A maize bundle sheath defective mutation mapped on chromosome 1 between SSR markers umc1395 and umc1603

Journal of Integrative Agriculture 2015, 14(10): 1949–1957 Available online at www.sciencedirect.com

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RESEARCH ARTICLE

A maize bundle sheath defective mutation mapped on chromosome 1 between SSR markers umc1395 and umc1603 PAN Yu1, 2*, CHEN Xu-qing1*, XIE Hua1, DENG Lei3, LI Xiang-long1, ZHANG Xiao-dong1, HAN Li-xin1, YANG Feng-ping1, XUE Jing1, ZHANG Li-quan1 1

Beijing Agro-Biotechnology Research Center, Beijing Academy of Agricultural and Forestry Science, Beijing 100097, P.R.China Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education/College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715, P.R.China 3 Bioengineering College, Chongqing University, Chongqing 400030, P.R.China 2

Abstract The bsd-pg (bundle sheath defective pale green) mutant is a novel maize mutation, controlled by a single recessive gene, which was isolated from offspring of maize plantlets regenerated from tissue callus of the maize inbred line 501. The characterization was that the biogenesis and development of the chloroplasts was mainly interfered in bundle sheath cells rather than in mesophyll cells. For mapping the bsd-pg, an F2 population was derived from a cross between the mutant bsd-pg and an inbred line Xianzao 17. Using specific locus amplified fragment sequencing (SLAF-Seq) technology, a total of 5 783 polymorphic SLAFs were analysed with 1 771 homozygous alleles between maternal and paternal parents. There were 49 SLAFs, which had a ratio of paternal to maternal alleles of 2:1 in bulked normal lines, and three trait-related candidate regions were obtained on chromosome 1 with a size of 3.945 Mb. For the fine mapping, new simple sequence repeats (SSRs) markers were designed by utilizing information of the B73 genome and the candidate regions were localized a size of 850 934 bp on chromosome 1 between umc1603 and umc1395, including 35 candidate genes. These results provide a foundation for the cloning of bsd-pg by map-based strategy, which is essential for revealing the functional differentiation and coordination of the two cell types, and helps to elucidate a comprehensive understanding of the C4 photosynthesis pathway and related processes in maize leaves. Keywords: maize, bsd-pg, SLAF, SSR assossiation analysis, fine mapping

1. Introduction Received 23 September, 2014 Accepted 6 July, 2015 PAN Yu, Tel: +86-23-68250974, Mobile: +86-18723494126, Fax: +86-23-68251274, E-mail: [email protected]; Correspondence CHEN Xu-qing, Tel: +86-10-51503868, Fax: +86-10-51503980, E-mail: [email protected] * These authors contributed equally to this study. © 2015, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61130-3

Maize is one of the most important crops in the world, as well as in China (Li et al. 2005). Similar to Arabidopsis, maize is also used as an important model organism for fundamental research (Schnable et al. 2009). In non-succulent vascular plants, the development of vegetative leaves is an indispensable feature in their life cycle, and the maize leaf was also an excellent system for the study of post-primordial differentiation events that were morphologically and

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biochemically distinct. At maturity, sheath and blade leaves are delimited by an epidermal fringe known as the ligule. Additionally, the parallel veins, contained in the sheath and blade, were concentric circles of bundle sheath cells (BSC) and mesophyll cells (MC), which could fixated the CO2 in C4 photosynthetic cycle (Nelson and Langdale 1992). Recently, the evidences suggested that the structurally and biochemically distinct between BSC and MC in mature leaves were the differences on the type of chloroplasts and their arranging (Bastías et al. 2013; Sharpe and Offermann 2014). In BSC and MC, the photosynthetic enzymes also showed differentiation which resulted in the cell-specific gene expression and plastid development (Bansal and Bogorad 1993; Langdale et al. 1988). A similar phenotype had been described in maize mutants bsd1 (now named g2) and bsd2 by Langdale’s labs (Roth et al. 1996; Hall et al. 1998a, b; Brutnell et al. 1999; Cribb et al. 2001; Rossini et al. 2001). Although researches has greatly increased our understanding of the structurally and biochemically distinct between the BSC and MC in the past few decades (Hall et al. 1998a; Brutnell et al. 1999), little is known about the biochemical reactions and the enzymes involved in the regulation of C4 system. With the advancement in genomics, the genomic sequences of several C3 and C4 model plants have become available. Also, a vast amount of genomic information were provided according to the draft genomic sequences of maize B73 (http://www.maizegdb.org/), and allowed us to perform detailed genetic analysis (Schnable et al. 2009). Nowadays, specific locus amplified fragment sequencing (SLAF-Seq) has also been used for an efficient method of large-scale genotyping, which could combine the locus-specific amplification and high-throughput sequencing to reduce the complexity of the genome (Sun et al. 2013). It allows researchers to design and develop specific molecular markers from the constructed SLAF-Seq libraries, and has been successfully used in crops (Chen et al. 2013; Sun et al. 2013; Xu et al. 2015). In this study, we have isolated and characterized a mutation from the offspring of selfed plantlets regenerated from the immature embryo callus of maize (Zea mays L.) inbred line 501 (Fig. 1). In this mutant, the biogenesis and development of chloroplasts could be impaired in BSC with no or few chloroplasts in BSC, but chloroplasts could develop normally in MC (Fig. 2). To elaborate the tightly coordinated development of BSC and MC cells, SLAF-Seq technology was first used to sequence the pale green and normal pools segregated DNA samples and two parental DNA samples by the Beijing Biomarker Technologies Co. Ltd., China (http://www.biomarker.com. cn/english/). Based on the results of SLAF-Seq genotyping data, a high-density genetic map was constructed and

used for mapping of bsd-pg gene. Hence, the objective of this study is to quickly identify candidate genes for the pale green trait, and then to fine mapping bsd-pg gene. These results will provide an important penetration into the mechanism of morphological variation between the BSC and MC in maize, and help us to understand the nucleic gene regulated chloroplast differentiation, the photosynthetic and

Fig. 1 Phenotypes of the control and bsd-pg mutant. The control inbred line 501 (left) and the bsd-pg mutant (right).

A

MC BSC

20 μm B

MC

BSC

20 μm

Fig. 2 Micrograph of Kranz structure of wild type (the male parent) and bsd-pg mutant by transmission electron microscope. A, Kranz structure of wild type (the male parent). B, no chloroplast developed in bundle sheath cell of bsd-pg leaf. MC, mesophyll cells; BSC, bundle sheath cells.

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other metabolic pathways in C4 plants.

Bsd-pg 501

2. Results

Huo 09-3

2.1. Phylogenetic tree of 12 maize inbred lines and bsd-pg mutant

X478 Ji 853 Jing 24

Polymorphism analysis of the parental plants is essential for the genetic map construction, and then the polymorphic of 12 maize inbred lines were analysed by 312 simple sequence repeats (SSRs). A total of 506 polymorphic bands were detected among 12 maize inbred lines and the mutant. The phylogenetic analysis showed that the bsd-pg mutant had the highest identity with inbred line 501, but had the larger genetic differences with Xianzao 17 and Han 21 (Fig. 3), confirmed the genetic origins of bsd-pg mutant, which was derived from inbred line 501. To fine mapping the bsd-pg gene, additionally, an F2 population were derived from a cross between Xianzao 17 and bsd-pg. Lastly, 9 of 13 F1 hybridized progenies (progeny -1, -2, -3, -5, -6, -8, -10, -11 and -13) were successfully identified using the the phi080 and phi065 markers, and used for selfing, respectively (Fig. 4). Then the F2 seeds were obtained by F1 self-pollinated, including 502 wildtype individual lines and 228 pale green individual lines, respectively. The expect ratio was 3:1 (χ2=0.24<3.841). Chi-square test indicated that the bsd-pg mutation was controlled by a single recessive gene.

Fig. 3 Phylogenic tree of 13 maize inbred lines.

2.2. SLAF-Seq data and polymorphic analysis

Fig. 4 F1 hybrids between bsd-pg mutant and Xianzao 17 checked by two simple sequence repeats (SSRs) phi080 and phi065.

Four libraries were used for high-throughput sequencing by SLAF technology, which were including two parental, bsd-pg (M) and Xianzao 17 (P), 50 pale green F2 progenies (ab) and 50 normal F2 progenies (aa) bulked as two pools. After sequencing, 4.421 Gb of raw data including 18 002 562 valid reads was procured with per read length of ~146 bp (Table 1). The numbers of SLAF tags was 54 788, and the average coverage for each tag was 116.81-fold. In addition, the produced 54 788 high-quality SLAF tags were divided into 6 types, 5 783 of which were polymorphic basing on the differences of gene sequences. Moreover, the polymorphic SLAFs were used as the markers with a polymorphism rate of 10.56%, mainly including the single nucleotide polymorphism (SNP), restrict enzyme position mutation (EPSNP)

Ji 44 B73 Chang 7-2 Huang C Xianzao 17 Han 21 0.26

0.34 Coefficient

0.42

0.50

M-up M-down Bsd-pg F1-1 F1-2 F1-3 F1-4 F1-5 F1-6 F1-7 F1-8 F1-9 F1-10 F1-11 F1-12 F1-13 Xianzao 17 M-down M-up

0.18

phi080 phi065

Table 1 Analysis of sequencing data in each sample Sample Female parent Male parent Wild pool Mutant pool

Sample ID P M aa ab

Read length (bp) 146 146 146 146

Read number 3 401 849 3 482 011 5 108 876 6 009 826

GC (%) 43.38 43.38 43.75 43.75

and nucleotide insert/delete (INDEL) markers, respectively (Table 2). Simultaneously, the SLAFs numbers on per chromosome were calculated using the SLAFs positioning of the genome (Table 3), and then constructed the each chromosomes (Fig. 5). The results showed that all SLAFs

Table 2 Statistic results of types of specific locus amplified fragments (SLAFs)1) Number Percent (%) 1)

SNP 5 967 10.39

EPSNP 12 0.02

INDEL 562 1.02

No. of polymorphism 47 257 86.25

Unknown 350 0.63

Repeat 910 1.66

SNP, single nucleotide polymorphism; EPSNP, restrict enzyme position mutation; INDEL, nucleotide insert/delete.

Total 54 788

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Table 3 Distribution of SLAF and markers on each chromosome in maize Chromosome ID Chromosome 1 Chromosome 2 Chromosome 3 Chromosome 4 Chromosome 5 Chromosome 6 Chromosome 7 Chromosome 8 Chromosome 9 Chromosome 10 Chromosome Mt Chromosome Pt Unknown Total 1)

SLAF no. 7 516 6 084 6 134 6 659 5 836 4 364 4 934 4 739 4 231 3 967 22 25 277 54 788

Marker no. 668 676 665 699 598 461 566 504 465 453 1 1 26 5 783

Diff_marker no.1) 29 2 3 1 0 3 1 5 2 2 1 0 0 49

Diff_marker no., the number of different markers.

equally distributed on per chromosome, indicating that the maize genome has been simplified in this study.

2.3. Association analysis In the F2 population, the pale green was controlled by a qualitative recessive gene. Hence, we hypothesized the genetic model, including the genotypes of P (Xianzao 17), M (bsd-pg), and with 50 individuals in per pool. Actually, the bsd-pg gene should have a high genotype ratio in ab, not in aa. Thus 5 783 polymorphic SLAF markers were obtained according to the sequencing of the 4 samples. A total of 1 771 SLAF markers with homozygous alleles were selected from maternal (bsd-pg) and paternal (Xianzao 17). Moreover, the associated markers should also be identitied with this segregation ratio, that is the ratio of Paa:Maa should be 2:1 among the associated markers, and Pab should be equal to 0. Finally, we obtained 49 Diff_markers through calculating the difference ratio of Paa and Maa groups. However, these Diff_markers were uneven distributed on all the chromosomes, such as 29 on chromosome 1, 5 on chromosome 8, 3 on chromosomes 3 and 6, etc. (Table 3). On chromosome 1, 3 trait-related candidate regions were identified according to the association analysis (Table 4 and Fig. 6), covering a size of 3.945 Mb. Eventually, we verified the position according to the B73 genome sequence, which were localized on chromosome 1 within positions 98 913 000– 100 239 000, 169 409 000–169 995 000 and 180 904 000– 182 937 000, respectively.

2.4. Molecular marker assays for bsd-pg analysis Based on the physical locations of IBM2 2008 markers and ISU integrated markers on B73 Refgen_v2 (http://www.

maizegdb.org/), there were 8 SSR markers (umc1515, umc2230, umc2233, umc1603, umc1601, umc1812, umc1590 and umc1811) around 3 association regions on chromosome 1 (Table 5). Three of them (umc2233, umc1603 and umc1601) were identified and closely linked with the pale green mutation in F2 population. Moreover only one cross was detected on F2 population by umc1603 and umc1601 (Fig. 7-A and B), but no cross was found by umc2233 (Fig. 7-C), indicating that the bsd-pg gene should be flanked downstream of umc1603 and umc1601. Similarly, we found 2 SSRs (umc2232 and umc1395) located on the upstream of umc2233, which were checked in the same F2 population. Only one cross was finally found by umc2232 and umc1395 (Fig. 7-D and E). However, no previously published SSRs had been found between umc1395 and umc2233. Therefore, we inferred the bsd-pg was localized on chromosome 1 between umcc1395 and umc2233, approximately 850 934 bp in distance from umc1395 to umc1603 in B73 genome, with 35 putative genes in this region based on B73 Refgen_v2 gene models (Table 6). Notably, it is essential for revealing the regulatory networks that control the functional differentiation of BSC and MC in maize by identifying the function of candidate genes in association regions in the future.

3. Discussion The bsd-pg is a novel mutant of maize, involved in the functional differentiation and coordination of the BSC and MC types in various metabolic pathways. Although it has some similarities in phenotype to the previously identified mutants, bsd1 (now named g2) and bsd2, as well as some differences. The leaf blades of bsd1 displayed a variegated phenotype with pale green and dark green sectors (Langdale and Kidner 1994), but the bsd2 had slightly pale green and grainy leaves (Brutnell et al. 1999), which were identified and characterized using different maize mutants (Roth et al. 1996; Hall et al. 1998a, b; Brutnell et al. 1999; Cribb et al. 2001; Rossini et al. 2001). In this study, the biogenesis and development of chloroplasts in BSC was impaired in bsd-pg mutant. In addition, there were no or few chloroplasts in BSC, but chloroplasts could develop normally in MC (Fig. 2). Therefore, the type and phenotype of bsd-pg mutant is obviously different from bsd1 and bsd2. Based on AGI’s B73 Refgen_v2 sequence, the locus bsd1 is within positions of 1 464 399–1 469 113 on chromosome 3, and bsd2 within 158 313 837–158 325 193 on chromosome 1 (http://www. maizegdb.org/) (Schnable et al. 2009). However, the new mutant is not allelic to bsd1 or bsd2, located on 164 555 621– 165 406 555 of chromosome 1. Hence, we speculate that the bsd-pg is a novel mutant of maize, enriching the type of maize leaf mutants.

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SLAF distribution on chromosome Chr.1

20

Chr.2 Chr.3 Chr.4 Chr.5 Chr.6 0 Chr.7 Chr.8 Chr.9 Chr.10

0M 10 M 20 M 30 M 40 M 50 M 60 M 70 M 80 M 90 M 100 M 110 M 120 M 130 M 140 M 150 M 160 M 170 M 180 M 190 M 200 M 210 M 220 M 230 M 240 M 250 M 260 M 270 M 280 M 290 M 300 M

Unknown

Marker distribution on chromosome Chr.1

5

Chr.2 Chr.3 Chr.4 Chr.5 Chr.6

0

Chr.7 Chr.8 Chr.9 Chr.10

0M 10 M 20 M 30 M 40 M 50 M 60 M 70 M 80 M 90 M 100 M 110 M 120 M 130 M 140 M 150 M 160 M 170 M 180 M 190 M 200 M 210 M 220 M 230 M 240 M 250 M 260 M 270 M 280 M 290 M 300 M

Unknown

Fig. 5 Distribution of specific locus amplified fragments (SLAFs) and marker on maize every chromosome. SLAFs and markers distribute randomly.

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Table 4 Information of association regions on chromosome ID 1 2 3

Chromosome Chromosome 1 Chromosome 1 Chromosome 1

Start 98 913 000 169 409 000 180 904 000

End 100 239 000 169 995 000 182 937 000

Additionally, to identify the genetic of bsd-pg gene, an F2 mapping population was constructed by crossing the bsdpg mutant with the inbred line Xianzao 17. The results of Chi-square test to F2 population indicated that the mutation is controlled by a single recessive gene. The SLAF-Seq technique, based on the next generation sequencing (NGS), can simultaneously develop markers and check the F2 population - thus polymorphic markers do not need to be developed in advance. SLAF-Seq can also develop a large number of polymorphic markers very rapidly (Sun et al. 2013). In present study, 43 562 SLAF tags were developed, and 5 783 polymorphic markers were characterized with quantity and quality met the requirements. All the SLAF tags are well distributed on per chromosome, displaying a high integrity and accuracy (Table 2 and Fig. 5). The 29 Diff_markers are mainly distributed on chromosome 1, providing datasets that are useful for mapping the candidate

Size (Mb) 1.326 0.586 2.033

Marker no. 3 3 3

–logP –1.5 –1.36 –0.67

associated regions into 1.326, 2.033 and 0.586 Mb in size, with 63 genes. For the further mapping study, the SSR markers were used by utilizing information of genomic sequences from B73 Refgen_v2 (http://www.maizegdb.org/), and the bsd-pg gene was localized between umc1395 and umc1603. Finally, 35 annotated genes will be acted as the candidate gene contributing to the further study in mapbased gene isolation. In maize, current models hypothesize that the distinction of MC and BSC are mainly focused on the different chloroplasts type and arrangement, and the functional differentiation of MC and BSC types is the key initiating factors responsible for the development or metabolite transport of C4 photosynthesis (Vičánková et al. 2005; Sharpe et al. 2011). Although some genes involved in the C4 physiology have been identified, mechanisms that control expression of the genes are still not well defined (Tausta et al. 2014). Further study on bsd-pg mutant would illustrate the mechanism of chloroplasts morphological differentiation in bundle sheath and mesophyll cells, and the closely associated markers can be used for marker-assisted selection in maize breeding.

4. Conclusion

Fig. 6 Associated region to bsd-pg. The bsd-pg associated to 3 regions on chromosom 1.

The bsd-pg mutant is assumed a novel mutant other than bsd1 and bsd2, etc., controlled by a single recessive gene. Then a total of 5 783 polymorphic SLAFs are analyzed and 49 Diff_markers are identified. Meanwhile, 3 trait-related candidate regions are detected on chromosome 1 using the association analysis, eventually localized within the region of 850 934 bp between umc1603 and umc1395 in chromosome 1. Using the B73 Refgen_v2 gene models, annotation of the

Table 5 Physical and genetic position of tested SSRs in maize Locus name umc1515 umc2230 umc2233 umc1603 umc1601 umc1812 umc1590 umc1811 umc2232 umc1395

Genome position (bp) coordinates on B73 RefGen_v2 based on sequence identity Start End Unknown Unknown 103 311 427 103 311 831 165 404 557 165 405 077 165 406 002 165 406 555 166 770 809 166 770 972 180 716 274 180 717 420 182 926 541 182 927 313 184 699 381 184 700 585 164 508 927 164 509 358 164 555 621 164 557 259

Genetic map position on genetic 1 Bin 1.05 1.05 1.05 1.05 1.05 1.06 1.06 1.06 1.05 1.05

Position (cM) 107.70 108.10 118.63 118.98 118.45 127.05 129.25 131.60 117.73 117.93

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Fig. 7 Gel pattern of 5 SSRs on F2 population. A, pattern of umc1603 in maternal, F2 pale green progenies and paternal. B, pattern of umc1601. C, pattern of umc2233. D, pattern of umc2232. E, pattern of umc1395. M, 20-bp DNA ladder; Maternal, bsd-pg mutant; Paternal, Xianzao 17; F2-1 to F2-94 were 94 pale green individual progenies from the same F2 population. The arrows showed that there was a cross-over between this SSR and bsd-pg mutation in this F2 progeny.

35 candidate genes contributes to the further study in mapbased gene isolation.

5. Materials and methods 5.1. Plant materials The maize mutation bsd (bundle sheath defective) pale green was isolated from offspring of regeneration seedlings of maize inbred line 501. All 12 maize inbred lines (501, Huo 09-3, X478, Ji 853, Jing 24, Zheng 58, Ji 44, B73, Chang 7-2, Huang C, Xianzao 17 and Han 21) were collected from Chinese maize breeders, which were grown in Beijing of China, and followed the normal agronomic procedures.

5.2. DNA extraction for SLAF-Seq Genomic DNA was extracted from the seedling of per individual in F2 population using a standard CTAB extraction protocol and purified by RNase A. The quality and purity

of DNA samples were measured by 1.0% agarose gel electrophoresis, and the sample DNA was diluted to 100 ng µL–1 with the 1.8–2.2 OD260/280 value. Finally, two DNA pools were constructed by equally mixing the 50 individual genomic DNAs from per line, respectively.

5.3. SLAF library construction for sequencing The SLAF library was constructed according to the the pre-designed scheme, and the procedures based on the previously described by Sun et al. (2013) with little modifications. In brief, two DNA pools and two parents were first treated with appropriate restriction enzyme combination, finally digested into DNA fragments with appropriate sizes (300–450 bp) used in PCR amplification. Then these fragments were excised and diluted for pair-end sequencing by Illumina High-Seq 2500 sequencing platform following the Illumina sample preparation guide (Illumina, Inc., San Diego, CA, USA) at Beijing Biomarker Technologies Corporation (http://www.biomarker.com.cn).

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Table 6 Thirty-five putative genes in association region between umc1395 to umc1603 in B73 genome No. 1 2 3 4 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Name of gene GRMZM2G131897 GRMZM2G131903 GRMZM2G131907 GRMZM2G131935 GRMZM2G561045 GRMZM2G554721 GRMZM2G172116 GRMZM2G510223 GRMZM2G585036 GRMZM2G034916 GRMZM2G501759 GRMZM2G501757 GRMZM2G034768 AC177939.3_FG004 GRMZM2G501744 GRMZM2G034690 GRMZM2G034645 GRMZM2G034174 GRMZM2G032008 GRMZM5G838511 AC191049.3_FG007 GRMZM2G065496 GRMZM2G065478 GRMZM2G065481 GRMZM2G065459 GRMZM2G522066 GRMZM2G119731 GRMZM2G034563 AC211566.3_FG002 GRMZM2G034672 GRMZM2G034680 GRMZM2G335438 GRMZM2G428216 GRMZM2G127739

Start site 164 644 881 164 649 689 164 639 295 164 661 029 164 666 125 164 743 983 164 795 235 164 796 127 164 796 819 164 852 675 164 864 628 164 877 877 164 962 077 164 962 166 164 950 370 164 967 275 164 972 064 164 974 147 165 028 153 165 028 590 165 036 796 165 085 207 165 089 891 165 090 739 165 092 695 165 099 882 165 106 192 165 151 519 165 155 238 165 211 482 165 220 642 165 227 930 165 420 890 165 422 830

End site 164 645 955 164 650 251 164 642 137 164 661 502 164 666 820 164 744 080 164 795 728 164 796 228 164 797 025 164 853 379 164 864 744 164 877 973 164 963 486 164 962 456 164 950 485 164 970 900 164 973 907 164 977 022 165 037 837 165 028 988 165 037 191 165 088 108 165 092 452 165 092 452 165 093 728 165 101 147 165 106 755 165 153 474 165 155 552 165 217 346 165 221 997 165 252 900 165 421 476 165 427 653

5.4. Genotyping and association analysis In this study, the male parent, female parent, wild pool and mutant pool were assigned by P, M, aa and ab, respectively. According to the barcode sequences, raw reads were de-multiplexed to individuals. Then all sequences mapped to the same position were defined as a SLAF loci according to the reference genomic sequences B73 Refgen_v2 (http:// www.maizegdb.org/). Compared by the BLAST-like alignment tool (BLAT) (Holmes 2010), sequences with over 90% identity were clustered in one SLAF locus. A large number of specific fragments were also selected for specific molecular markers development by the method described by Sun et al. (2013). Moreover, a high quality SLAF markers were found for which has less than 3 SNP and average depth of each sample above 3. Finally, the high-density genetic map was constructed by SLAF markers with parental homozygous. The trait-related candidate regions were identified by 3 or more consecutive Diff_markers (ratio_ab≥2). The Maa

Direction Forward Forward Forward Forward Forward Reverse Forward Reverse Forward Forward Reverse Reverse Forward Reverse Forward Forward Reverse Forward Forward Reverse Reverse Forward Forward Reverse Reverse Forward Reverse Forward Reverse Reverse Reverse Reverse Forward Reverse

Description Dof domain, zinc finger Unknown Adenine phosphoribosyltransferase Unknown Unknown Not characterization Not characterization Not characterization Not characterization Not characterization Not characterization Not characterization Unknown Unknown Not characterization Molybdopterin cofactor sulfurase (MOSC) Unknown Unknown Unknown Unknown Unknown B3 DNA binding domain B3 DNA binding domain Not characterization Not characterization B3 DNA binding domain Not characterization Myb-like DNA-binding domain Unknown Family not named Unknown CBP/P300-related Unknown GTP-binding protein alpha subunit

and Paa indicates the depth of aa population derived from M and P, Mab and Pab stands for depth of ab population derived from M and P. Then the ratio of aa is Paa/Maa, and the ratio of ab is Mab/Pab. We defined that ratio_aa or ratio_ab had a value of 1 000 with Maa or Mab equalled 0.

5.5. Narrowed the association regions by SSR assays Based on the physical locations of IBM2 2008 markers and ISU integrated markers on B73 Refgen_v2 (http://www. maizegdb.org/), a total of SSR markers were obtained and synthesized by Sangon Biotech Co. Ltd., Shanghai, China. The PCR reactions was performed in 96-well plates with a volume of 20 μL, including 100 ng of DNA template, 1 pmol of each primer, 2× master mixture buffer (Premix Taq® version 2.0, product code D334C, Thermo Fisher Scientific, Shanghai). And the reaction procedure was carried out in PTC-200 thermal cycle (TaKaRa Biotechnology (Dalian) Co., Ltd., China) as following: 94°C for 5 min; 33 cycles with 94°C for

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30 s; 55°C annealing for 30 s and 72°C elongation for 90 s. Then the 72°C elongation maintained 10 min and dropped to 4°C. All PCR products were evaluated by denaturing polyacrylamide gel electrophoresis (6% polyacrylamide) used PowerPacTM (Bio-Rad, USA) and electrophoresis cell and silver staining (Beyer et al. 1997).

Acknowledgements We thank Dr. Hu Bin from the Department of Biochemistry, University of Oxford, UK, for his advice and English editing. This research was supported by the National Natural Science Foundation of China (30700476 and 31071057) and the Beijing Natural Science Foundation, China (5083021).

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