Corresponding author information - plantphysiol.org · RTN) has been confirmed ... formation and seed coat color (Zhang et al., 2009). ... in the coding sequences of the candidate

1

Short title: Functional SNPs for leaf hair number 1

Corresponding author information: 2

Yuke He 3

Address: Fenglin Road 300, Shanghai 200032, China 4

E-mail: [email protected] 5

Title: Identification of functional single-nucleotide polymorphisms affecting leaf 6

hair number in Brassica rapa 7

All authors’ names and affiliations: 8

Wenting Zhang, Shirin Mirlohi, Xiaorong Li and Yuke He 9

National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in 10

Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, 11

Chinese Academy of Sciences, Fenglin Road 300, Shanghai 200032, China 12

13

Contribution of authors: 14

Y.H. conceived the project and research plan. W. Z. performed the experiments. W.Z. 15

and S.M. analyzed the data and wrote the article with contributions from all the 16

authors. X.L. co-supervised and complemented the writing. 17

18

One sentence summary: Functional SNPs for leaf hair number in Brassica rapa 19

were selected and non-functional SNPs excluded by intensive mutagenesis and 20

genetic transformation. 21

22

Funding information: 23

This work was supported by National Programs for Science and Technology Development 24

of China (Grant No. 2016YFD0101900) and Natural Science Foundation of China 25

(Grant No. 31571261) 26

27

Corresponding author email: [email protected] 28

29

Plant Physiology Preview. Published on April 25, 2018, as DOI:10.1104/pp.18.00025

Copyright 2018 by the American Society of Plant Biologists

www.plantphysiol.orgon August 26, 2018 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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2

30

Identification of functional single-nucleotide polymorphisms 31

affecting leaf hair number in Brassica rapa 32

33

Wenting Zhang1,2,3, Shirin Mirlohi1,2,3, Xiaorong Li1,2,3and Yuke He1,3* 34

35 1National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, 36

Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China 37 2Graduate School of the Chinese Academy of Sciences, Shanghai, China 38

3CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and 39

Ecology, Chinese Academy of Sciences, Shanghai, China 40

41

*Corresponding author. E-mail: [email protected] 42

43

44

ABSTRACT 45

Leaf traits affect plant agronomic performance; for example, leaf hair number 46

provides a morphological indicator of drought and insect resistance. Brassica rapa 47

crops have diverse phenotypes and many B. rapa single-nucleotide polymorphisms 48

(SNPs) have been identified and used as molecular markers for plant breeding. 49

However, which SNPs are functional for leaf hair traits and therefore effective for 50

breeding purposes remains unknown. Here we identify a set of SNPs in the B. rapa 51

ssp. pekinenesis candidate gene BrpHAIRY LEAVES1 (BrpHL1) and a number of 52

SNPs of BrpHL1 in a natural population of 210 B. rapa accessions that have hairy, 53

margin-only hairy, and hairless leaves. BrpHL1 genes and their orthologs and 54

paralogs have many SNPs. By intensive mutagenesis and genetic transformation, we 55

selected the functional SNPs for leaf hairs by exclusion of non-functional SNPs and 56

the orthologous and paraologous genes. The residue Trp92

of BrpHL1a was essential 57

for direct interaction with GLABROUS3 (BrpGL3) and thus necessary for formation 58

of leaf hairs. The accessions with the functional SNP leading to substitution of the 59

Trp92

residue had hairless leaves. The orthologous BrcHL1b from B. rapa ssp. 60

chinensis regulates hair formation on leaf margins rather than leaf surfaces. The 61

selected SNP for the hairy phenotype could be adopted as a molecular marker for 62



3

insect resistance in Brassica crops. Moreover, the procedures optimized here can be 63

used to explain the molecular mechanisms of natural variation and to facilitate 64

molecular breeding of many crops. 65

66

Key words: Brassica rapa; functional SNPs; leaf hairs; natural variation; SNPs; 67

molecular breeding by designing; trichome; 68

69

INTRODUCTION 70

Brassica is one of the most important branches of the Brassicaceae family, including 71

many varieties of common vegetable crops. By means of natural and artificial 72

selection through time, many crops in Brassica rapa have evolved that show obvious 73

differences in leaf traits such as heading Chinese cabbage (B. rapa ssp. pekinenesis), 74

non-heading Chinese cabbage (B. rapa ssp. chinensis), turnip (B. rapa ssp. rapifera), 75

and yellow sarson (B. rapa ssp. trilocularis). The Brassica database (BRAD) 76

website (http://brassicadb.org/brad/) has released the complete genome sequence of 77

several Brassica crops (Cheng et al., 2011). Traditionally, leaf shape, size and 78

curvature are the main traits in these crops that have been genetically selected for 79

improved yield and quality. Hence, studying the diversity of the leaf traits in 80

Brassica could provide valuable information to help understand leaf development 81

and leaf variation and how to genetically manipulate these vegetable crops in the 82

future. 83

A leaf hair (trichome) is an epidermal hair that serve as a physical barrier on 84

plant surfaces against biotic and abiotic stress, including insect herbivores, 85

pathogenic microorganisms, UV light, excessive transpiration, freezing, etc. (Harada 86

et al., 2010; Hegebarth et al., 2016; Nafisi et al., 2015; Van Cutsem et al., 2011). The 87

adaptive significance of leaf hairs for arid land plants has been documented 88

(Ehleringer and Mooney 1978). 89

Density and localization of leaf hairs vary with crops within B. rapa. In 90

Arabidopsis (Arabidopsis thaliana), leaf hairs usually exist throughout the whole 91

plant, except for the cotyledons and epicotyls. A range of mutants defining specific 92



4

aspects of trichome development have been found in Arabidopsis. The genetic 93

analysis of these mutants has revealed a number of key genes controlling this 94

patterning process, and the mechanism by which trichome differentiation is triggered 95

in individual cells has been best studied in Arabidopsis (Galway et al., 1994; 96

Oppenheimer et al., 1991; Payne et al., 2000; Rerie et al., 1994; Wada et al., 1997). 97

As most biological traits are genetically complicated, mapping quantitative trait 98

loci (QTL) is a powerful means for estimating the framework of the genetic 99

architecture for a trait and potentially identifying the genes responsible for a specific 100

phenotype. Recombinant inbred lines (RILs) of two Arabidopsis ecotypes, hairy 101

Columbia and less hairy Landsberg erecta, have been constructed to identify QTL 102

contributing to trichome number, and a major locus named REDUCED TRICHOME 103

NUMBER (RTN) has been confirmed (Larkin et al., 1996). In another study, four 104

recombinant inbred mapping populations based on six Arabidopsis ecotypes have 105

been used to reveal QTL controlling trichome density, and nine QTL have been 106

identified as responsible for trichome initiation and development (Symonds et al., 107

2005). 108

Some studies report that the leaf hairs in Chinese cabbage are controlled by a 109

single dominant gene, whereas others have shown that leaf hairs are a quantitative 110

phenotype, controlled by several major QTL (Song et al., 1995; Zhang et al., 2009). 111

The mechanisms of trichome development in Brassica crops and in Arabidopsis 112

might be highly conserved (Alahakoon et al., 2016; Nayidu et al., 2014). A gene 113

(Bra009770) located on chromosome A06 in B. rapa is homologous with 114

TRANSPARENT TESTA GLABRA1 (TTG1) in Arabidopsis, and it controls trichome 115

formation and seed coat color (Zhang et al., 2009). Moreover, nucleotide 116

polymorphisms of four alleles in the GLABROUS1 (GL1) ortholog (BrGL1) are 117

associated with hairless leaves (Li et al., 2011). In addition, a 5 bp deletion in Brtri1 118

(BrGL1) is related to a glabrous phenotype (Ye et al., 2016). 119

The molecular understanding of the functional consequences of genetic 120

variation is critical for application of single nucleotide polymorphisms (SNPs) to 121

plant breeding. Progress towards this goal has been mostly successful when the 122



5

genetic variation falls within a coding region. Unfortunately, most SNPs identified in 123

plants are located within large introns or are distal to coding regions. 124

SNPs have a wide distribution and can be found in any region of a gene, mRNA 125

or intergenic region. Although identification of SNPs is an important first step in 126

understanding the relationship between variation and phenotypes, a major challenge 127

in the post-GWAS era is to understand the functional significance of the identified 128

SNPs and to apply these SNPs to plant breeding. Usually, GWAS SNPs (SNPs at 129

QTL) in the coding sequences of the candidate genes are used for designing DNA 130

markers. In practice, many GWAS SNPs are not effective for selection of objective 131

traits and many breeders have experienced failures in the practice of molecular 132

marker-aided selection with GWAS SNPs. Among these SNPs, some are functional 133

for developmental events and traits because they affect the levels of gene expression 134

or translation, splicing, efficiency to enhance or inhibit mRNA stability and protein 135

function. Many polyploid plants have multiple gene copies and many SNPs. 136

Selection of the causal SNPs for certain traits largely depends on the exclusion of 137

non-functional SNPs. Identifying functional SNPs for objective traits from a large 138

number of SNPs presents a bottleneck in the process. Furthermore, successful 139

molecular breeding of crops relies largely on the accuracy of functional SNPs. 140

Recently, many SNPs have been identified in B. rapa (Kim et al., 2016; 141

Tanhuanpaa et al., 2016; Yu et al., 2016). However, which SNPs are responsible for 142

leaf hairs remain unknown. In this study, we took advantage of recent advances in 143

genome resequencing to perform QTL mapping using 150 RILs derived from the 144

cross between the hairy genotype Bre (B. rapa ssp. pekinensis) and the hairless 145

genotype Wut (B. rapa ssp. chinensis) of Chinese cabbage (Yu et al., 2012). We 146

identified functional SNPs from numerous SNPs in the candidate genes and their 147

duplicated copies, and optimized the procedures for selection of functional SNPs for 148

agronomic traits. The selected SNP for leaf hair is a molecular marker of insect 149

resistance in Brassica crops, and the optimized procedures can be used to explain the 150

molecular mechanism of natural variation and to manipulate molecular breeding of 151

many crops. 152



6

153

RESULTS 154

155

Genetic control of leaf hair number 156

The species B. rapa includes heading Chinese cabbage (B. rapa ssp. pekinensis), 157

non-heading Chinese cabbage (B. rapa ssp. chinensis), turnip (B. rapa ssp. rapa), 158

and yellow sarson (B. rapa ssp. trilocularis). Non-heading Chinese cabbage consists 159

of many crop types: baicai, caixin, caitai, purple caitai, taicai, wutacai. These crops 160

and crop types are characterized by their specialized product organs: curved leaves, 161

leafy heads, fleshy petioles, fleshy stems and fleshy roots. 162

To analyze the leaf variation of B. rapa, we collected 210 accessions of B. rapa. 163

The leaf hairs on young leaves of these accessions were observed under a 164

binocular stereo microscope. There were three types of leaves with regard to leaf 165

hairs (Table 1). Most of the accessions belonged to the "all hairy" leaves in which 166

the leaf hairs were visible on the surfaces and margins. Some accessions belonged to 167

the "margin-only" leaves in which the leaf hairs were visible only on the leaf 168

margins, and the rest of the accessions had hairless leaves. Among the 210 169

accessions observed, 99 showed the all-hairy phenotype while 16 displayed the 170

margin-only hairy phenotype (Table 1). Bre is a representative of the all-hairy 171

phenotype and Wut is a representative of the margin-only hairy phenotype: 172

numerous hairs were detected on the leaf surface and leaf margins of Bre whereas 173

only a few leaf hairs were seen on the leaf margins of Wut. (Fig. 1AB). Leaf hairs of 174

all-hairy and margin-only hairy phenotypes were not branched, in contrast with the 175

branched trichomes of Arabidopsis (Fig. 1CD). Most heading Chinese cabbage 176

accessions showed the all-hairy phenotype (Table 1, Fig. 1E, F, I, J), and most 177

non-heading Chinese cabbage accessions displayed a hairless or margin-only hairy 178

phenotype (Table 1, Fig. 1G, H, K, L). 179

180

Positional cloning of the HAIRY LEAVES1 (HL1a) loci 181

In the course of previous studies, the inbred lines of Bre and Wut were crossed to 182



7

construct RIL populations for inheritance analysis and chromosomal mapping of the 183

related QTL. One major QTL, qTr, located on chromosome 6, was identified as a 184

locus for hairy leaves, and Bra025311 in the reference genome of Chiifu-401-42 was 185

selected as a candidate gene (Yu et al., 2013). Bra025311 belongs to the MYB gene 186

family (Supplementary Fig. 1) and is homologous to Arabidopsis GL1 with 59% 187

amino acid identity (Supplementary Fig. 2A). GL1 encodes an R2R3-MYB 188

transcription factor with a central function in the leaf hair patterning pathway, and is 189

involved in epidermal cell fate specification in leaves, promoting leaf hair formation 190

and endoreplication (Szymanski et al., 1998). 191

We named the candidate gene Bra025311 in Bre and Wut as BrpHL1a (B. rapa 192

ssp. pekinensis HL1a) and BrcHL1a (B. rapa ssp. chinensis HL1a), respectively. The 193

gene body (5' UTR, exons, introns and 3' UTR) and cDNAs of Bre BrpHL1a and 194

Wut BrcHL1a were cloned on the basis of genomic resequencing. Bre BrpHL1a 195

showed 56 SNPs in the gene body compared with that of Bra025311 of 196

Chiifu-401-42 (Supplementary Fig. 3). Fifty-five SNPs were identified in Wut 197

BrcHL1a (Supplementary Fig. 4). Compared to Chiifu-401-42 BrpHL1a, 11 SNPs 198

were detected in the exons of Bre BrpHL1a, causing 7 nonsynonymous substitutions; 199

and 13 SNPs were found in the Wut BrcHL1a gene, leading to 9 nonsynonymous 200

substitutions. Compared to Bre BrpHL1a, Wut BrcHL1a had 2 more SNPs in the 201

exons: 274T/C (274th dTMP of the coding sequence was changed to dCMP) and 202

403T/G (403rd T to dGMP) (Fig. 2A). SNPs 274T/C and 403T/G of Wut BrcHL1a 203

caused two nonsynonymous substitutions W92R and Y135D while 256C/T (256th 204

dCMP to dTMP) of Wut BrcHL1b leads to one nonsynonymous substitution Y86H. 205

B. rapa is a mesohexaploid and has more duplicated genes than Arabidopsis. To 206

check the other copies of the BrpHL1a gene, we searched for the genome sequences 207

of a collection of B. rapa accessions. BrpHL1b and BrcHL1b were detected on 208

chromosome 9 in Bre and Wut, respectively. The alignment shows that BrpHL1b and 209

BrcHL1b are homologous with GL1 of Arabidopsis (Supplementary Fig. 2B). Thus, 210

four members of the BrpHL1 gene family were related to the hairy phenotype of B. 211



8

rapa. Our aim was to first discover which SNPs in the candidate BrpHL1a gene 212

were functional or causal for all hairy or hairless phenotypes. Then we aimed to 213

determine whether and how contributions of the other three members of the BrpHL1 214

gene family could be ruled out. 215

216

SNP analysis of BrpHL1a and BrpHL1b alleles in B. rapa 217

Compared to Chiifu-401-42 BrpHL1b, Bre BrpHL1b has an A/G SNP 85 bp 218

prior to the start codon, causing a shifted reading frame and prolongation of the first 219

exon. This gene also shows a 4.5 kb insertion in its second exon (Fig. 2B, 220

Supplementary Fig. 5A), which suggests that BrpHL1b was not functional in Bre. In 221

contrast, Wut BrcHL1b seemed to be functional as the A/G SNP and the large 222

insertion were not detected. 223

To find all the SNPs in B. rapa, we cloned and sequenced BrpHL1a and 224

BrpHL1b genes of 13 representative genotypes (Supplementary Table 1 and 2). A 225

subset of SNPs was detected. In total, 169 nucleotides (9%) of Chiifu-401-42 226

BrpHL1a and 27 nucleotides (2%) of Chiifu-401-42 BrpHL1b were substituted by 227

the other nucleotides of various BrpHL1a and BrpHL1b alleles, respectively. To 228

confirm the accuracy of SNPs, we cloned the full-length cDNA sequences of 229

BrpHL1a and BrpHL1b genes. Sequence analysis of these clones confirmed the 230

accuracy of the genomic sequences of BrpHL1a and BrpHL1b genes. 231

We then analyzed the association between SNPs of BrpHL1 genes and the hairy 232

phenotype. For BrpHL1a alleles, all the genotypes (Wut, Ripposinica and Qincai) 233

with SNP 274C showed the hairless phenotype (Supplementary Table 1), revealing a 234

association between 274C and the hairless phenotype. Among 10 genotypes with 235

274T, 7 showed all hairy phenotypes. Two of 7 genotypes with SNP 403G showed 236

the hairless phenotype. Four of 6 genotypes with 403T showed the all-hairy 237

phenotype. For BrpHL1b alleles, 2 of 3 genotypes with 255C/T showed the hairless 238

phenotype (Supplementary Table 2). Therefore, we were not certain that these SNPs 239

were associated with all-hairy phenotypes. 240

To clarify the relationship between SNPs and leaf hairs, we extended the SNP 241



9

calling to a natural population of 210 B. rapa accessions. The re-sequencing of these 242

accessions generated two paired-end libraries with 150-bp reads (Supplementary 243

Table 3). According to the reference genome of B. rapa v1.5 (Ref), the sequencing 244

depth of the parental lines was more than 10-fold in each accession, and the mapped 245

depth was about 9-20. Each SNP supported by fewer than 4 reads was filtered out, 246

leading to 0.5~1.69 million high-quality SNPs (Supplementary Table 4). These SNPs 247

include many nucleotide substitutions, insertions and deletions. 248

Based on the genomic data in the BRAD, the SNPs were used to update the 249

genomic sequences of the BrpHL1a alleles (Supplementary Table 5) and separately 250

estimated by grouping the 210 accessions. Among 28 accessions with SNP 274C, 24 251

were hairless while 3 were margin-only hairy (Table 2; Supplementary Table 6), thus 252

showing the high association between SNP 274C and the hairless phenotype. Among 253

184 accessions with SNP 274T, 96 showed the all-hairy phenotype, revealing that 254

nearly half of the accessions with SNP 274T failed to show the all-hairy phenotype. 255

Surprisingly, the accessions with SNP 274T included a large proportion of hairless 256

accessions and a small proportion of margin-only hairy accessions. 257

258

Expression patterns of BrpHL1 genes 259

RT-qPCR and RT-PCR were used to examine the differences in expression of 260

BrpHL1a/BrcHL1a between Bre and Wut using the same pair of primers. The 261

expression level of BrpHL1a/BrcHL1a in Wut leaves was considerably higher than 262

that of Bre leaves (Fig. 3A). A similar result was obtained using RT-qPCR (Fig. 3B). 263

To investigate the expression patterns of BrpHL1a in the plants, we fused BrpHL1a 264

and BrcHL1a with the β-glucuronidase (GUS) gene. In the seedlings of the resultant 265

transgenic plants, the GUS signals of BrpHL1a::GUS and BrcHL1a::GUS were 266

visible in all organs, especially in cotyledons and rosette leaves (Fig. 3C). RT-PCR 267

showed that BrpHL1a and BrcHL1a genes were expressed in the leaf, stem, 268

cotyledon and root of Bre and Wut (Figure 3D). These results show that the temporal 269

and spatial expression patterns of two BrHL1a genes in Bre and Wut are similar. 270

271



10

Functional analysis of the SNPs in BrpHL1a genes 272

To examine the GWAS SNPs of BrpHL1a alleles, we aligned gene bodies of 273

BrpHL1a with Wut BrcHL1a identified by GWAS. There were 44 GWAS SNPs in 274

BrpHL1a alleles (Supplementary Table 1), most of which were located in the introns. 275

We found that the two SNPs, 274T/C and/or 403T/G, cause non-synonymous 276

substitutions. To determine SNPs functional for leaf hairs, we mutagenized BrpHL1a 277

genomic DNA with 274C and/or 403G and BrcHL1a-g genomic DNA with 274T 278

and/or 403G and constructed a series of binary vectors of the mutated genes under 279

their control of the native promoters (Table 3). We then transferred them into the null 280

gl1 mutants of Arabidopsis that are deficient in trichome formation (Fig. 3E-F). 281

Firstly, the genomic BrpHL1a-g completely rescued the phenotype in terms of 282

trichome formation whereas the genomic BrcHL1a-g was unable to rescue the 283

phenotypes of the gl1 mutants (Table 3), revealing that Wut BrcHL1a-g is deficient 284

in formation of leaf hairs. Secondly, the C274T mutagenesis in BrcHL1a-g274C/T

285

plants completely rescued the gl1 phenotype, thus indicating that 274T/C is the 286

functional SNP for leaf hair. Thirdly, 403T/G mutagenesis in BrpHL1a-g403T/G

plants 287

also rescued the gl1 phenotype, showing that 403T/G is dispensable for leaf hair. 288

To exclude the possible effects of introns and the 3′-noncoding region on 289

function of BrcHL1a, we constructed cDNA sequences of BrpHL1a and BrcHL1a 290

under the control of their native promoters and the 3′-noncoding region and then 291

transferred them into the gl1 mutants. As expected, both BrpHL1a-c and BrcHL1a-c 292

were expressed equally at the transcriptional levels in all the transgenic lines (Fig. 293

3H). BrcHL1a-c did not rescue the phenotypes of the gl1 mutants, whereas 294

BrpHL1a-c completely rescued the phenotype in terms of trichome formation. 295

BrcHL1a-c274C/T

completely rescued the gl1 phenotype, while BrpHL1a-c274T/C

did 296

not rescue the gl1 phenotype. The phenotypic outcomes of BrpHL1a-c, BrcHL1a-c, 297

and BrcHL1a-c274C/T

in gl1 mutants were the same as the ones of BrpHL1a-g, 298

BrcHL1a-g, and BrcHL1a-g274C/T

, respectively (Fig. 3G). We conclude that the SNPs 299

in the intron and the 3′-noncoding regions of BrcHL1a are not the reason for the 300

alteration of leaf hairs. 301



11

302

Effects of SNP 274T/C on direct interaction of BrcHL1a with BrpGL3 303

In Arabidopsis, a network of three classes of proteins consisting of TTG1 (a WD40 304

repeat protein), GL3 (a bHLH transcription factor) and GL1 (a MYB transcription 305

factor), activates trichome initiation and patterning (Zhang et al., 2003). GL3 306

functions together with GL1 and TTG1 to form a MYB-bHLH-WD40 (MBW) 307

activator complex. GL3 participates in the physical interactions with GL1, TTG1, 308

and itself, but GL1 and TTG1 do not interact. GL1 has the conserved 309

[DE]Lx2[RK]x3Lx6Lx3R amino acid signature in the R3 domain of R2R3 MYBs, 310

which is the structural basis for interaction between MYB and R/B-like bHLH 311

proteins (Zimmermann et al., 2004). We found that the W92R amino acid 312

substitution was within this conserved sequence. To further examine whether the 313

Trp92

mutation interferes with the interaction of BrcHL1a and GL3, we performed 314

pull-down assays. The result showed that the interaction of Arabidopsis 315

Maltose-binding protein (MBP)-AtGL3 with glutathione S-transferase 316

(GST)-BrpHL1a and GST-BrcHL1aR92W

(Fig. 4A) was strong, while that of 317

MBP-AtGL3 with GST-BrcHL1a and GST-BrpHL1aW92R

was weak. These results 318

suggest that Trp92

plays a critical role in direct interaction between BrpHL1a and 319

BrpGL3. 320

To further confirm the function of Trp92

, we performed a bimolecular 321

fluorescence complementation (BiFC) assay based on enhanced yellow fluorescent 322

protein (EYFP). The full-length coding sequences of AtGL3, BrpHL1a, BrcHL1a, 323

BrcHL1aR92W

and BrpHL1aW92R

were fused to the N- or C-terminal halves of EYFP. 324

Both types of fusion proteins were transiently introduced into mesophyll protoplasts 325

of Arabidopsis. The protein–protein interaction between the tester proteins resulted 326

in the proper folding of EYFP leading to its subsequent fluorescence in the 327

co-infiltrated protoplasts. The strong EYFP signals between BrpHL1a and AtGL3 328

and between BrcHL1aR92W

and AtGL3 were observed in the nucleus (Fig. 4B), 329

whereas much weaker BiFC signals were observed between BrpHL1aW92R

and 330

AtGL3 and between BrcHL1a and AtGL3. This result reveals that the interaction 331


http://www.plantcell.org/content/26/4/1764.long#def-12


12

between BrcHL1a and AtGL3 was weak, thus confirming the critical role of Trp92

in 332

formation of leaf hairs. 333

334

Activation of BrpGL2 by BrpHL1a 335

In Arabidopsis, GL2 is required for normal trichome development. GL2 expression 336

is regulated by GL1 (Rerie et al., 1994). GL1 and GL3 bind directly to 337

the GL2 promoter (Wang and Chen, 2008). Dai et al., (2016) report that the 338

substitution of the 92nd

serine to phenylalanine (S92F) in the R3 domain of 339

Arabidopsis GL1 does not affect the interaction of GL1 and GL3 but affects the 340

binding of GL1 to the promoter of GL2. In Bre BrpHL1a, the 92nd

tryptophan 341

corresponds to the 91st tryptophan rather than the 92

nd serine in Arabidopsis GL1. 342

We supposed that the expression level of BrcGL2 in Wut would be reduced 343

compared with that of BrpGL2 in Bre if W92R in BrcHL1a was responsible for the 344

interaction between BrcHL1a and BrcGL3. To address this deduction, we performed 345

RT-qPCR using the same pair of primers whose sequences are conserved in Bre and 346

Wut. BrcGL2 expression was considerably lower than BrpGL2 expression in Bre 347

(Fig. 5A). 348

To confirm the role of the 92nd

tryptophan in the relevance of BrpHL1a to 349

BrpGL2, we analyzed the expression levels of GL2 in the Arabidopsis gl1 mutants 350

with exogenous BrpHL1a and BrcHL1a. GL2 expression was up-regulated in 351

pBrpHL1a::BrpHL1a plants, but not in pBrcHL1a::BrcHL1a plants (Fig. 5B), 352

indicating that BrcHL1a was not able to activate GL2. 353

354

Analysis of BrpHL1b gene functions 355

Considering that BrpHL1a functions in formation of leaf hairs in Bre while BrcHL1a 356

does not in Wut, we wondered whether and how BrpHL1b and BrcHL1b function in 357

formation of leaf hairs. So we analyzed the sequences of these two genes and found 358

that a 4.5 kb insertion was detected in the second exon of Bre BrpHL1b but not in 359

Wut BrcHL1b. RT-PCR result showed no expression of BrpHL1b in Bre (Fig. 5C), 360

meaning that BrpHL-2 is not functional. Also, BrpHL1a was the only functional 361



13

gene of BrpHL1 genes in Bre. BrcHL1b of Wut did not contain any 362

insertions/deletions (InDels) that interrupt the protein sequence. If BrcHL1b of Wut 363

is functional for leaf hair, its hairless phenotype could be difficult to explain. To 364

verify the function of Wut BrcHL1b, we cloned the BrcHL1b gene body including 365

the promoter from Wut plants, and transferred pBrcHL1b::BrcHL1b (under the 366

control of the native promoter) and pAA6::BrcHL1b (under the control of 367

constitutive promoter AA6) constructs (Wang et al., 2014) into gl1 mutants. 368

Although no trichomes were observed on the leaf surface of pBrcHL1b::BrcHL1b 369

plants, a few trichomes were seen on the leaf margin. pAA6::BrcHL1b plants also 370

showed more trichomes on leaf margins than pBrcHL1b::BrcHL1b plants (Table 3; 371

Figure 5D). All together, these results indicated that BrcHL1b regulates hair 372

formation on leaf margins rather than the leaf surface. 373

374

DISCUSSION 375

376

Natural variation at the BrpHL1 locus is extensive 377

Genetic variation is brought about by mutation. Fundamentally, the numbers and 378

density of SNPs in a genome reflect the extent of natural variation in this species. In B. 379

rapa, our natural population of 210 accessions showed 0.5~1.69 million high-quality 380

SNPs compared with the reference genome of Chiifu-401. BrpHL1a on chromosome 381

6 of Bre shows 169 nucleotides that are substituted by its alleles of 13 representative 382

crop types, revealing that natural variation at BrpHL1 locus is extensive. However, 383

most of the SNPs are located in introns and may not be functional. The -85A/G 384

substitution of BrpHL1b on chromosome 9 in some accessions should change the start 385

codon at the 5' side and could thus affect the function of BrpHL1b. On the other hand, 386

BrpHL1b (the second copy of BrpHL1a) of Bre has a 4.5-kb insertion in the second 387

exon compared with BrcHL1b of Wut. This insertion causes a frame shift and 388

truncation of BrpHL1b in Bre. Although the SNP -85A/G and 4.5-kb insertion are not 389

GWAS SNPs, they substantially affect the functions of BrpHL1b in Bre. 390



14

The SNPs of BrcHL1a and BrcHL1b in Wut may be related to the hairless 391

phenotype as Wut leaves are hairless. Among these SNPs, 403T/G is not a causal 392

element for leaf hair since T-to-C mutation in BrcHL1a is not able to rescue the gl1 393

mutant phenotype of Arabidopsis. Expression of BrcHL-2 under the control of its 394

native promoter causes marginal trichomes on the gl1 mutant, thus showing the 395

margin-specific expression of BrcHL-2. In this way, the contribution of the SNPs 396

to all hairy phenotypes is excluded except for SNP 274T/C in the BrcHL1a allele. 397

274T/C is a functional SNP for leaf hairs 398

In recent years, with the rapid development of next-generation sequencing 399

technologies and bioinformatics methods, crop breeding theory and technology has 400

undergone major changes. Numerous studies on genetic map construction and 401

marker-assisted selection have been carried out in Brassica crops. Genomic 402

resequencing is a method designed to sequence all regions of the genome aimed at 403

simplifying genome complexity. Marker-assisted selection is an effective 404

technology for obtaining large numbers of molecular markers and has been widely 405

used for high-throughput SNP discovery and for genotyping in different organisms 406

which are now widely used for large-scale high-throughput SNP genotyping, 407

particularly for de novo SNP discovery. In addition to the advantage of high density 408

and high throughput, our GWAS analysis of SNPs for leaf hairs in B. rapa was 409

effective. One major advantage of using the RIL populations is that researchers can 410

identify some QTL for the specific traits using low-covered resequencing. 411

Compared to GWAS in a natural population, GWAS in biparental cross populations 412

is more efficient and accurate (Yu et al., 2013). The QTL obtained in this way are 413

suitable for selection of candidate genes and GWAS SNPs relevant to leaf hairs, and 414

thereby establish the relationship between SNPs and leaf hairs in a natural 415

population. 416

The identification of causal SNPs is more difficult and should be combined with 417

the exclusion of other SNPs and the relevant alleles. The four members of the BrpHL1 418

gene family are relevant to leaf hairs. We resequenced 210 accessions of B. rapa and 419



15

identified a SNP 274T/C in the candidate BrpHL1a gene. The function of the 420

candidate genes in two parents was identified by transgenic plants of suitable mutants. 421

Through point mutagenesis of SNPs in BrpHL1a genes and functional analysis of 422

these SNPs in the gl1 mutants of Arabidopsis, we excluded some SNPs in introns and 423

nonsynonymous SNPs in coding sequences. This selection criterion is more advanced 424

than that reported in many other previous studies. Nonetheless, a functional test in 425

Arabidopsis is not necessarily a proof of function in B. rapa. Therefore, we suggest 426

that the SNPs in the coding regions of B. rapa genes could be identified accurately by 427

gene transfer into Brassica crops. 428

The number of functional SNPs reported remains very limited, and some 429

functional SNPs should be embedded in the resequencing data. Thus, a great deal of 430

work is still needed to improve the SNP calling and QTL mapping accuracy by using 431

high-throughput sequencing technologies and making full use of the reference 432

Chinese cabbage genome. 433

434

Trp92

is essential for direct interaction with GL3 435

In Arabidopsis, a network of three classes of proteins consisting of TTG1 (a WD40 436

repeat protein), GL3 (a bHLH factor) and GL1 (a MYB transcription factor), 437

activates trichome initiation and patterning (Zhang et al., 2003). As positive 438

regulators, these three proteins form a MBW activator complex. GL3 participates in 439

physical interactions with GL1, TTG1, and itself, but GL1 and TTG1 do not interact 440

with each other. We also found the Trp92

in BrpHL1a is critical for interaction with 441

GL3. The interaction would be disrupted as soon as the critical amino acids in 442

BrpHL1a are mutated. In our BiFC experiments, the interactions between BrcHL1a 443

and GL3 and between BrpHL1aW92R

and GL3 were hardly detectable while 444

interactions between BrpHL1a and GL3 and between BrcHL1aR92W

and GL3 were 445

strong. In pull-down assays, the relative interaction strengths were similar. The hairy 446

phenotype of pBrpHL1a::BrpHL1a plants and hairless pBrcHL1a::BrcHL1a plants 447

in the gl1 background demonstrated that Trp92

of pBrpHL1a is essential for 448

formation of leaf hairs in B. rapa. Analyses of loss-of-function mutants reveal that 449



16

single-repeat R3 MYB transcription factors act as negative regulators (Gan et al., 450

2011; Schellmann et al., 2002; Schnittger et al., 1999). In Arabidopsis, intron 1 and 451

the 3′-noncoding region of GL1 have been shown to be important for the expression 452

of GL1 (Larkin et al., 1993; Wang et al., 2004). Chromatin immune precipitation 453

results show that the single-repeat R3 MYB transcription factor TRICHOMELESS 1 454

(TCL1) can be recruited to the cis-acting regulatory elements of GL1, negatively 455

regulating trichome cell specification by directly suppressing the transcription of 456

GL1 (Wang et al., 2007). The importance of Trp92

for plant phenotype reveals that 457

intron 1 and the 3′-non-coding region of BrpHL1a are not as essential as Trp92

for 458

hair formation on the leaf surface. 459

The MYB (GL1)-bHLH(GL3/EGL3)-WDR(TTG1) proteins form a trimeric 460

MBW complex that activates the expression of the homo domain protein, GL2, 461

which, in turn, induces trichome formation (Rerie et al., 1994). Here, we noticed that 462

the expression of GL2 was reduced in Trp92

mutant plants, suggesting that blocking 463

the interaction between BrHL1a and GL3 affects hair formation on the leaf surface. 464

465

BrpHL1a regulates hair formation on both the leaf surface and leaf margin 466

while BrcHL1b functions only on the leaf margin 467

Usually, there are many SNPs in one gene, which makes it difficult for researchers to 468

select functional SNPs. It is very important to exclude the non-functional GWAS 469

SNPs, especially when duplicated genes are predicted to have biological functions. 470

Although the exclusion of non-functional SNPs is time-consuming and 471

labor-intensive, it is necessary for us to explain the complicated genetic process of 472

agronomic traits. Bre is a representative hairy crop type as the surface and margins of 473

leaves are hairy. By contrast, Wut is regarded as a representative hairless crop type as 474

hair is not seen on the surface of leaves and only a few hairs are detected on leaf 475

margins. The hairy phenotype in Bre corresponds to 274T and 403T in BrpHL1a and a 476

shift in the reading frame and a large insertion in the second exon in BrpHL1b. In 477

contrast, the hairless phenotype is concurrent with 274C and 403G in BrcHL1a and a 478

normal reading frame in BrpHL1b. By genetic transformation, we confirmed that 479



17

BrpHL1a regulates hair formation on both the leaf surface and leaf margin while 480

BrpHL1b does not. The point mutation of BrpHL1a and BrcHL1a genes shows that 481

274T of BrpHL1a or Trp92

of BrpHL1a is essential for hair formation while 403T or 482

Tyr135

is dispensable. Comparison of intronic and non-intronic BrpHL1a transgenes 483

reveals that the first intron and 3’ UTR of BrpHL1a is not essential for its function, in 484

contrast with Arabidopsis GL1 whose first intron and 3’ UTR play roles in trichome 485

formation by interaction of GL1 with GL2 and GL3, respectively (Larkin et al., 1993; 486

Wang et al., 2004). Young developing gl3 leaves lack marginal trichomes, a 487

phenotype further enhanced in the tt8gl3 double mutant, indicating that both 488

TRANSPARENT TESTA8 (TT8) and GL3 are essential for trichome development on 489

leaf margins (Maes et al., 2008). 490

The function of BrpHL1b and BrcHL1b should be considered when that of 491

BrpHL1a and BrcHL1a is clarified. BrpHL1b in Bre is not functional due to the shift 492

in the reading frame and the large insertion in the coding region. However, BrcHL1b 493

in Wut is functional because its exogenous expression in gl1 mutant rescues the 494

trichome formation on leaf margins. Interestingly, hair formation on leaf margins in B. 495

rapa is not attributable to the promoter region of BrcHL1b as its native and 496

constitutive promoters generate leaf hairs on the same regions of leaf margins. 497

498

Functional SNPs are useful for molecular breeding by design 499

Traditional breeding is based on phenotype, and therefore depends primarily on 500

breeders’ experience. Since many traits of crops, such as disease resistance and yield, 501

cannot be observed easily, traditional breeding faces challenges and demands 502

high-throughput genotyping platforms. Molecular breeding by design is considered 503

the best option for breeders to improve their breeding efficiency. With the progress 504

in functional genomics research, increasing numbers of genes and QTL responsible 505

for agriculturally important traits have been identified, which provide valuable 506

genetic resources for molecular breeding. Resequencing and SNP genotyping are 507

two key strategies used in GWAS research and development of molecular markers to 508

target agronomic traits. To be suitable for molecular breeding by designing, we 509



18

optimized the procedures for selection of functional SNPs for agronomic traits (Fig. 510

6). The segregation populations including RIL or doubled haploid lines are suitable 511

for QTL identification in which the genomic resequencing of different lines 512

generates the saturated SNPs. The SNPs located at the QTL are regarded as GWAS 513

SNPs because the candidate genes at the QTL locus are predicted according to 514

GWAS analysis. On the other hand, natural populations include major cultivars, 515

inbred lines and mutants, and, therefore, are very useful for variation analysis of 516

many agronomic traits. SNP genotyping on the basis of genomic resequencing 517

provides a strong tool for detection of SNPs in the large accession collections. 518

Through the comparison of GWAS SNPs from segregation and natural populations, 519

the candidates of functional SNPs are selected. They may be from exon-intron 520

junctures, DNA-RNA binding sites, protein-DNA binding sites, protein-protein 521

interaction domains, miRNAs and miRNA-target complementary sites. The 522

functional identification of the candidate SNPs is important but time-consuming. All 523

the binary vectors dedicated for genetic transformation should be designed to 524

exclude all of the non-functional SNPs and to select the functional SNPs. The null 525

mutants of the genes examined should be chosen for phenotypic rescue. The 526

molecular mechanism underlying the functional SNPs for agronomic traits could be 527

clarified. 528

In rice (Oryza sativa), a high-density SNP array with 51,478 markers has been 529

developed on the Illumina Infinium platform for use in functional genomics studies 530

and molecular breeding (Chen et al., 2014). However, many molecular makers 531

designed according to the GWAS SNPs are not effective in actual crop breeding, 532

largely due to non-functional SNPs. Among our accessions with SNP 274T/C, a 533

large proportion of accessions with 274T that are expected to have all hairy 534

phenotype show the hairless phenotype. One explanation is that some genes 535

downstream of BrpHL1 genes are mutated. Secondly, some cis- and trans-elements 536

exert effects on BrpHL1a. If 274T was used to design the molecular marker for leaf 537

hair, the high proportion of the false breeding materials would be selected. In 538

contrast, almost all accessions with 274C display the hairless phenotype. Therefore, 539



19

274C is a functional SNP for designing the effective molecular markers to select the 540

hairless breeding materials. The application of the functional SNPs to designing 541

molecular markers will facilitate the selection of germplasms, parents and hybrids. 542

Further, the functional SNPs must be verified for their effectiveness in breeding of 543

new varieties with desirable traits. 544

545

MATERIALS AND METHODS 546

547

Plant materials 548

210 accessions of Brassica rapa were used in this study to survey leaf hairs. They 549

include many subspecies and varieties such as B. rapa subsp. chinensis, B. rapa var. 550

parachinensis, B. rapa var. purpuraria, B. rapa subsp. oleifera, B. rapa subsp. 551

narinosa, B. rapa var. perviridis, and B. rapa subsp. nipposinica. The seeds of these 552

crop types were sown in the field at the SIPPE Farm Station in Shanghai during 553

August 20-25 of 2008, 2009 and 2010. 554

Arabidopsis (Arabidopsis thaliana) gl1 (SALK_039478) mutants were kindly 555

provided by Prof. Xiaoya Chen (Wang et al., 2004). For phenotypic observation, 556

seeds were sown in pots with peat soil and incubated at 4°C in darkness for 3-4 days 557

and then moved to a growth chamber with 22°C temperature and 16/8 h of light/dark. 558

559

Phenotyping of leaf hairs 560

The third leaves at seedling stages were fixed and observed and the leaf hairs were 561

observed under an anatomical microscope, and the numbers of leaf hairs on surfaces 562

and margins of blades and petioles were observed. The mean value of the numbers of 563

leaf hairs per leaf in 10 plants was calculated. Plants with 1 or 2 hairs that were too 564

short to be recognized were regarded as hairless plants while plants with more than 2 565

hairs were considered hairy plants. 566

567

Sequencing data and alignment with reference genome 568

The DNA samples were sent to Novogene for sequencing by an Illumina 569



20

hiseq-XTEN system, which produced the paired-end libraries with 2×150 bp read 570

length. All data were submitted to The Sequence Read Archive with BioProject ID 571

PRJNA421038. 572

After cutting adapters, the mean of the quality scores and the GC proportion of raw 573

reads were calculated. The first whole genome sequence of the Brassica A genome 574

species (B. rapa ssp. pekinensis vs Chiifu-401-42) was used as the reference 575

(http://brassicadb.org). The raw paired-end libraries of the 210 accessions were 576

aligned to the reference genome using SOAPalligner (SOAP2) software with the 577

parameter ‘‘-l 32 -s 40 -v 5 -m 10 -x 1000 -r 2’’, as well as bwa/samtools with the 578

default parameter. The effective depth of sequencing was calculated as follows: the 579

total length of clean reads minus that of the filter reads that could not match to the 580

reference genome, all divided by the length of the reference genome. 581

582

SNP calling and filtering 583

Based on the alignment file of SOAPalligner, the reads of genomic resequencing that 584

aligned with the 10 different chromosomes were separated into 10 files, and ordered 585

according to the physical location of the chromosome. SAMtools 586

(http://samtools.sourceforge.net) was used for SNP and InDel detection of each 587

chromosome using Bayesian theory. 588

The true SNPs were selected based on the following criteria: (1) no second 589

heterozygous base existed; (2) there was a quality score over 20; and (3) there were at 590

least five supported reads. The genes containing SNPs and short InDels were selected 591

by comparing the location of SNP and InDel with those of all Brassica gene models 592

v1.5 (http://blast.ncbi.nlm.nih.gov). SNPs were further determined as per whether 593

they were located in exon regions, and whether they caused synonymous/ 594

nonsynonymous mutation, premature termination, or abnormal termination. 595

596

Gene cloning and genetic transformation 597

The BrpHL1a promoter region (1824 bp upstream of the translation start site), 3’ 598

UTR region (1608 bp upstream of the translation termination site) and a full-length 599


https://www.ncbi.nlm.nih.gov/bioproject/PRJNA421038

http://brassicadb.org/

http://brassicadb.org/


21

coding sequence (1596 bp) were amplified from Bre. Meanwhile, the BrcHL1a 600

promoter region (1886 bp upstream of the translation start site), 3’ UTR region 601

(1622 bp upstream of the translation termination site) and a full-length coding 602

sequence (1224 bp) were amplified from Wut. The promoter, 3’ UTR region, and a 603

full-length coding sequence were cloned into pCAMBIA1301 binary vectors to 604

obtain the pBrpHL1a::BrpHL1a and pBrcHL1a::BrcHL1a constructs, respectively. 605

To verify the function of mutation site, site-directed mutagenesis was performed. 606

The C274T nucleotide substitution of the BrcHL1a coding sequence resulted in the 607

mutated BrcHL1aR92W

while the T403G nucleotide substitution of the BrpHL1a 608

coding sequence gave rise to the mutated BrpHL1aY135D

. The primers used for 609

polymerase chain reactions (PCRs) are listed in Supplemental Table 7. 610

The Arabidopsis plants were transformed using the floral-dip method (Clough 611

and Bent, 1998). For selection of transgenic plants, the seeds were sterilized and 612

germinated on agar medium containing 50 mg/ml hygromycin. Seedlings conferring 613

resistance to the hygromycin were transplanted in a greenhouse and grown at 22°C 614

under an 8-h light regimen. 615

616

RNA analysis 617

For reverse transcription quantitative PCR (RT-qPCR), total RNA was extracted 618

using Trizol (Invitrogen) and treated with DNas I (TaKaRa), followed by a 619

phenol/chloroform extraction to remove contaminating DNA. Approximately 4 μg of 620

purified RNA was used for first-strand complementary DNA (cDNA) synthesis 621

using PrimeScript® Reverse Transcriptase (TaKaRa) with oligo (dT) primers. 622

RT-qPCR was performed using the specific primer pairs (Supplementary Table 7) in 623

the MyiQ2 Two-color Real-time PCR Detection System (Bio-Rad, Hercules, CA, 624

USA). The comparative threshold cycle (Ct) method was used to determine relative 625

transcript levels (MyiQ2 two-color real-time PCR detection system; Bio-Rad). 626

Expression was normalized relative to that of ACTIN. Two developing leaves in one 627

B. rapa seedling and ten shoots of Arabidopsis seedlings were harvested for RNA 628

sampling. Three biological replicates and three technical replicates were performed. 629



22

Error bars indicate standard deviation. 630

631

GUS Staining 632

GUS staining was performed on 14-d-old plants. Seedlings of the transgenic plants 633

were placed in staining solution (50 mM Na3PO4, pH 7.0, 0.5 mM X-gluc 634

[5-bromo-4-chloro-3-indolyl glucuronide], and 20% [v/v] methanol), vacuum 635

infiltrated, and incubated at 37°C overnight. After staining, tissues were fixed in 636

alcohol for further analysis. 637

638

BiFC Assays 639

Paired cYFP-tagged and nYFP-tagged constructs were cotransformed into 640

Arabidopsis protoplasts. After incubation at 22°C in darkness for 12 h, GFP and 641

YFP fluorescence signals were excited with 488 or 514 nm argon laser lines, with an 642

emission band of 495–540 nm for GFP detection, 520–560 nm for YFP detection, 643

and 675–765 nm for chlorophyll autofluorescence by confocal microscopy. 644

645

In vitro pull-down assays 646

For MBP pull-down assays, MBP-tagged proteins were bound to amylose resin 647

(NEB) in binding buffer containing 25 mMTris, pH 7.4, 1 mM EDTA, 0.01% NP-40 648

and 2 M NaCl, and incubated with GST-tagged proteins overnight at 4°C. Then the 649

resin was washed 10 times in the binding buffer and eluted by boiling in sodium 650

dodecyl sulfate (SDS)-PAGE loading dye. Aliquots of eluents (20 μl) were resolved 651

on SDS-PAGE gels for immunoblotting with the GST antibody. 652

653

654

ACKNOWLEDGEMENT 655

The gl1 mutants were obtained from Dr. Wang (Wang et al., 2004). This work was 656

supported by National Programs for Science and Technology Development of China (Grant 657

No. 2016YFD0101900) and Natural Science Foundation of China (Grant No. 658

31571261) 659



23

660

ACCESSION NUMBERS 661

Arabidopsis thaliana GL1:AT3G27920; GL2:AT1G79840 GL3:AT5G41315. 662

Brassica rapa BrpHL1a:Bra025311; BrpHL1b:Bra039065; BrpGL2:Bra003535. 663

664

665

SUPPLEMENTARY MATERIALS 666

Supplementary Fig. 1. Alignment of BrpHL1a with homologs in other plant 667

species. 668

Supplementary Fig. 2. Alignment of BrpHL1a and BrpHL1b amino acid 669

sequences with GL1. 670

Supplementary Fig. 3. Alignment of cloned BrpHL1a with a reference gene 671

sequence. 672

Supplementary Fig. 4. Alignment of cloned BrcHL1a with a reference gene 673

sequence. 674

Supplementary Fig. 5. Schematic diagram of BrpHL1b on chromosome 9 in Bre 675

and Wut. 676

677

Supplementary Table 1. Genomic sequences of the BrpHL1a alleles cloned from 678

13 Brassica rapa genotypes (inbred lines). 679

Supplementary Table 2. Genomic sequences of the BrpHL1b alleles cloned from 680

13 Brassica rapa genotypes (inbred lines). 681

Supplementary Table 3. Summary of genome resequencing and mapping data 682

in 210 Brassica rapa accessions. 683

Supplementary Table 4. Summary of SNP calling data from 210 Brassica rapa 684

accessions. 685

Supplementary Table 5. SNP genotyping of BrpHL1a alleles from genome 686

resequencing of 210 Brassica rapa accessions. 687

Supplementary Table 6. Association between SNPs 274T/C and 403T/G of 688

BrpHL1a with hairy phenotypes in 210 Brassica rapa accessions. 689

Supplementary Table 7. Primer sequences used in this study. 690

691

Figure legends 692

693

Figure 1. Leaf hairs of different crop types in Brassica rapa. 694

(A, B) Plants of Bre (A) and Wut (B) at the seedling stage. 695

(C, D) Scanning electron microscopy showing the leaf hairs of Bre (C; scale bar=500 µm) and 696

Wut (D; scale bar=200 µm). 697

(E, F) Hair distribution on adaxial surfaces of leaves in Da38 (E) and Da15 (F). 698

(G, H) Hair distribution on leaf margins in B26 (G) and W12 (H). 699

(I, J) Hair distribution on abaxial surfaces of leaves in Da102 (I) and Da203 (J). 700



24

(K, L) The leaf surface without hairs in B55 (K) and B61 (L). 701

702

Figure 2. cDNA and amino acid sequences of BrpHL1a and BrpHL1b. 703

(A) SNPs of BrpHL1a and nonsynonymous substitutions of BrpHL1a between Bre, Wut and 704

Chiifu-401-42. 705

(B) SNPs of BrpHL1b and nonsynonymous substitutions of BrpHL1b between Bre, Wut and 706

Chiifu-401-42. 707

SNPs are shown on a white background. Nonsynonymous substitutions are boxed. Black lines 708

indicate 5’UTR, exon or 3’UTR; Red arrows show R2 and R3 domains of MYB transcription 709

factors. 710

711

Figure 3. Temporal and spatial expression of BrpHL1a and BrpHL1b and the phenotypic 712

rescue of Arabidopsis gl1 by BrpHL1a and BrpHL1b and their mutated versions. 713

(A, B) RT-PCR (A) and RT-qPCR (B) showing the expression of BrpHL1a/BrcHL1a in Bre and 714

Wut. 715

(C, D) GUS fusion signals in Arabidopsis (C) and RT-PCR (D) in B. rapa showing the 716

expression patterns of BrpHL1a and BrcHL1a in 20-day-old seedlings. Bars=5 mm. 717

(E) Seedling phenotypes of the wild-type (Col) and gl1 mutants of Arabidopsis. Bars=10 mm. 718

(F, G) Seedling phenotypes of gl1 mutants transgenic for genomic BrpHL1a-g and BrcHL1a-g (F) 719

and BrpHL1a-c and BrcHL1a-c cDNAs (G) under the control of the native promoters. Bars=10 720

mm. 721

(H) RT-qPCR showing the expression level of BrpHL1a/BrcHL1a in gl1 mutants and all 722

transgenic Arabidopsis lines. Three biological replicates were used. Error bars indicate standard 723

deviation. 724

725

Figure 4. Physical interaction between BrpHL1a and GL3 proteins. 726

(A) Pull-down assay showing protein–protein interaction between BrpHL1a versions and GL3 727

tagged with GST and MBP respectively. 728

(B) BiFC analysis showing protein–protein interaction between BrpHL1a versions and GL3 in 729

protoplasts. Number of cells with GFP is shown in the table beneath. 730

731

Figure 5. BrpGL2 expression activation by BrpHL1a. 732

(A) RT-qPCR showing the relative expression of BrGL2 in Bre and Wut (5-to-8th leaves). 733

(B) RT-qPCR showing the relative expression of AtGL2 in gl1 mutants and transgenic lines of 734

Arabidopsis. 735

(C) RT-PCR showing expression of BrcHL1b/BrcHL1b in Bre and Wut. 736

(D) Seedling phenotypes of gl1 mutants transgenic for genomic BrcHL1b under the control of 737

the native promoter and PAA6 promoter. Bars=10 mm. 738

Three biological replicates were used for PCR. Error bars indicate standard deviation. 739

740

Figure 6. Procedures optimized for selection of functional SNPs. (1) Building of 741

segregation populations and natural populations. (2) Phenotyping, DNA 742

resequencing, SNP calling with segregation populations and variation analysis of 743

agronomic traits with natural populations. (3) QTLs identification with segregation 744



25

populations and DNA resequencing with natural populations. (4) Selection of 745

GWAS SNPs with natural populations and SNP genotyping with natural 746

populations．(5) Functional identification of the candidate genes and SNPs. (6) 747

Prediction of functional SNPs on the basis of SNP mutagenesis and phenotype 748

rescue. (7) Exclusion of non-functional SNPs. (8) Selection of functional SNPs. (9) 749

Designing of effective markers. (10) Molecular breeding by design. DH, Doubled 750

haploid lines; GWAS, genome-wide association study; QTL, Quantitative trait locus; 751

SNP, Single nucleotide polymorphism; RIL, Recombinant inbred lines. 752

753

754



26

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884



1

Table 1. Number of accessions with and without leaf hairs in a collection of 210

Brassica rapa accessions.

Crop types

Number of accessions

Total All

hairy

Margin-only

hairy Hairless

Heading Chinese cabbage 116 85 12 19

Non-heading Chinese

cabbage

Baicai 70 10 2 58

Caitai 5 1 0 4

Caixin 8 0 0 8

Taicai 2 0 0 2

Tacai 4 0 1 3

Turnip 3 1 1 1

Yellow sarson 2 2 0 0

Total 210 99 16 95



1

Table 2. Correlation between the accessions with the SNPs 274T/C and 403T/G of

BrpHL1a and hairy phenotypes.

Genotype Number of accessions

274 site 403 site Total All hairy Margin-only

hairy

Hairless

T T 111 63 7 41

T G 67 31 5 31

C T 0 0 0 0

C G 28 1 3 24

T X 4 2 1 1

Total 210 97 16 97

Note: “X”indicates the unknown nucleotide.



1

Table 3. Phenotypic rescue of Arabidopsis gl1 mutants by BrpHL1 genes of

Brassica rapa.

Genes Transgenic lines Phenotype rescue

BrpHL1a_g pBrpHL1a::BrpHL1a_g +

BrcHL1a_g pBrcHL1a::BrcHL1a_g -

BrpHL1a_g403T/G

pBrpHL1a::BrpHL1a_g403T/G

+

BrcHL1a_g274C/T

pBrcHL1a::BrcHL1a_g274C/T

+

BrpHL1a_c pBrpHL1a::BrpHL1a_c +

BrcHL1a_c pBrcHL1a::BrcHL1a_c -

BrpHL1a_c274T/G

pBrpHL1a::BrpHL1a_c274T/C

-

BrcHL1a_c274C/T

pBrcHL1a::BrcHL1a_c274C/T

+

BrcHL1b pBrcHL1b::BrcHL1b Partial on leaf

margin



1

1 Figure 1. Leaf hairs of different crop types in Brassica rapa. 2

(A, B) Plants of Bre (A) and Wut (B) at the seedling stage. 3

(C, D) Scanning electron microscopy showing the leaf hairs of Bre (C; scale bar=500 µm) and 4

Wut (D; scale bar=200 µm). 5

(E, F) Hair distribution on adaxial surfaces of leaves in Da38 (E) and Da15 (F). 6

(G, H) Hair distribution on leaf margins in B26 (G) and W12 (H). 7

(I, J) Hair distribution on abaxial surfaces of leaves in Da102 (I) and Da203 (J). 8

(K, L) The leaf surface without hairs in B55 (K) and B61 (L). 9

10



1

Figure 2. cDNA and amino acid sequences of BrpHL1a and BrpHL1b.

(A) SNPs of BrpHL1a and nonsynonymous substitutions of BrpHL1a between Bre, Wut and

Chiifu-401-42.

(B) SNPs of BrpHL1b and nonsynonymous substitutions of BrpHL1b between Bre, Wut and

Chiifu-401-42.

SNPs are shown on a white background. Nonsynonymous substitutions are boxed. Black lines

indicate 5’UTR, exon or 3’UTR; Red arrows show R2 and R3 domains of MYB transcription

factors.



2



1

Figure 3. Temporal and spatial expression of BrpHL1a and BrpHL1b and the phenotypic

rescue of Arabidopsis gl1 by BrpHL1a and BrpHL1b and their mutated versions.

(A, B) RT-PCR (A) and RT-qPCR (B) showing the expression of BrpHL1a/BrcHL1a in Bre and

Wut.

(C, D) GUS fusion signals in Arabidopsis (C) and RT-PCR (D) in B. rapa showing the expression

patterns of BrpHL1a and BrcHL1a in 20-day-old seedlings. Bars=5 mm.

(E) Seedling phenotypes of the wild-type (Col) and gl1 mutants of Arabidopsis. Bars=10 mm.

(F, G) Seedling phenotypes of gl1 mutants transgenic for genomic BrpHL1a-g and BrcHL1a-g (F)

and BrpHL1a-c and BrcHL1a-c cDNAs (G) under the control of the native promoters. Bars=10

mm.

(H) RT-qPCR showing the expression level of BrpHL1a/BrcHL1a in gl1 mutants and all

transgenic Arabidopsis lines. Three biological replicates were used. Error bars indicate standard

deviation.



1

Figure 4. Physical interaction between BrpHL1a and GL3 proteins.

(A) Pull-down assay showing protein–protein interaction between BrpHL1a versions and GL3

tagged with GST and MBP respectively.

(B) BiFC analysis showing protein–protein interaction between BrpHL1a versions and GL3 in

protoplasts. Number of cells with GFP is shown in the table beneath.



1

Figure 5. BrpGL2 expression activation by BrpHL1a.

(A) RT-qPCR showing the relative expression of BrGL2 in Bre and Wut (5-to-8th leaves).

(B) RT-qPCR showing the relative expression of AtGL2 in gl1 mutants and transgenic lines of

Arabidopsis.



1

Figure 6. Procedures optimized for selection of functional SNPs. (1) Building of

segregation populations and natural populations. (2) Phenotyping, DNA resequencing,

SNP calling with segregation populations and variation analysis of agronomic traits

with natural populations. (3) QTLs identification with segregation populations and

DNA resequencing with natural populations. (4) Selection of GWAS SNPs with

natural populations and SNP genotyping with natural populations. (5) Functional

identification of the candidate genes and SNPs. (6) Prediction of functional 745 SNPs

on the basis of SNP mutagenesis and phenotype rescue. (7) Exclusion of

non-functional SNPs. (8) Selection of functional SNPs. (9) Designing of effective

markers. (10) Molecular breeding by design. DH, Doubled haploid lines; GWAS,

genome-wide association study; QTL, Quantitative trait locus; SNP, Single nucleotide

polymorphism; RIL, Recombinant inbred lines.



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http://www.ncbi.nlm.nih.gov/pubmed?term=Kim%2C J%2E%2C Kim%2C D%2ES%2E%2C Park%2C S%2E%2C Lee%2C H%2EE%2E%2C Ahn%2C Y%2EK%2E%2C Kim%2C J%2EH%2E%2C Yang%2C H%2EB%2E%2C Kang%2C B%2EC%2E%2C 2016%2E Development of a high%2Dthroughput SNP marker set by transcriptome sequencing to accelerate genetic background selection in Brassica rapa%2E Hortic Environ Biote 57%2C 280%2D290%2E&dopt=abstract

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Li, F., Kitashiba, H., Nishio, T., 2011. Association of sequence variation in Brassica GLABRA1 orthologs with leaf hairiness. MolBreeding 28, 577-584.


Maes, L., Inze, D., Goossens, A., 2008. Functional specialization of the TRANSPARENT TESTA GLABRA1 network allows differentialhormonal control of laminal and marginal trichome initiation in Arabidopsis rosette leaves. Plant physiology 148, 1453-1464.


Nafisi, M., Stranne, M., Fimognari, L., Atwell, S., Martens, H.J., Pedas, P.R., Hansen, S.F., Nawrath, C., Scheller, H.V., Kliebenstein, D.J.,Sakuragi, Y., 2015. Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plantstress responses. Frontiers in plant science 6.


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Oppenheimer, D.G., Herman, P.L., Sivakumaran, S., Esch, J., Marks, M.D., 1991. A Myb Gene Required for Leaf Trichome Differentiationin Arabidopsis Is Expressed in Stipules. Cell 67, 483-493.


Payne, C.T., Zhang, F., Lloyd, A.M., 2000. GL3 encodes a bHLH protein that regulates trichome development in arabidopsis throughinteraction with GL1 and TTG1. Genetics 156, 1349-1362.


Rerie, W.G., Feldmann, K.A., Marks, M.D., 1994. The Glabra2 Gene Encodes a Homeo Domain Protein Required for Normal TrichomeDevelopment in Arabidopsis. Gene Dev 8, 1388-1399.


Schellmann, S., Schnittger, A., Kirik, V., Wada, T., Okada, K., Beermann, A., Thumfahrt, J., Jurgens, G., Hulskamp, M., 2002.TRIPTYCHON and CAPRICE mediate lateral inhibition during trichome and root hair patterning in Arabidopsis. The EMBO journal 21,5036-5046.


Schnittger, A., Folkers, U., Schwab, B., Jurgens, G., Hulskamp, M., 1999. Generation of a spacing pattern: the role of triptychon intrichome patterning in Arabidopsis. The Plant cell 11, 1105-1116.


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http://www.ncbi.nlm.nih.gov/pubmed?term=Li%2C F%2E%2C Kitashiba%2C H%2E%2C Nishio%2C T%2E%2C 2011%2E Association of sequence variation in Brassica GLABRA1 orthologs with leaf hairiness%2E Mol Breeding 28%2C 577%2D584%2E&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Maes%2C L%2E%2C Inze%2C D%2E%2C Goossens%2C A%2E%2C 2008%2E Functional specialization of the TRANSPARENT TESTA GLABRA1 network allows differential hormonal control of laminal and marginal trichome initiation in Arabidopsis rosette leaves%2E Plant physiology 148%2C 1453%2D1464%2E&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Maes, L., Inze, D., Goossens, A., 2008. Functional specialization of the TRANSPARENT TESTA GLABRA1 network allows differential hormonal control of laminal and marginal trichome initiation in Arabidopsis rosette leaves. Plant physiology 148, 1453-1464.

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http://www.ncbi.nlm.nih.gov/pubmed?term=Nafisi%2C M%2E%2C Stranne%2C M%2E%2C Fimognari%2C L%2E%2C Atwell%2C S%2E%2C Martens%2C H%2EJ%2E%2C Pedas%2C P%2ER%2E%2C Hansen%2C S%2EF%2E%2C Nawrath%2C C%2E%2C Scheller%2C H%2EV%2E%2C Kliebenstein%2C D%2EJ%2E%2C Sakuragi%2C Y%2E%2C 2015%2E Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses%2E Frontiers in plant science 6%2E&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Nafisi, M., Stranne, M., Fimognari, L., Atwell, S., Martens, H.J., Pedas, P.R., Hansen, S.F., Nawrath, C., Scheller, H.V., Kliebenstein, D.J., Sakuragi, Y., 2015. Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses. Frontiers in plant science 6.

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http://www.ncbi.nlm.nih.gov/pubmed?term=Nayidu%2C N%2EK%2E%2C Kagale%2C S%2E%2C Taheri%2C A%2E%2C Withana%2DGamage%2C T%2ES%2E%2C Parkin%2C I%2EA%2EP%2E%2C Sharpe%2C A%2EG%2E%2C Gruber%2C M%2EY%2E%2C 2014%2E Comparison of Five Major Trichome Regulatory Genes in Brassica villosa with Orthologues within the Brassicaceae%2E Plos One 9%2E&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Oppenheimer%2C D%2EG%2E%2C Herman%2C P%2EL%2E%2C Sivakumaran%2C S%2E%2C Esch%2C J%2E%2C Marks%2C M%2ED%2E%2C 1991%2E A Myb Gene Required for Leaf Trichome Differentiation in Arabidopsis Is Expressed in Stipules%2E Cell 67%2C 483%2D493%2E&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Rerie%2C W%2EG%2E%2C Feldmann%2C K%2EA%2E%2C Marks%2C M%2ED%2E%2C 1994%2E The Glabra2 Gene Encodes a Homeo Domain Protein Required for Normal Trichome Development in Arabidopsis%2E Gene Dev 8%2C 1388%2D1399%2E&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Schellmann%2C S%2E%2C Schnittger%2C A%2E%2C Kirik%2C V%2E%2C Wada%2C T%2E%2C Okada%2C K%2E%2C Beermann%2C A%2E%2C Thumfahrt%2C J%2E%2C Jurgens%2C G%2E%2C Hulskamp%2C M%2E%2C 2002%2E TRIPTYCHON and CAPRICE mediate lateral inhibition during trichome and root hair patterning in Arabidopsis%2E The EMBO journal 21%2C 5036%2D5046%2E&dopt=abstract

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http://scholar.google.com/scholar?as_q=TRIPTYCHON and CAPRICE mediate lateral inhibition during trichome and root hair patterning in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schellmann,&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Schnittger%2C A%2E%2C Folkers%2C U%2E%2C Schwab%2C B%2E%2C Jurgens%2C G%2E%2C Hulskamp%2C M%2E%2C 1999%2E Generation of a spacing pattern%3A the role of triptychon in trichome patterning in Arabidopsis%2E The Plant cell 11%2C 1105%2D1116%2E&dopt=abstract

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http://scholar.google.com/scholar?as_q=Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in Brassica rapa&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in Brassica rapa&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zimmermann%2C I%2EM%2E%2C Heim%2C M%2EA%2E%2C Weisshaar%2C B%2E%2C Uhrig%2C J%2EF%2E%2C 2004%2E Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R%2FB%2Dlike BHLH proteins%2E Plant Journal 40%2C 22%2D34%2E&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zimmermann, I.M., Heim, M.A., Weisshaar, B., Uhrig, J.F., 2004. Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins. Plant Journal 40, 22-34.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zimmermann,&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zimmermann,&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1


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Corresponding author information - plantphysiol.org · RTN) has been confirmed ... formation and seed coat color (Zhang et al., 2009). ... in the coding sequences of the candidate