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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 23, 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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Identification of functional single-nucleotide polymorphisms 31

affecting leaf hair number in Brassica rapa 32

33

Wenting Zhang1, Shirin Mirlohi1, Xiaorong Li1and Yuke He1* 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

38

*Corresponding author. E-mail: [email protected] 39

40

41

ABSTRACT 42

Leaf traits affect plant agronomic performance; for example, leaf hair number 43

provides a morphological indicator of drought and insect resistance. Brassica rapa 44

crops have diverse phenotypes and many B. rapa single-nucleotide polymorphisms 45

(SNPs) have been identified and used as molecular markers for plant breeding. 46

However, which SNPs are functional for leaf hair traits and therefore effective for 47

breeding purposes remains unknown. Here we identify a set of SNPs in the B. rapa 48

ssp. pekinenesis candidate gene BrpHAIRY LEAVES1 (BrpHL1) and a number of 49

SNPs of BrpHL1 in a natural population of 210 B. rapa accessions that have hairy, 50

margin-only hairy, and hairless leaves. BrpHL1 genes and their orthologs and 51

paralogs have many SNPs. By intensive mutagenesis and genetic transformation, we 52

selected the functional SNPs for leaf hairs by exclusion of non-functional SNPs and 53

the orthologous and paraologous genes. The residue Trp92

of BrpHL1a was essential 54

for direct interaction with GLABROUS3 (BrpGL3) and thus necessary for formation 55

of leaf hairs. The accessions with the functional SNP leading to substitution of the 56

Trp92

residue had hairless leaves. The orthologous BrcHL1b from B. rapa ssp. 57

chinensis regulates hair formation on leaf margins rather than leaf surfaces. The 58

selected SNP for the hairy phenotype could be adopted as a molecular marker for 59

insect resistance in Brassica crops. Moreover, the procedures optimized here can be 60

used to explain the molecular mechanisms of natural variation and to facilitate 61



3

molecular breeding of many crops. 62

63

Key words: Brassica rapa; functional SNPs; leaf hairs; natural variation; SNPs; 64

molecular breeding by designing; trichome; 65

66

INTRODUCTION 67

Brassica is one of the most important branches of the Brassicaceae family, including 68

many varieties of common vegetable crops. By means of natural and artificial 69

selection through time, many crops in Brassica rapa have evolved that show obvious 70

differences in leaf traits such as heading Chinese cabbage (B. rapa ssp. pekinenesis), 71

non-heading Chinese cabbage (B. rapa ssp. chinensis), turnip (B. rapa ssp. rapifera), 72

and yellow sarson (B. rapa ssp. trilocularis). The Brassica database (BRAD) 73

website (http://brassicadb.org/brad/) has released the complete genome sequence of 74

several Brassica crops (Cheng et al., 2011). Traditionally, leaf shape, size and 75

curvature are the main traits in these crops that have been genetically selected for 76

improved yield and quality. Hence, studying the diversity of the leaf traits in 77

Brassica could provide valuable information to help understand leaf development 78

and leaf variation and how to genetically manipulate these vegetable crops in the 79

future. 80

A leaf hair (trichome) is an epidermal hair that serve as a physical barrier on 81

plant surfaces against biotic and abiotic stress, including insect herbivores, 82

pathogenic microorganisms, UV light, excessive transpiration, freezing, etc. (Harada 83

et al., 2010; Hegebarth et al., 2016; Nafisi et al., 2015; Van Cutsem et al., 2011). The 84

adaptive significance of leaf hairs for arid land plants has been documented 85

(Ehleringer and Mooney 1978). 86

Density and localization of leaf hairs vary with crops within B. rapa. In 87

Arabidopsis (Arabidopsis thaliana), leaf hairs usually exist throughout the whole 88

plant, except for the cotyledons and epicotyls. A range of mutants defining specific 89

aspects of trichome development have been found in Arabidopsis. The genetic 90

analysis of these mutants has revealed a number of key genes controlling this 91



4

patterning process, and the mechanism by which trichome differentiation is triggered 92

in individual cells has been best studied in Arabidopsis (Galway et al., 1994; 93

Oppenheimer et al., 1991; Payne et al., 2000; Rerie et al., 1994; Wada et al., 1997). 94

As most biological traits are genetically complicated, mapping quantitative trait 95

loci (QTL) is a powerful means for estimating the framework of the genetic 96

architecture for a trait and potentially identifying the genes responsible for a specific 97

phenotype. Recombinant inbred lines (RILs) of two Arabidopsis ecotypes, hairy 98

Columbia and less hairy Landsberg erecta, have been constructed to identify QTL 99

contributing to trichome number, and a major locus named REDUCED TRICHOME 100

NUMBER (RTN) has been confirmed (Larkin et al., 1996). In another study, four 101

recombinant inbred mapping populations based on six Arabidopsis ecotypes have 102

been used to reveal QTL controlling trichome density, and nine QTL have been 103

identified as responsible for trichome initiation and development (Symonds et al., 104

2005). 105

Some studies report that the leaf hairs in Chinese cabbage are controlled by a 106

single dominant gene, whereas others have shown that leaf hairs are a quantitative 107

phenotype, controlled by several major QTL (Song et al., 1995; Zhang et al., 2009). 108

The mechanisms of trichome development in Brassica crops and in Arabidopsis 109

might be highly conserved (Alahakoon et al., 2016; Nayidu et al., 2014). A gene 110

(Bra009770) located on chromosome A06 in B. rapa is homologous with 111

TRANSPARENT TESTA GLABRA1 (TTG1) in Arabidopsis, and it controls trichome 112

formation and seed coat color (Zhang et al., 2009). Moreover, nucleotide 113

polymorphisms of four alleles in the GLABROUS1 (GL1) ortholog (BrGL1) are 114

associated with hairless leaves (Li et al., 2011). In addition, a 5 bp deletion in Brtri1 115

(BrGL1) is related to a glabrous phenotype (Ye et al., 2016). 116

The molecular understanding of the functional consequences of genetic 117

variation is critical for application of single nucleotide polymorphisms (SNPs) to 118

plant breeding. Progress towards this goal has been mostly successful when the 119

genetic variation falls within a coding region. Unfortunately, most SNPs identified in 120

plants are located within large introns or are distal to coding regions. 121



5

SNPs have a wide distribution and can be found in any region of a gene, mRNA 122

or intergenic region. Although identification of SNPs is an important first step in 123

understanding the relationship between variation and phenotypes, a major challenge 124

in the post-GWAS era is to understand the functional significance of the identified 125

SNPs and to apply these SNPs to plant breeding. Usually, GWAS SNPs (SNPs at 126

QTL) in the coding sequences of the candidate genes are used for designing DNA 127

markers. In practice, many GWAS SNPs are not effective for selection of objective 128

traits and many breeders have experienced failures in the practice of molecular 129

marker-aided selection with GWAS SNPs. Among these SNPs, some are functional 130

for developmental events and traits because they affect the levels of gene expression 131

or translation, splicing, efficiency to enhance or inhibit mRNA stability and protein 132

function. Many polyploid plants have multiple gene copies and many SNPs. 133

Selection of the causal SNPs for certain traits largely depends on the exclusion of 134

non-functional SNPs. Identifying functional SNPs for objective traits from a large 135

number of SNPs presents a bottleneck in the process. Furthermore, successful 136

molecular breeding of crops relies largely on the accuracy of functional SNPs. 137

Recently, many SNPs have been identified in B. rapa (Kim et al., 2016; 138

Tanhuanpaa et al., 2016; Yu et al., 2016). However, which SNPs are responsible for 139

leaf hairs remain unknown. In this study, we took advantage of recent advances in 140

genome resequencing to perform QTL mapping using 150 RILs derived from the 141

cross between the hairy genotype Bre (B. rapa ssp. pekinensis) and the hairless 142

genotype Wut (B. rapa ssp. chinensis) of Chinese cabbage (Yu et al., 2012). We 143

identified functional SNPs from numerous SNPs in the candidate genes and their 144

duplicated copies, and optimized the procedures for selection of functional SNPs for 145

agronomic traits. The selected SNP for leaf hair is a molecular marker of insect 146

resistance in Brassica crops, and the optimized procedures can be used to explain the 147

molecular mechanism of natural variation and to manipulate molecular breeding of 148

many crops. 149

150

RESULTS 151



6

152

Genetic control of leaf hair number 153

The species B. rapa includes heading Chinese cabbage (B. rapa ssp. pekinensis), 154

non-heading Chinese cabbage (B. rapa ssp. chinensis), turnip (B. rapa ssp. rapa), 155

and yellow sarson (B. rapa ssp. trilocularis). Non-heading Chinese cabbage consists 156

of many crop types: baicai, caixin, caitai, purple caitai, taicai, wutacai. These crops 157

and crop types are characterized by their specialized product organs: curved leaves, 158

leafy heads, fleshy petioles, fleshy stems and fleshy roots. 159

To analyze the leaf variation of B. rapa, we collected 210 accessions of B. rapa. 160

The leaf hairs on young leaves of these accessions were observed under a 161

binocular stereo microscope. There were three types of leaves with regard to leaf 162

hairs (Table 1). Most of the accessions belonged to the "all hairy" leaves in which 163

the leaf hairs were visible on the surfaces and margins. Some accessions belonged to 164

the "margin-only" leaves in which the leaf hairs were visible only on the leaf 165

margins, and the rest of the accessions had hairless leaves. Among the 210 166

accessions observed, 99 showed the all-hairy phenotype while 16 displayed the 167

margin-only hairy phenotype (Table 1). Bre is a representative of the all-hairy 168

phenotype and Wut is a representative of the margin-only hairy phenotype: 169

numerous hairs were detected on the leaf surface and leaf margins of Bre whereas 170

only a few leaf hairs were seen on the leaf margins of Wut. (Fig. 1AB). Leaf hairs of 171

all-hairy and margin-only hairy phenotypes were not branched, in contrast with the 172

branched trichomes of Arabidopsis (Fig. 1CD). Most heading Chinese cabbage 173

accessions showed the all-hairy phenotype (Table 1, Fig. 1E, F, I, J), and most 174

non-heading Chinese cabbage accessions displayed a hairless or margin-only hairy 175

phenotype (Table 1, Fig. 1G, H, K, L). 176

177

Positional cloning of the HAIRY LEAVES1 (HL1a) loci 178

In the course of previous studies, the inbred lines of Bre and Wut were crossed to 179

construct RIL populations for inheritance analysis and chromosomal mapping of the 180

related QTL. One major QTL, qTr, located on chromosome 6, was identified as a 181



7

locus for hairy leaves, and Bra025311 in the reference genome of Chiifu-401-42 was 182

selected as a candidate gene (Yu et al., 2013). Bra025311 belongs to the MYB gene 183

family (Supplementary Fig. 1) and is homologous to Arabidopsis GL1 with 59% 184

amino acid identity (Supplementary Fig. 2A). GL1 encodes an R2R3-MYB 185

transcription factor with a central function in the leaf hair patterning pathway, and is 186

involved in epidermal cell fate specification in leaves, promoting leaf hair formation 187

and endoreplication (Szymanski et al., 1998). 188

We named the candidate gene Bra025311 in Bre and Wut as BrpHL1a (B. rapa 189

ssp. pekinensis HL1a) and BrcHL1a (B. rapa ssp. chinensis HL1a), respectively. The 190

gene body (5' UTR, exons, introns and 3' UTR) and cDNAs of Bre BrpHL1a and 191

Wut BrcHL1a were cloned on the basis of genomic resequencing. Bre BrpHL1a 192

showed 56 SNPs in the gene body compared with that of Bra025311 of 193

Chiifu-401-42 (Supplementary Fig. 3). Fifty-five SNPs were identified in Wut 194

BrcHL1a (Supplementary Fig. 4). Compared to Chiifu-401-42 BrpHL1a, 11 SNPs 195

were detected in the exons of Bre BrpHL1a, causing 7 nonsynonymous substitutions; 196

and 13 SNPs were found in the Wut BrcHL1a gene, leading to 9 nonsynonymous 197

substitutions. Compared to Bre BrpHL1a, Wut BrcHL1a had 2 more SNPs in the 198

exons: 274T/C (274th dTMP of the coding sequence was changed to dCMP) and 199

403T/G (403rd T to dGMP) (Fig. 2A). SNPs 274T/C and 403T/G of Wut BrcHL1a 200

caused two nonsynonymous substitutions W92R and Y135D while 256C/T (256th 201

dCMP to dTMP) of Wut BrcHL1b leads to one nonsynonymous substitution Y86H. 202

B. rapa is a mesohexaploid and has more duplicated genes than Arabidopsis. To 203

check the other copies of the BrpHL1a gene, we searched for the genome sequences 204

of a collection of B. rapa accessions. BrpHL1b and BrcHL1b were detected on 205

chromosome 9 in Bre and Wut, respectively. The alignment shows that BrpHL1b and 206

BrcHL1b are homologous with GL1 of Arabidopsis (Supplementary Fig. 2B). Thus, 207

four members of the BrpHL1 gene family were related to the hairy phenotype of B. 208

rapa. Our aim was to first discover which SNPs in the candidate BrpHL1a gene 209

were functional or causal for all hairy or hairless phenotypes. Then we aimed to 210



8

determine whether and how contributions of the other three members of the BrpHL1 211

gene family could be ruled out. 212

213

SNP analysis of BrpHL1a and BrpHL1b alleles in B. rapa 214

Compared to Chiifu-401-42 BrpHL1b, Bre BrpHL1b has an A/G SNP 85 bp 215

prior to the start codon, causing a shifted reading frame and prolongation of the first 216

exon. This gene also shows a 4.5 kb insertion in its second exon (Fig. 2B, 217

Supplementary Fig. 5A), which suggests that BrpHL1b was not functional in Bre. In 218

contrast, Wut BrcHL1b seemed to be functional as the A/G SNP and the large 219

insertion were not detected. 220

To find all the SNPs in B. rapa, we cloned and sequenced BrpHL1a and 221

BrpHL1b genes of 13 representative genotypes (Supplementary Table 1 and 2). A 222

subset of SNPs was detected. In total, 169 nucleotides (9%) of Chiifu-401-42 223

BrpHL1a and 27 nucleotides (2%) of Chiifu-401-42 BrpHL1b were substituted by 224

the other nucleotides of various BrpHL1a and BrpHL1b alleles, respectively. To 225

confirm the accuracy of SNPs, we cloned the full-length cDNA sequences of 226

BrpHL1a and BrpHL1b genes. Sequence analysis of these clones confirmed the 227

accuracy of the genomic sequences of BrpHL1a and BrpHL1b genes. 228

We then analyzed the association between SNPs of BrpHL1 genes and the hairy 229

phenotype. For BrpHL1a alleles, all the genotypes (Wut, Ripposinica and Qincai) 230

with SNP 274C showed the hairless phenotype (Supplementary Table 1), revealing a 231

association between 274C and the hairless phenotype. Among 10 genotypes with 232

274T, 7 showed all hairy phenotypes. Two of 7 genotypes with SNP 403G showed 233

the hairless phenotype. Four of 6 genotypes with 403T showed the all-hairy 234

phenotype. For BrpHL1b alleles, 2 of 3 genotypes with 255C/T showed the hairless 235

phenotype (Supplementary Table 2). Therefore, we were not certain that these SNPs 236

were associated with all-hairy phenotypes. 237

To clarify the relationship between SNPs and leaf hairs, we extended the SNP 238

calling to a natural population of 210 B. rapa accessions. The re-sequencing of these 239

accessions generated two paired-end libraries with 150-bp reads (Supplementary 240



9

Table 3). According to the reference genome of B. rapa v1.5 (Ref), the sequencing 241

depth of the parental lines was more than 10-fold in each accession, and the mapped 242

depth was about 9-20. Each SNP supported by fewer than 4 reads was filtered out, 243

leading to 0.5~1.69 million high-quality SNPs (Supplementary Table 4). These SNPs 244

include many nucleotide substitutions, insertions and deletions. 245

Based on the genomic data in the BRAD, the SNPs were used to update the 246

genomic sequences of the BrpHL1a alleles (Supplementary Table 5) and separately 247

estimated by grouping the 210 accessions. Among 28 accessions with SNP 274C, 24 248

were hairless while 3 were margin-only hairy (Table 2; Supplementary Table 6), thus 249

showing the high association between SNP 274C and the hairless phenotype. Among 250

184 accessions with SNP 274T, 96 showed the all-hairy phenotype, revealing that 251

nearly half of the accessions with SNP 274T failed to show the all-hairy phenotype. 252

Surprisingly, the accessions with SNP 274T included a large proportion of hairless 253

accessions and a small proportion of margin-only hairy accessions. 254

255

Expression patterns of BrpHL1 genes 256

RT-qPCR and RT-PCR were used to examine the differences in expression of 257

BrpHL1a/BrcHL1a between Bre and Wut using the same pair of primers. The 258

expression level of BrpHL1a/BrcHL1a in Wut leaves was considerably higher than 259

that of Bre leaves (Fig. 3A). A similar result was obtained using RT-qPCR (Fig. 3B). 260

To investigate the expression patterns of BrpHL1a in the plants, we fused BrpHL1a 261

and BrcHL1a with the β-glucuronidase (GUS) gene. In the seedlings of the resultant 262

transgenic plants, the GUS signals of BrpHL1a::GUS and BrcHL1a::GUS were 263

visible in all organs, especially in cotyledons and rosette leaves (Fig. 3C). RT-PCR 264

showed that BrpHL1a and BrcHL1a genes were expressed in the leaf, stem, 265

cotyledon and root of Bre and Wut (Figure 3D). These results show that the temporal 266

and spatial expression patterns of two BrHL1a genes in Bre and Wut are similar. 267

268

Functional analysis of the SNPs in BrpHL1a genes 269

To examine the GWAS SNPs of BrpHL1a alleles, we aligned gene bodies of 270



10

BrpHL1a with Wut BrcHL1a identified by GWAS. There were 44 GWAS SNPs in 271

BrpHL1a alleles (Supplementary Table 1), most of which were located in the introns. 272

We found that the two SNPs, 274T/C and/or 403T/G, cause non-synonymous 273

substitutions. To determine SNPs functional for leaf hairs, we mutagenized BrpHL1a 274

genomic DNA with 274C and/or 403G and BrcHL1a-g genomic DNA with 274T 275

and/or 403G and constructed a series of binary vectors of the mutated genes under 276

their control of the native promoters (Table 3). We then transferred them into the null 277

gl1 mutants of Arabidopsis that are deficient in trichome formation (Fig. 3E-F). 278

Firstly, the genomic BrpHL1a-g completely rescued the phenotype in terms of 279

trichome formation whereas the genomic BrcHL1a-g was unable to rescue the 280

phenotypes of the gl1 mutants (Table 3), revealing that Wut BrcHL1a-g is deficient 281

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

282

plants completely rescued the gl1 phenotype, thus indicating that 274T/C is the 283

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

plants 284

also rescued the gl1 phenotype, showing that 403T/G is dispensable for leaf hair. 285

To exclude the possible effects of introns and the 3′-noncoding region on 286

function of BrcHL1a, we constructed cDNA sequences of BrpHL1a and BrcHL1a 287

under the control of their native promoters and the 3′-noncoding region and then 288

transferred them into the gl1 mutants. As expected, both BrpHL1a-c and BrcHL1a-c 289

were expressed equally at the transcriptional levels in all the transgenic lines (Fig. 290

3H). BrcHL1a-c did not rescue the phenotypes of the gl1 mutants, whereas 291

BrpHL1a-c completely rescued the phenotype in terms of trichome formation. 292

BrcHL1a-c274C/T

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

did 293

not rescue the gl1 phenotype. The phenotypic outcomes of BrpHL1a-c, BrcHL1a-c, 294

and BrcHL1a-c274C/T

in gl1 mutants were the same as the ones of BrpHL1a-g, 295

BrcHL1a-g, and BrcHL1a-g274C/T

, respectively (Fig. 3G). We conclude that the SNPs 296

in the intron and the 3′-noncoding regions of BrcHL1a are not the reason for the 297

alteration of leaf hairs. 298

299

Effects of SNP 274T/C on direct interaction of BrcHL1a with BrpGL3 300



11

In Arabidopsis, a network of three classes of proteins consisting of TTG1 (a WD40 301

repeat protein), GL3 (a bHLH transcription factor) and GL1 (a MYB transcription 302

factor), activates trichome initiation and patterning (Zhang et al., 2003). GL3 303

functions together with GL1 and TTG1 to form a MYB-bHLH-WD40 (MBW) 304

activator complex. GL3 participates in the physical interactions with GL1, TTG1, 305

and itself, but GL1 and TTG1 do not interact. GL1 has the conserved 306

[DE]Lx2[RK]x3Lx6Lx3R amino acid signature in the R3 domain of R2R3 MYBs, 307

which is the structural basis for interaction between MYB and R/B-like bHLH 308

proteins (Zimmermann et al., 2004). We found that the W92R amino acid 309

substitution was within this conserved sequence. To further examine whether the 310

Trp92

mutation interferes with the interaction of BrcHL1a and GL3, we performed 311

pull-down assays. The result showed that the interaction of Arabidopsis 312

Maltose-binding protein (MBP)-AtGL3 with glutathione S-transferase 313

(GST)-BrpHL1a and GST-BrcHL1aR92W

(Fig. 4A) was strong, while that of 314

MBP-AtGL3 with GST-BrcHL1a and GST-BrpHL1aW92R

was weak. These results 315

suggest that Trp92

plays a critical role in direct interaction between BrpHL1a and 316

BrpGL3. 317

To further confirm the function of Trp92

, we performed a bimolecular 318

fluorescence complementation (BiFC) assay based on enhanced yellow fluorescent 319

protein (EYFP). The full-length coding sequences of AtGL3, BrpHL1a, BrcHL1a, 320

BrcHL1aR92W

and BrpHL1aW92R

were fused to the N- or C-terminal halves of EYFP. 321

Both types of fusion proteins were transiently introduced into mesophyll protoplasts 322

of Arabidopsis. The protein–protein interaction between the tester proteins resulted 323

in the proper folding of EYFP leading to its subsequent fluorescence in the 324

co-infiltrated protoplasts. The strong EYFP signals between BrpHL1a and AtGL3 325

and between BrcHL1aR92W

and AtGL3 were observed in the nucleus (Fig. 4B), 326

whereas much weaker BiFC signals were observed between BrpHL1aW92R

and 327

AtGL3 and between BrcHL1a and AtGL3. This result reveals that the interaction 328

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

in 329

formation of leaf hairs. 330


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


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331

Activation of BrpGL2 by BrpHL1a 332

In Arabidopsis, GL2 is required for normal trichome development. GL2 expression 333

is regulated by GL1 (Rerie et al., 1994). GL1 and GL3 bind directly to 334

the GL2 promoter (Wang and Chen, 2008). Dai et al., (2016) report that the 335

substitution of the 92nd

serine to phenylalanine (S92F) in the R3 domain of 336

Arabidopsis GL1 does not affect the interaction of GL1 and GL3 but affects the 337

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

tryptophan 338

corresponds to the 91st tryptophan rather than the 92

nd serine in Arabidopsis GL1. 339

We supposed that the expression level of BrcGL2 in Wut would be reduced 340

compared with that of BrpGL2 in Bre if W92R in BrcHL1a was responsible for the 341

interaction between BrcHL1a and BrcGL3. To address this deduction, we performed 342

RT-qPCR using the same pair of primers whose sequences are conserved in Bre and 343

Wut. BrcGL2 expression was considerably lower than BrpGL2 expression in Bre 344

(Fig. 5A). 345

To confirm the role of the 92nd

tryptophan in the relevance of BrpHL1a to 346

BrpGL2, we analyzed the expression levels of GL2 in the Arabidopsis gl1 mutants 347

with exogenous BrpHL1a and BrcHL1a. GL2 expression was up-regulated in 348

pBrpHL1a::BrpHL1a plants, but not in pBrcHL1a::BrcHL1a plants (Fig. 5B), 349

indicating that BrcHL1a was not able to activate GL2. 350

351

Analysis of BrpHL1b gene functions 352

Considering that BrpHL1a functions in formation of leaf hairs in Bre while BrcHL1a 353

does not in Wut, we wondered whether and how BrpHL1b and BrcHL1b function in 354

formation of leaf hairs. So we analyzed the sequences of these two genes and found 355

that a 4.5 kb insertion was detected in the second exon of Bre BrpHL1b but not in 356

Wut BrcHL1b. RT-PCR result showed no expression of BrpHL1b in Bre (Fig. 5C), 357

meaning that BrpHL-2 is not functional. Also, BrpHL1a was the only functional 358

gene of BrpHL1 genes in Bre. BrcHL1b of Wut did not contain any 359

insertions/deletions (InDels) that interrupt the protein sequence. If BrcHL1b of Wut 360



13

is functional for leaf hair, its hairless phenotype could be difficult to explain. To 361

verify the function of Wut BrcHL1b, we cloned the BrcHL1b gene body including 362

the promoter from Wut plants, and transferred pBrcHL1b::BrcHL1b (under the 363

control of the native promoter) and pAA6::BrcHL1b (under the control of 364

constitutive promoter AA6) constructs (Wang et al., 2014) into gl1 mutants. 365

Although no trichomes were observed on the leaf surface of pBrcHL1b::BrcHL1b 366

plants, a few trichomes were seen on the leaf margin. pAA6::BrcHL1b plants also 367

showed more trichomes on leaf margins than pBrcHL1b::BrcHL1b plants (Table 3; 368

Figure 5D). All together, these results indicated that BrcHL1b regulates hair 369

formation on leaf margins rather than the leaf surface. 370

371

DISCUSSION 372

373

Natural variation at the BrpHL1 locus is extensive 374

Genetic variation is brought about by mutation. Fundamentally, the numbers and 375

density of SNPs in a genome reflect the extent of natural variation in this species. In B. 376

rapa, our natural population of 210 accessions showed 0.5~1.69 million high-quality 377

SNPs compared with the reference genome of Chiifu-401. BrpHL1a on chromosome 378

6 of Bre shows 169 nucleotides that are substituted by its alleles of 13 representative 379

crop types, revealing that natural variation at BrpHL1 locus is extensive. However, 380

most of the SNPs are located in introns and may not be functional. The -85A/G 381

substitution of BrpHL1b on chromosome 9 in some accessions should change the start 382

codon at the 5' side and could thus affect the function of BrpHL1b. On the other hand, 383

BrpHL1b (the second copy of BrpHL1a) of Bre has a 4.5-kb insertion in the second 384

exon compared with BrcHL1b of Wut. This insertion causes a frame shift and 385

truncation of BrpHL1b in Bre. Although the SNP -85A/G and 4.5-kb insertion are not 386

GWAS SNPs, they substantially affect the functions of BrpHL1b in Bre. 387

The SNPs of BrcHL1a and BrcHL1b in Wut may be related to the hairless 388

phenotype as Wut leaves are hairless. Among these SNPs, 403T/G is not a causal 389

element for leaf hair since T-to-C mutation in BrcHL1a is not able to rescue the gl1 390



14

mutant phenotype of Arabidopsis. Expression of BrcHL-2 under the control of its 391

native promoter causes marginal trichomes on the gl1 mutant, thus showing the 392

margin-specific expression of BrcHL-2. In this way, the contribution of the SNPs 393

to all hairy phenotypes is excluded except for SNP 274T/C in the BrcHL1a allele. 394

274T/C is a functional SNP for leaf hairs 395

In recent years, with the rapid development of next-generation sequencing 396

technologies and bioinformatics methods, crop breeding theory and technology has 397

undergone major changes. Numerous studies on genetic map construction and 398

marker-assisted selection have been carried out in Brassica crops. Genomic 399

resequencing is a method designed to sequence all regions of the genome aimed at 400

simplifying genome complexity. Marker-assisted selection is an effective 401

technology for obtaining large numbers of molecular markers and has been widely 402

used for high-throughput SNP discovery and for genotyping in different organisms 403

which are now widely used for large-scale high-throughput SNP genotyping, 404

particularly for de novo SNP discovery. In addition to the advantage of high density 405

and high throughput, our GWAS analysis of SNPs for leaf hairs in B. rapa was 406

effective. One major advantage of using the RIL populations is that researchers can 407

identify some QTL for the specific traits using low-covered resequencing. 408

Compared to GWAS in a natural population, GWAS in biparental cross populations 409

is more efficient and accurate (Yu et al., 2013). The QTL obtained in this way are 410

suitable for selection of candidate genes and GWAS SNPs relevant to leaf hairs, and 411

thereby establish the relationship between SNPs and leaf hairs in a natural 412

population. 413

The identification of causal SNPs is more difficult and should be combined with 414

the exclusion of other SNPs and the relevant alleles. The four members of the BrpHL1 415

gene family are relevant to leaf hairs. We resequenced 210 accessions of B. rapa and 416

identified a SNP 274T/C in the candidate BrpHL1a gene. The function of the 417

candidate genes in two parents was identified by transgenic plants of suitable mutants. 418

Through point mutagenesis of SNPs in BrpHL1a genes and functional analysis of 419



15

these SNPs in the gl1 mutants of Arabidopsis, we excluded some SNPs in introns and 420

nonsynonymous SNPs in coding sequences. This selection criterion is more advanced 421

than that reported in many other previous studies. Nonetheless, a functional test in 422

Arabidopsis is not necessarily a proof of function in B. rapa. Therefore, we suggest 423

that the SNPs in the coding regions of B. rapa genes could be identified accurately by 424

gene transfer into Brassica crops. 425

The number of functional SNPs reported remains very limited, and some 426

functional SNPs should be embedded in the resequencing data. Thus, a great deal of 427

work is still needed to improve the SNP calling and QTL mapping accuracy by using 428

high-throughput sequencing technologies and making full use of the reference 429

Chinese cabbage genome. 430

431

Trp92

is essential for direct interaction with GL3 432

In Arabidopsis, a network of three classes of proteins consisting of TTG1 (a WD40 433

repeat protein), GL3 (a bHLH factor) and GL1 (a MYB transcription factor), 434

activates trichome initiation and patterning (Zhang et al., 2003). As positive 435

regulators, these three proteins form a MBW activator complex. GL3 participates in 436

physical interactions with GL1, TTG1, and itself, but GL1 and TTG1 do not interact 437

with each other. We also found the Trp92

in BrpHL1a is critical for interaction with 438

GL3. The interaction would be disrupted as soon as the critical amino acids in 439

BrpHL1a are mutated. In our BiFC experiments, the interactions between BrcHL1a 440

and GL3 and between BrpHL1aW92R

and GL3 were hardly detectable while 441

interactions between BrpHL1a and GL3 and between BrcHL1aR92W

and GL3 were 442

strong. In pull-down assays, the relative interaction strengths were similar. The hairy 443

phenotype of pBrpHL1a::BrpHL1a plants and hairless pBrcHL1a::BrcHL1a plants 444

in the gl1 background demonstrated that Trp92

of pBrpHL1a is essential for 445

formation of leaf hairs in B. rapa. Analyses of loss-of-function mutants reveal that 446

single-repeat R3 MYB transcription factors act as negative regulators (Gan et al., 447

2011; Schellmann et al., 2002; Schnittger et al., 1999). In Arabidopsis, intron 1 and 448

the 3′-noncoding region of GL1 have been shown to be important for the expression 449



16

of GL1 (Larkin et al., 1993; Wang et al., 2004). Chromatin immune precipitation 450

results show that the single-repeat R3 MYB transcription factor TRICHOMELESS 1 451

(TCL1) can be recruited to the cis-acting regulatory elements of GL1, negatively 452

regulating trichome cell specification by directly suppressing the transcription of 453

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

for plant phenotype reveals that 454

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

for 455

hair formation on the leaf surface. 456

The MYB (GL1)-bHLH(GL3/EGL3)-WDR(TTG1) proteins form a trimeric 457

MBW complex that activates the expression of the homo domain protein, GL2, 458

which, in turn, induces trichome formation (Rerie et al., 1994). Here, we noticed that 459

the expression of GL2 was reduced in Trp92

mutant plants, suggesting that blocking 460

the interaction between BrHL1a and GL3 affects hair formation on the leaf surface. 461

462

BrpHL1a regulates hair formation on both the leaf surface and leaf margin 463

while BrcHL1b functions only on the leaf margin 464

Usually, there are many SNPs in one gene, which makes it difficult for researchers to 465

select functional SNPs. It is very important to exclude the non-functional GWAS 466

SNPs, especially when duplicated genes are predicted to have biological functions. 467

Although the exclusion of non-functional SNPs is time-consuming and 468

labor-intensive, it is necessary for us to explain the complicated genetic process of 469

agronomic traits. Bre is a representative hairy crop type as the surface and margins of 470

leaves are hairy. By contrast, Wut is regarded as a representative hairless crop type as 471

hair is not seen on the surface of leaves and only a few hairs are detected on leaf 472

margins. The hairy phenotype in Bre corresponds to 274T and 403T in BrpHL1a and a 473

shift in the reading frame and a large insertion in the second exon in BrpHL1b. In 474

contrast, the hairless phenotype is concurrent with 274C and 403G in BrcHL1a and a 475

normal reading frame in BrpHL1b. By genetic transformation, we confirmed that 476

BrpHL1a regulates hair formation on both the leaf surface and leaf margin while 477

BrpHL1b does not. The point mutation of BrpHL1a and BrcHL1a genes shows that 478

274T of BrpHL1a or Trp92

of BrpHL1a is essential for hair formation while 403T or 479



17

Tyr135

is dispensable. Comparison of intronic and non-intronic BrpHL1a transgenes 480

reveals that the first intron and 3’ UTR of BrpHL1a is not essential for its function, in 481

contrast with Arabidopsis GL1 whose first intron and 3’ UTR play roles in trichome 482

formation by interaction of GL1 with GL2 and GL3, respectively (Larkin et al., 1993; 483

Wang et al., 2004). Young developing gl3 leaves lack marginal trichomes, a 484

phenotype further enhanced in the tt8gl3 double mutant, indicating that both 485

TRANSPARENT TESTA8 (TT8) and GL3 are essential for trichome development on 486

leaf margins (Maes et al., 2008). 487

The function of BrpHL1b and BrcHL1b should be considered when that of 488

BrpHL1a and BrcHL1a is clarified. BrpHL1b in Bre is not functional due to the shift 489

in the reading frame and the large insertion in the coding region. However, BrcHL1b 490

in Wut is functional because its exogenous expression in gl1 mutant rescues the 491

trichome formation on leaf margins. Interestingly, hair formation on leaf margins in B. 492

rapa is not attributable to the promoter region of BrcHL1b as its native and 493

constitutive promoters generate leaf hairs on the same regions of leaf margins. 494

495

Functional SNPs are useful for molecular breeding by design 496

Traditional breeding is based on phenotype, and therefore depends primarily on 497

breeders’ experience. Since many traits of crops, such as disease resistance and yield, 498

cannot be observed easily, traditional breeding faces challenges and demands 499

high-throughput genotyping platforms. Molecular breeding by design is considered 500

the best option for breeders to improve their breeding efficiency. With the progress 501

in functional genomics research, increasing numbers of genes and QTL responsible 502

for agriculturally important traits have been identified, which provide valuable 503

genetic resources for molecular breeding. Resequencing and SNP genotyping are 504

two key strategies used in GWAS research and development of molecular markers to 505

target agronomic traits. To be suitable for molecular breeding by designing, we 506

optimized the procedures for selection of functional SNPs for agronomic traits (Fig. 507

6). The segregation populations including RIL or doubled haploid lines are suitable 508

for QTL identification in which the genomic resequencing of different lines 509



18

generates the saturated SNPs. The SNPs located at the QTL are regarded as GWAS 510

SNPs because the candidate genes at the QTL locus are predicted according to 511

GWAS analysis. On the other hand, natural populations include major cultivars, 512

inbred lines and mutants, and, therefore, are very useful for variation analysis of 513

many agronomic traits. SNP genotyping on the basis of genomic resequencing 514

provides a strong tool for detection of SNPs in the large accession collections. 515

Through the comparison of GWAS SNPs from segregation and natural populations, 516

the candidates of functional SNPs are selected. They may be from exon-intron 517

junctures, DNA-RNA binding sites, protein-DNA binding sites, protein-protein 518

interaction domains, miRNAs and miRNA-target complementary sites. The 519

functional identification of the candidate SNPs is important but time-consuming. All 520

the binary vectors dedicated for genetic transformation should be designed to 521

exclude all of the non-functional SNPs and to select the functional SNPs. The null 522

mutants of the genes examined should be chosen for phenotypic rescue. The 523

molecular mechanism underlying the functional SNPs for agronomic traits could be 524

clarified. 525

In rice (Oryza sativa), a high-density SNP array with 51,478 markers has been 526

developed on the Illumina Infinium platform for use in functional genomics studies 527

and molecular breeding (Chen et al., 2014). However, many molecular makers 528

designed according to the GWAS SNPs are not effective in actual crop breeding, 529

largely due to non-functional SNPs. Among our accessions with SNP 274T/C, a 530

large proportion of accessions with 274T that are expected to have all hairy 531

phenotype show the hairless phenotype. One explanation is that some genes 532

downstream of BrpHL1 genes are mutated. Secondly, some cis- and trans-elements 533

exert effects on BrpHL1a. If 274T was used to design the molecular marker for leaf 534

hair, the high proportion of the false breeding materials would be selected. In 535

contrast, almost all accessions with 274C display the hairless phenotype. Therefore, 536

274C is a functional SNP for designing the effective molecular markers to select the 537

hairless breeding materials. The application of the functional SNPs to designing 538

molecular markers will facilitate the selection of germplasms, parents and hybrids. 539



19

Further, the functional SNPs must be verified for their effectiveness in breeding of 540

new varieties with desirable traits. 541

542

MATERIALS AND METHODS 543

544

Plant materials 545

210 accessions of Brassica rapa were used in this study to survey leaf hairs. They 546

include many subspecies and varieties such as B. rapa subsp. chinensis, B. rapa var. 547

parachinensis, B. rapa var. purpuraria, B. rapa subsp. oleifera, B. rapa subsp. 548

narinosa, B. rapa var. perviridis, and B. rapa subsp. nipposinica. The seeds of these 549

crop types were sown in the field at the SIPPE Farm Station in Shanghai during 550

August 20-25 of 2008, 2009 and 2010. 551

Arabidopsis (Arabidopsis thaliana) gl1 (SALK_039478) mutants were kindly 552

provided by Prof. Xiaoya Chen (Wang et al., 2004). For phenotypic observation, 553

seeds were sown in pots with peat soil and incubated at 4°C in darkness for 3-4 days 554

and then moved to a growth chamber with 22°C temperature and 16/8 h of light/dark. 555

556

Phenotyping of leaf hairs 557

The third leaves at seedling stages were fixed and observed and the leaf hairs were 558

observed under an anatomical microscope, and the numbers of leaf hairs on surfaces 559

and margins of blades and petioles were observed. The mean value of the numbers of 560

leaf hairs per leaf in 10 plants was calculated. Plants with 1 or 2 hairs that were too 561

short to be recognized were regarded as hairless plants while plants with more than 2 562

hairs were considered hairy plants. 563

564

Sequencing data and alignment with reference genome 565

The DNA samples were sent to Novogene for sequencing by an Illumina 566

hiseq-XTEN system, which produced the paired-end libraries with 2×150 bp read 567

length. All data were submitted to The Sequence Read Archive with BioProject ID 568

PRJNA421038. 569


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


20

After cutting adapters, the mean of the quality scores and the GC proportion of raw 570

reads were calculated. The first whole genome sequence of the Brassica A genome 571

species (B. rapa ssp. pekinensis vs Chiifu-401-42) was used as the reference 572

(http://brassicadb.org). The raw paired-end libraries of the 210 accessions were 573

aligned to the reference genome using SOAPalligner (SOAP2) software with the 574

parameter ‘‘-l 32 -s 40 -v 5 -m 10 -x 1000 -r 2’’, as well as bwa/samtools with the 575

default parameter. The effective depth of sequencing was calculated as follows: the 576

total length of clean reads minus that of the filter reads that could not match to the 577

reference genome, all divided by the length of the reference genome. 578

579

SNP calling and filtering 580

Based on the alignment file of SOAPalligner, the reads of genomic resequencing that 581

aligned with the 10 different chromosomes were separated into 10 files, and ordered 582

according to the physical location of the chromosome. SAMtools 583

(http://samtools.sourceforge.net) was used for SNP and InDel detection of each 584

chromosome using Bayesian theory. 585

The true SNPs were selected based on the following criteria: (1) no second 586

heterozygous base existed; (2) there was a quality score over 20; and (3) there were at 587

least five supported reads. The genes containing SNPs and short InDels were selected 588

by comparing the location of SNP and InDel with those of all Brassica gene models 589

v1.5 (http://blast.ncbi.nlm.nih.gov). SNPs were further determined as per whether 590

they were located in exon regions, and whether they caused synonymous/ 591

nonsynonymous mutation, premature termination, or abnormal termination. 592

593

Gene cloning and genetic transformation 594

The BrpHL1a promoter region (1824 bp upstream of the translation start site), 3’ 595

UTR region (1608 bp upstream of the translation termination site) and a full-length 596

coding sequence (1596 bp) were amplified from Bre. Meanwhile, the BrcHL1a 597

promoter region (1886 bp upstream of the translation start site), 3’ UTR region 598

(1622 bp upstream of the translation termination site) and a full-length coding 599


http://brassicadb.org/

http://brassicadb.org/


21

sequence (1224 bp) were amplified from Wut. The promoter, 3’ UTR region, and a 600

full-length coding sequence were cloned into pCAMBIA1301 binary vectors to 601

obtain the pBrpHL1a::BrpHL1a and pBrcHL1a::BrcHL1a constructs, respectively. 602

To verify the function of mutation site, site-directed mutagenesis was performed. 603

The C274T nucleotide substitution of the BrcHL1a coding sequence resulted in the 604

mutated BrcHL1aR92W

while the T403G nucleotide substitution of the BrpHL1a 605

coding sequence gave rise to the mutated BrpHL1aY135D

. The primers used for 606

polymerase chain reactions (PCRs) are listed in Supplemental Table 7. 607

The Arabidopsis plants were transformed using the floral-dip method (Clough 608

and Bent, 1998). For selection of transgenic plants, the seeds were sterilized and 609

germinated on agar medium containing 50 mg/ml hygromycin. Seedlings conferring 610

resistance to the hygromycin were transplanted in a greenhouse and grown at 22°C 611

under an 8-h light regimen. 612

613

RNA analysis 614

For reverse transcription quantitative PCR (RT-qPCR), total RNA was extracted 615

using Trizol (Invitrogen) and treated with DNas I (TaKaRa), followed by a 616

phenol/chloroform extraction to remove contaminating DNA. Approximately 4 μg of 617

purified RNA was used for first-strand complementary DNA (cDNA) synthesis 618

using PrimeScript® Reverse Transcriptase (TaKaRa) with oligo (dT) primers. 619

RT-qPCR was performed using the specific primer pairs (Supplementary Table 7) in 620

the MyiQ2 Two-color Real-time PCR Detection System (Bio-Rad, Hercules, CA, 621

USA). The comparative threshold cycle (Ct) method was used to determine relative 622

transcript levels (MyiQ2 two-color real-time PCR detection system; Bio-Rad). 623

Expression was normalized relative to that of ACTIN. Two developing leaves in one 624

B. rapa seedling and ten shoots of Arabidopsis seedlings were harvested for RNA 625

sampling. Three biological replicates and three technical replicates were performed. 626

Error bars indicate standard deviation. 627

628

GUS Staining 629



22

GUS staining was performed on 14-d-old plants. Seedlings of the transgenic plants 630

were placed in staining solution (50 mM Na3PO4, pH 7.0, 0.5 mM X-gluc 631

[5-bromo-4-chloro-3-indolyl glucuronide], and 20% [v/v] methanol), vacuum 632

infiltrated, and incubated at 37°C overnight. After staining, tissues were fixed in 633

alcohol for further analysis. 634

635

BiFC Assays 636

Paired cYFP-tagged and nYFP-tagged constructs were cotransformed into 637

Arabidopsis protoplasts. After incubation at 22°C in darkness for 12 h, GFP and 638

YFP fluorescence signals were excited with 488 or 514 nm argon laser lines, with an 639

emission band of 495–540 nm for GFP detection, 520–560 nm for YFP detection, 640

and 675–765 nm for chlorophyll autofluorescence by confocal microscopy. 641

642

In vitro pull-down assays 643

For MBP pull-down assays, MBP-tagged proteins were bound to amylose resin 644

(NEB) in binding buffer containing 25 mMTris, pH 7.4, 1 mM EDTA, 0.01% NP-40 645

and 2 M NaCl, and incubated with GST-tagged proteins overnight at 4°C. Then the 646

resin was washed 10 times in the binding buffer and eluted by boiling in sodium 647

dodecyl sulfate (SDS)-PAGE loading dye. Aliquots of eluents (20 μl) were resolved 648

on SDS-PAGE gels for immunoblotting with the GST antibody. 649

650

651

ACKNOWLEDGEMENT 652

The gl1 mutants were obtained from Dr. Wang (Wang et al., 2004). This work was 653

supported by National Programs for Science and Technology Development of China (Grant 654

No. 2016YFD0101900) and Natural Science Foundation of China (Grant No. 655

31571261) 656

657

ACCESSION NUMBERS 658



23

Arabidopsis thaliana GL1:AT3G27920; GL2:AT1G79840 GL3:AT5G41315. 659

Brassica rapa BrpHL1a:Bra025311; BrpHL1b:Bra039065; BrpGL2:Bra003535. 660

661

662

SUPPLEMENTARY MATERIALS 663

Supplementary Fig. 1. Alignment of BrpHL1a with homologs in other plant 664

species. 665

Supplementary Fig. 2. Alignment of BrpHL1a and BrpHL1b amino acid 666

sequences with GL1. 667

Supplementary Fig. 3. Alignment of cloned BrpHL1a with a reference gene 668

sequence. 669

Supplementary Fig. 4. Alignment of cloned BrcHL1a with a reference gene 670

sequence. 671

Supplementary Fig. 5. Schematic diagram of BrpHL1b on chromosome 9 in Bre 672

and Wut. 673

674

Supplementary Table 1. Genomic sequences of the BrpHL1a alleles cloned from 675

13 Brassica rapa genotypes (inbred lines). 676

Supplementary Table 2. Genomic sequences of the BrpHL1b alleles cloned from 677

13 Brassica rapa genotypes (inbred lines). 678

Supplementary Table 3. Summary of genome resequencing and mapping data 679

in 210 Brassica rapa accessions. 680

Supplementary Table 4. Summary of SNP calling data from 210 Brassica rapa 681

accessions. 682

Supplementary Table 5. SNP genotyping of BrpHL1a alleles from genome 683

resequencing of 210 Brassica rapa accessions. 684

Supplementary Table 6. Association between SNPs 274T/C and 403T/G of 685

BrpHL1a with hairy phenotypes in 210 Brassica rapa accessions. 686

Supplementary Table 7. Primer sequences used in this study. 687

688

Figure legends 689

690

Figure 1. Leaf hairs of different crop types in Brassica rapa. 691

(A, B) Plants of Bre (A) and Wut (B) at the seedling stage. 692

(C, D) Scanning electron microscopy showing the leaf hairs of Bre (C; scale bar=500 µm) and 693

Wut (D; scale bar=200 µm). 694

(E, F) Hair distribution on adaxial surfaces of leaves in Da38 (E) and Da15 (F). 695

(G, H) Hair distribution on leaf margins in B26 (G) and W12 (H). 696

(I, J) Hair distribution on abaxial surfaces of leaves in Da102 (I) and Da203 (J). 697

(K, L) The leaf surface without hairs in B55 (K) and B61 (L). 698

699

Figure 2. cDNA and amino acid sequences of BrpHL1a and BrpHL1b. 700



24

(A) SNPs of BrpHL1a and nonsynonymous substitutions of BrpHL1a between Bre, Wut and 701

Chiifu-401-42. 702

(B) SNPs of BrpHL1b and nonsynonymous substitutions of BrpHL1b between Bre, Wut and 703

Chiifu-401-42. 704

SNPs are shown on a white background. Nonsynonymous substitutions are boxed. Black lines 705

indicate 5’UTR, exon or 3’UTR; Red arrows show R2 and R3 domains of MYB transcription 706

factors. 707

708

Figure 3. Temporal and spatial expression of BrpHL1a and BrpHL1b and the phenotypic 709

rescue of Arabidopsis gl1 by BrpHL1a and BrpHL1b and their mutated versions. 710

(A, B) RT-PCR (A) and RT-qPCR (B) showing the expression of BrpHL1a/BrcHL1a in Bre and 711

Wut. 712

(C, D) GUS fusion signals in Arabidopsis (C) and RT-PCR (D) in B. rapa showing the 713

expression patterns of BrpHL1a and BrcHL1a in 20-day-old seedlings. Bars=5 mm. 714

(E) Seedling phenotypes of the wild-type (Col) and gl1 mutants of Arabidopsis. Bars=10 mm. 715

(F, G) Seedling phenotypes of gl1 mutants transgenic for genomic BrpHL1a-g and BrcHL1a-g (F) 716

and BrpHL1a-c and BrcHL1a-c cDNAs (G) under the control of the native promoters. Bars=10 717

mm. 718

(H) RT-qPCR showing the expression level of BrpHL1a/BrcHL1a in gl1 mutants and all 719

transgenic Arabidopsis lines. Three biological replicates were used. Error bars indicate standard 720

deviation. 721

722

Figure 4. Physical interaction between BrpHL1a and GL3 proteins. 723

(A) Pull-down assay showing protein–protein interaction between BrpHL1a versions and GL3 724

tagged with GST and MBP respectively. 725

(B) BiFC analysis showing protein–protein interaction between BrpHL1a versions and GL3 in 726

protoplasts. Number of cells with GFP is shown in the table beneath. 727

728

Figure 5. BrpGL2 expression activation by BrpHL1a. 729

(A) RT-qPCR showing the relative expression of BrGL2 in Bre and Wut (5-to-8th leaves). 730

(B) RT-qPCR showing the relative expression of AtGL2 in gl1 mutants and transgenic lines of 731

Arabidopsis. 732

(C) RT-PCR showing expression of BrcHL1b/BrcHL1b in Bre and Wut. 733

(D) Seedling phenotypes of gl1 mutants transgenic for genomic BrcHL1b under the control of 734

the native promoter and PAA6 promoter. Bars=10 mm. 735

Three biological replicates were used for PCR. Error bars indicate standard deviation. 736

737

Figure 6. Procedures optimized for selection of functional SNPs. (1) Building of 738

segregation populations and natural populations. (2) Phenotyping, DNA 739

resequencing, SNP calling with segregation populations and variation analysis of 740

agronomic traits with natural populations. (3) QTLs identification with segregation 741

populations and DNA resequencing with natural populations. (4) Selection of 742

GWAS SNPs with natural populations and SNP genotyping with natural 743

populations．(5) Functional identification of the candidate genes and SNPs. (6) 744



25

Prediction of functional SNPs on the basis of SNP mutagenesis and phenotype 745

rescue. (7) Exclusion of non-functional SNPs. (8) Selection of functional SNPs. (9) 746

Designing of effective markers. (10) Molecular breeding by design. DH, Doubled 747

haploid lines; GWAS, genome-wide association study; QTL, Quantitative trait locus; 748

SNP, Single nucleotide polymorphism; RIL, Recombinant inbred lines. 749

750

751



26

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881



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.



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Gan, L.J., Xia, K., Chen, J.G., Wang, S.C., 2011. Functional characterization of TRICHOMELESS2, a new single-repeat R3 MYBtranscription factor in the regulation of trichome patterning in Arabidopsis. Bmc Plant Biol 11.


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Larkin, J.C., Young, N., Prigge, M., Marks, M.D., 1996. The control of trichome spacing and number in Arabidopsis. Development 122,997-1005.


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.


Nayidu, N.K., Kagale, S., Taheri, A., Withana-Gamage, T.S., Parkin, I.A.P., Sharpe, A.G., Gruber, M.Y., 2014. Comparison of Five MajorTrichome Regulatory Genes in Brassica villosa with Orthologues within the Brassicaceae. Plos One 9.


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.


Song, K., Slocum, M.K., Osborn, T.C., 1995. Molecular marker analysis of genes controlling morphological variation in Brassica rapa(syn. campestris). TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik 90, 1-10.


Symonds, V.V., Godoy, A.V., Alconada, T., Botto, J.F., Juenger, T.E., Casal, J.J., Lloyd, A.M., 2005. Mapping quantitative trait loci inmultiple populations of Arabidopsis thaliana identifies natural allelic variation for trichome density. Genetics 169, 1649-1658.



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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://scholar.google.com/scholar?as_q=Association of sequence variation in Brassica GLABRA1 orthologs with leaf hairiness&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Li,&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

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://www.ncbi.nlm.nih.gov/pubmed?term=Song%2C K%2E%2C Slocum%2C M%2EK%2E%2C Osborn%2C T%2EC%2E%2C 1995%2E Molecular marker analysis of genes controlling morphological variation in Brassica rapa %28syn%2E campestris%29%2E TAG%2E Theoretical and applied genetics%2E Theoretische und angewandte Genetik 90%2C 1%2D10%2E&dopt=abstract

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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.

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corresponding author information - plantphysiol.org · rtn) has been confirmed (larkin ......

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