SNP loci and key genes associated with hemicellulose, pectin, and hydrotrope were identified. And the genetic regulation sites of the ramie colloidal matter were found mainly concentrated on chromosome 1, 11, and 13. The identified loci and genes may be promising targets for genetic engineering and selection for modulating the colloidal matters in ramie.

Acknowledgements

We thank the Natural Science Foundation of Hunan Province (2021JJ30767), the Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IBFC04), the Ramie Breeding Station of the National Fiber Industry Technology System (CARS-16-E03), Department of Science and Technology of Hunan Province, the National Breeding Center of Bast Fiber Crops, and the Key Laboratory of Genetic Breeding and Microbial Processing for Bast Fiber Crop Product of Hunan Province.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This research was supported by the Natural Science Foundation of Hunan Province (2021JJ30767), Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IBFC04), the Ramie Breeding Station of the National Fiber Industry Technology System (CARS-16-E03), and the Key Research and Discovery Projects in the Field of Agriculture in Hunan Province of China (2019NK2061).