Identication of two duplicate etiolation genes (py1, py2) in pakchoi (Brassica rapa L. ssp. chinensis)

We identied a stably inherited yellow leaf mutant derived from ‘Huaguan’ pakchoi variety via isolated microspore culture and designated as pylm . This mutant displayed yellow leaves after germination. Its etiolated phenotype was nonlethal and stable during the whole growth period. Its growth was weak and its hypocotyls were markedly elongated. Two recessive nuclear genes named py1 and py2 had a duplicate effect on etiolation. BSR-Seq revealed that py1 and py2 were mapped on chromosomes A09 and A07, respectively. The genes were single Mendelian factors in F 3:4 populations based on a 3:1 phenotypic segregation ratio. The py1 was localized to a 258.3-kb interval on a 34-gene genome. The differentially expressed gene BraA09004189 was detected in the py1 mapping region and regulated heme catabolism. One single-nucleotide polymorphism of BraA09004189 occurred in pylm. A candidate gene-specic SNP marker in 1,520 F 3:4 yellow-colored individuals co-segregated with py1 . For py2 , 1,860 recessive homozygous F 3:4 individuals were investigated and localized py2 to a 4.4-kb interval. Of the ve genes in this region, BraA07001774 was predicted as a candidate for py2. It encoded an embryo defective 1187 and a phosphotransferase related to chlorophyll deciency and hypocotyl elongation. One single-nucleotide polymorphism of BraA07001774 occurred in pylm. It caused a single amino acid mutation from Asp to Asn. According to qRT-PCR, BraA07001774 was downregulated in pylm. Thus, BraA09004189 and BraA07001774 are candidate genes for py1 and py2 . These ndings will elucidate the molecular mechanisms of the gene interactions controlling pakchoi etiolation.


Introduction
The photosynthetic pigment chlorophyll (Chl) is ubiquitous in cyanobacteria and the chloroplasts of higher plants. Chl converts the energy of sunlight into bioavailable chemical energy which drives carbohydrate biosynthesis (Fromme et al. 2003). Chl is an essential component of leaf color which in uences dry matter accumulation and crop yield. In general, leaves appear green because Chl predominates and has a vital role in them. When the Chl content changes in plants, various leaf color mutant phenotypes result including chlorina, virescent, albino, yellow-green, and stay-green (Kurata et al. 2005). Leaf color mutants develop from the inhibition of genes regulating Chl biosynthesis and chloroplast development. Downregulation of these genes directly or indirectly in uences Chl synthesis and degradation and produces the leaf color mutations (Schultes et al. 2000;Motohashi et al. 2003;Song et al. 2018). Thus, leaf color mutants may be ideally suited for the elucidation of the mechanisms of photosynthesis, Chl biosynthesis, chloroplast development, and the expression and regulation of the genes associated with these processes (Stern et al. 2004;Kusaba et al. 2007;Bang et al. 2008;Chen et al. 2018). Leaf color mutants have been characterized in Arabidopsis (Jarvis et al. 2000), rice (Zhang et al. 2006;Moon et al. 2008), wheat (Hui et al. 2012), Brassica napus (Zhu et al. 2014), Brassica oleracea (Liu et al. 2016), barley (Mueller et al. 2012), kale (Zhou et al. 2013), tobacco , soybean (Fang et al. 2014), cotton (Song et al. 2012), and cucumber (Gao et al. 2016;Song et al. 2018). Much research has been invested in the analysis of the genetics, physiology, and molecular mechanisms of Chl biosynthesis and chloroplast development via leaf color mutants.
Several studies in genetic analysis have categorized leaf color mutation inheritance as nuclear and cytoplasmic. Most leaf color mutations are recessively inherited and conferred by a single nuclear gene (Wang et al. 2010;Ansari et al. 2013;Gao et al. 2016;Wang et al. 2018). Leaf color mutations involving two recessive genes are rare. Moreover, their inheritance is complex and it is di cult to apply genetic analysis and gene mapping on them. Wu et al. (2014) identi ed the light color mutant ws1 in Nicotiana tabacum and determined that this phenotype was controlled by the recessive nuclear genes ws1a and ws1b localized by different BC 1 F 2 groups to linkage groups 5 and 24, respectively. BnChd1-1 and BnChd1-2 are responsible for the light green leaf mutant phenotype in Brassica napus. Fine mapping of BnChd1-1 was achieved using the BC 3 F 1 population. Candidate gene prediction suggested that BnChd1-1 encodes a subunit of the NAD(P)H complex in the thylakoid lumenZhao et al. 2014). Chlde cient mutant phenotypes in durum wheat  and Brassica juncea  are also controlled by two recessive genes. Cytoplasmic mutants are uncommon compared to nuclear mutants. However, they have been reported for tobacco (Barak et al. 2000), barley (Colombo et al. 2008), and Brassica campestris (Tang et al. 2018).
In plants, Chl biosynthesis comprises 15 enzymatic steps regulated by at least 27 genes (Nagata et al. 2005). Inactivation mutations of the Chl biosynthetic genes usually results in Chl-de cient mutants (Frick et al. 2003;Zhang et al. 2006;Wang et al. 2010;Sheng et al. 2017). Mutations in the genes governing Chl degradation metabolism generally produce stay-green mutants which retain their green leaf phenotype even during senescence (Yoo et al. 2007;Morita et al. 2009;Wang et al. 2018). Chl and heme biosynthesis are two types of tetrapyrrole formation and share a common metabolic pathway from 5aminolevulinic acid (ALA) to protoporphyrin IX (Proto IX) (Weller et al. 1996). Heme is essential for both respiration and photosynthesis. In contrast, excessive heme accumulation inhibits glutamyl-tRNA reductase activity and ALA synthesis, reduces the rate of tetrapyrrole biosynthesis, and affects Chl biosynthesis (Terry and Kendrick 1999). Leaf color mutants arising from abnormal heme metabolism were identi ed for Arabidopsis , rice Chen et al. 2013;), pea (Linley et al. 2006, and maize (Shi et al. 2013).
In a previous study, we developed a pakchoi (Brassica rapa L. ssp. chinensis) yellow leaf mutant (pylm) derived from the 'Huaguan' pakchoi variety by isolated microspore culture. This strain is a double haploid (DH) with a stable yellow leaf phenotype (Zhang et al. 2017). In the present study, we conducted genetic analysis on pylm usingBSR-Seq with linkage analysis to map the corresponding genes. Then, the candidate genes associated with the mutant phenotype were predicted. The information derived from this work may help facilitate the cloning of etiolation genes and elucidate the molecular mechanisms of gene interactions.

Plant materials and mapping population development
The DH line pylm was obtained by isolated microspore culture of the 'Huaguan' pakchoi variety introduced from Japan musashino seed company. This strain was characterized by yellow leaves and elongated hypocotyls (Zhang et al. 2017). The pylm and the green Chinese cabbage DH line 'FT' were used as parents for the segregating population. The pylm was reciprocally crossed with 'FT' to produce the F 1 , F 2 , and BC 1 generations. Twenty F 2 individuals with green leaves were self-pollinated to produce F 2:3 progenies. Eight green F 2:3 individuals per group were randomly selected from the corresponding populations and self-pollinated to produce the F 3:4 populations. Those with character segregation were used in linkage analysis and gene mapping. All plants were grown in the greenhouse at Shenyang Agricultural University, China.

Detection of variations by BSR-Seq
One hundred individuals with extreme leaf color phenotype were separately collected from the F 2 progeny and pooled for RNA extraction (RNAprep Pure Plant Kit; Tiangen, China). The RNA concentration and integrity were analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The extreme mixed pools green-leaf (G-pool) and yellow-leaf (Y-pool) were constructed by mixing equal amounts of each RNA sample. RNA-Seq was run on an Illumina HiSeq 2500 by GENEWIZ Suzhou Biological Technology Co., Ltd., China.
The quality of the raw RNA-Seq reads was assessed with FastQC (v. 0.10.1). Adapter sequences and lowquality reads containing N and < 70 were deleted from the raw reads with Cutadapt (v. 1.9.1). Low-quality bases at the 5' or 3' end were ltered out. Those with mean quality < 20 were trimmed by the 4-bp sliding window method. Clean data were aligned to the Brassica database (http://brassicadb.org/brad/) with Hisat v. 2.0.14 (Kim et al. 2015). Candidate SNPs between the pools were obtained with the Mpileup module in SAM Tools (v. 0.1.18) SNP loci with depth coverage > 3× were screened for differential SNP analysis in the mutant and wild type pools. Euclidean distances (ED) for the differential SNP loci were calculated. The ED for each differential SNP locus was raised to the power ve, namely, ED 5 , to eliminate background noise (Su et al. 2016). All ED 5 were sorted and the differential SNP loci with ED 5 in the top 1% were screened and mapped to speci c chromosome regions based on the SNP locus distributions. Chromosome regions associated with the target traits were predicted according to the distributions of the ED 5 for the differential SNP loci on the chromosomes.

Differential gene expression analysis
To detect differentially expressed genes (DEGs) between the pools, a gene expression level analysis was performed with Htseq (v. 0.6.1). The reads per kilobases per million mapped reads (RPKM) were calculated (Mortazavi et al. 2008). DEGs were screened using a preset threshold (|log 2 fold change| ≥ 1 and false discovery rate (FDR) ≤ 0.05). DNA isolation, polymerase chain reaction (PCR), and linkage analysis A modi ed CTAB method (Murray and Thompson 1980) was used to extract genomic DNA from young leaves of the parental and F 3:4 populations with a phenotypic segregation ratio of 3:1. The primers for polymorphism analysis were designed in Primer Premier (v. 5.0). PCR ampli cations were performed following the instruction as described by Wang et al. (2018). PCR products were separated on 5% (w/v) denaturing polyacrylamide gels and examined by silver staining. The genetic linkage map was constructed by Join Map v. 4.0 (Van Ooijen 2006) using the segregation data. Map distances were calculated and reported in centimorgans (cM) according to Kosambi's mapping function (Kosambi 1944).

Candidate gene prediction and qRT-PCR validation
Using the chromosome location of the target gene, all adjacent genes were annotated with the Brassica database (http://brassicadb.org/brad/). Candidate genes were predicted according to gene annotation. PCR primers were designed to span the entire putative gene length between the mutant pylm and wild type 'CK-51'. Candidate genes were cloned following the method referenced from Huang et al. (2016). Sequences were determined by GENEWIZ Biological Technology Co. Ltd. (Suzhou, China) and aligned with DNAMAN software.

Phenotypic characterization of mutant pylm
The wild type 'CK-51' and pylm were both obtained from isolated microspore culture of the 'Huaguan' pakchoi variety. However, the latter displayed yellow leaves at germination and this phenotype was stable throughout its lifetime (Fig. 1). The mutant had a slender phenotype and weak growth. However, its yellow leaf color was nonlethal. Moreover, relative to 'CK-51', pylm displayed an elongated hypocotyl at the seedling stage ( Fig. 1c) and early owering at the bolting stage (Fig. 1b).
Genetic analysis of mutant pylm F 1 and F 2 populations were constructed from crosses between pylm (Fig. S1a)and the Chinese cabbage DH line 'FT' (Fig. S1b). The F 1 individuals from the reciprocal crosses had the same green leaf phenotype as 'FT'. Therefore, inheritance of the etiolation phenotype in pylm is nuclear rather than cytoplasmic. Segregation statistics data for the green and yellow leaf phenotypes of the F 2 population accorded with the expected Mendelian ratio of 15:1 ( 2 < 2 0.05 = 3.84). Thus, the Chl de ciency trait is controlled by two duplicate recessive nuclear genes. The BC 1 progeny was obtained from F 1 separately backcrossed with pylm and 'FT'. Segregation statistics data for the green and yellow leaf phenotypes of the BC 1 F 1 population from the cross of F 1 with pylm tted the expected Mendelian ratio of 3:1 ( 2 < 2 0.05 = 3.84). This nding con rms that the mutant trait is conferred by two duplicate recessive nuclear genes. They were designated as py1 and py2.Neither gene alone can induce the yellow leaf phenotype. Phenotypic data for all generations are listed in Table 1.

Segregation of py1 and py2
According to the genetic analysis of pylm, the reduced-Chl phenotype is controlled by the recessive nuclear genes py1 and py2 with duplicate effects on etiolation. In consequence, the F 2 and BC 1 F 1 populations could not be used to map these genes separately. To separate py1 from py2, F 2 individuals with green leaves may be randomly selected and self-pollinated to produce F 2:3 progeny. Green-colored F 2:3 plants with a statistical segregation ratio of 3:1 (green-colored:yellow-colored) may be self-pollinated to generate F 3:4 progeny. In theory, ~2/3 of the F 3:4 families should display the expected Mendelian segregation ratio of 3:1. Some of these could map py1 while the others could map py2 (Fig. 2).
Twenty green-colored individuals from F 2 were randomly selected and self-pollinated to produce F 2:3 . For the twenty F 2:3 families, phenotypic segregations were investigated. There were three distinct groups.

BSR-Seq analysis
A total of 47,526,126 and 49,119,466 raw reads (150-bp) were generated from the G-pool and Y-pool, respectively. After quality evaluation and data ltering, 97% of the read pairs (46,456,174 for the G-pool and 47,581,728 for the Y-pool) remained. Clean reads were mapped against the Brassica reference genome with Hisat v. 2.0.14. Of these, > 66% were uniquely mapped in both pools.
Relative to the reference genome, 154,863 and 157,022 SNPs were detected in the G-pool and Y-pool, respectively. Differential SNP loci were screened for ED 5 calculation and 412 target differential SNP loci were obtained between the pools according to the top 1% ED 5 threshold. Two distinct peaks were observed on chromosomes A07 and A09 (Fig. 3). This nding was consistent with the hypothesis that themutant trait is controlled by two duplicate recessive nuclear genes. Thus, it was predicted that the etiolation genes were located on chromosomes A07 and A09 within ve chromosome regions (Table 2).
Identi cation of differentially expressed genes RPKM was used to measure gene expression level. By setting RPKM ≥ 0.1, 55,250 genes were detected. These were divided into six RPKM distribution intervals (Table S3). There were 181 DEGs between the Gpool and Y-pool according to the constraint (|log 2 fold change| ≥ 1 and FDR ≤ 0.05). Ninety genes were upregulated and the others were downregulated when the G-pool was compared with the Y-pool (Fig. S2).
The DEGs are shown in Table S4.

Fine mapping of py1
Ninety-six SSR markers were developed around the three predicted chromosome regions on chromosome A09. They were used to detect polymorphisms between pylm and 'FT'. After screening, thirty-seven SSR markers displayed polymorphisms between parents. They were used to test twelve green-colored and yellow-colored individuals each from the No. 1 F 3:4 family. SSRzk5 and SSRzk12 were located near the 23,811,435-27,563,122 region on chromosome A09 and showed linkage to py1 on the opposite side.
A total of 1,520 yellow-colored individuals of the No. 1 F 3:4 family were selected as the py1 mapping population. A linkage analysis disclosed that py1 was located between SSRzk5 and SSRzk12 at estimated genetic distances of 3.2 cM and 1.8 cM, respectively (Fig. 4a). To identify the molecular markers tightly linked to py1 and narrow the py1 mapping interval, new SSR and Indel markers were developed between SSRzk5 and SSRzk12. The polymorphic markers SSRzk17, SSRzk28, SSRzk29, SSRzk36, Indelzk72, and Indelzk125 were linked to py1 (Table S5). SSRzk17, Indelzk72, and Indelzk125 were located on one side of py1 as SSRzk5 while SSRzk28, SSRzk29, and SSRzk36 were located on the other side of py1 as SSRzk12. The py1 was mapped between Indelzk125 and SSRzk36 at 0.13 cM and 0.2 cM, respectively (Fig. 4b). Therefore, py1 was mapped in a 258.3-kb region between the most tightly linked markers (Fig. 4c).

Candidate py1 analysis
The target DNA sequences of the 258.3-kb region between Indelzk125 and SSRzk36 were obtained from the Brassica database. A genomic sequence analysis revealed that the candidate region contained 34 genes (Fig. 4c, Table S6). Differential gene expression analysis disclosed only BraA09004189 in the py1 mapping region. BraA09004189 is a heme oxygenase (HO1) which participates in heme catabolism. Mutants with yellow leaf phenotype induced by defective HOs were reported in earlier studies (Chen et al. 2013;. BraA09004189 was predicted to be the most probable candidate py1 gene. To con rm this hypothesis, two pairs of primers were designed to sequence BraA09004189 in pylm and 'CK-51' ( Table S7). The BraA sequence did not differ between parents whereas the BraB sequence in pylm presented with one single nucleotide polymorphism (SNP) (Fig. 5). Based on the position of BraA09004189, an SNP marker was designed to screen 1,520 yellow-colored individuals from the No. 1 F 3:4 family. The bands of whole mapping individuals co-segregated with py1.
A qRT-PCR was performed to determine BraA09004189 expression in pylm and 'CK-51'. In accordance with the differential gene expression analysis, the results indicated that BraA09004189 expression level was much higher in 'CK-51' than that in pylm (Fig. 6). This nding con rms that BraA09004189 is the candidate gene for py1.

Fine mapping of py2
Considering the constructed populations size, we screened the Nos. 2-5 F 3:4 families using the same research strategy applied for SSRzk5 and SSRzk12 linked to py1. The etiolation gene py1 was identi ed in the Nos. 2, 4, and 5 F 3:4 families. In theory, then, the No. 3 F 3:4 family may be used to establish the py2 locus.
Forty-eight SSR markers were developed around the two predicted regions on chromosome A07 to detect polymorphisms between pylm and 'FT'. After screening, eleven SSR markers displayed polymorphisms between the parents. They were used to test twelve green-colored and twelve yellow-colored individuals of the No. 3 F 3:4 family. SSR84 and SSR103 were located around the region 11,166,810-15,034,483 on chromosome A07 and presented with linkages to py2 on the opposite sideFig. 7a; Table S8).
There were 1,860 yellow-colored individuals from the No. 3 F 3:4 family selected as the py2 mapping population. The py2 was located between SSR11 and SSR15 at estimated genetic distances of 0.24 cM and 0.03 cM, respectively (Fig. 7b). To narrow the py2 mapping interval and identify the molecular markers tightly linked to py2, new single-nucleotide variant (SNV) markers were developed between SSR11 and SSR15. Only the polymorphic marker SNV11 was linked to py2. Based on the recombinant individuals, the py2 interval was narrowed to 14,851,951-14,896,902 and contained ve genes (Fig. 7c).

Candidate py2 analysis
Annotation data for the ve candidate genes in the py2 target region were obtained from the Brassica database (Table S9). Primers were designed to cover the cDNA for each gene and predict the candidate genes (Table S10). There were no differences between pylm and 'CK-51' in terms of BraA07001775, BraA07001776, or BraA07001777. After PCR ampli cation, the BraA07001773 sequence was disordered and the sequence comparisons were inconsistent over serial repetitions. There was SNP variation between parents for the rst exonin BraA07001774 (Fig. 8). It caused a single amino acid mutation from Asp (GAT) in the wild type to Asn (AAT) in pylm (Fig. 9). Therefore, BraA07001774 was taken as the most probable candidate gene for py2.
BraA07001774 is an embryo defective 1187 (emb 1187) and a phosphotransferase. The albino mutants (pds1, pds2) phenotypes in Arabidopsis thaliana may be caused by emb 71 (Franzmann et al. 1995). For Arabidopsis seeds with silique defects, hypocotyl elongation was characterized during the development of F 2 generation mutant seedlings (Patton et al. 1991). We proposed that the mutant phenotype is determined by mutations in BraA07001774. To validate our prediction, BraA07001774 expression in pylm and 'CK-51' was analyzed by qRT-PCR. BraA07001774 was dramatically downregulated in pylm (Fig.  10). Thus, it probably is the candidate gene for py2.

Discussion
Mutations in leaf color are widespread in nature. The main type of leaf color mutation is Chl de ciency. Dwar sm, retarded growth, attenuated photosynthetic capacity, low yield, and death are associated with this defect (Jung et al. 2003;Wu et al. 2007;Zhao et al. 2014). Here, we identi ed the pakchoi yellow leaf mutant pylm from isolated microspore culture. Unlike previously reported Chl de cient mutants, pylm presented with substantially elongated hypocotyls at the seedling stage and early owering at the bolting stage. The etiolation phenotype in pylm was nonlethal and stable throughout the growth period. The Chl de ciency in pylm was controlled by two duplicate recessive genes. These characteristics suggested that pylm was of high value for research in revealing the Chl biosynthesis mechanism regulated by gene interactions.
Map-based cloning is an effective gene isolation strategy. It has been extensively used for plant gene function analysis (Jander and Last 2002;Sang et al. 2010;Xie et al. 2018). However, it is contingent upon ne mapping of the target gene. For most leaf color mutants, the traits are recessively inherited and controlled by a single nuclear gene. The F 2 populations are instinctively applied to map the target gene (Wu et al. 2007;Song et al. 2018). With regard to the character conferred by two recessive nuclear genes, F 2 populations may also be used in preliminary mapping. An e cient way to isolate allele pairs from each other and separately map them is to construct advanced backcrosses and other populations. The recessive white stem (ws) loci in Nicotiana tabacum and the Chl de ciency (Bnchd1) loci in Brassica napus were successfully mapped using constructed BC 1 F 2 and BC 3 F 1 populations, respectively Zhao et al. 2014). In the present study, genetic analysis revealed that the duplicate recessive nuclear genes designated as py1 and py2 were responsible for the etiolation trait. We successfully segregated py1 from py2 and constructed an inheritance model for the Chl de ciency trait in pakchoi. Twenty F 3:4 families with a phenotypic segregation ratio of 3:1 were constructed. Various F 3:4 families were successfully used to map the py1 and py2 loci separately. Compared to using advanced backcross populations to map pairs of recessive nuclear genes, creating and using F 2:3 or F 3:4 families avoid the selection errors and interference in genetic analysis caused by the incomplete emasculation of Brassica rapa.
BSR-Seq e ciently combines the respective superiorities of bulk segregation analysis (BSA) and RNA sequencing (RNA-Seq) for rapid gene mapping (Trick et al. 2012;Lee et al. 2014;Lu et al. 2014;Ramirez-Gonzalez et al. 2015). BSR-Seq is targeted at the mRNA level. It selects phenotypically opposite individuals from segregated populations and constructs two RNA mixing pools to nd SNPs at the transcript level. The transcriptome data localize the target genes and detect potentially associated DEGs (Schneeberger and Weigel 2011). BSR-Seq has been extensively applied to map the causal genes linked to a single target trait (Nestler et al. 2014;Tang et al. 2014;Wang et al. 2018). Two independently inherited traits may also be localized by BSR-Seq. Tan et al. (2019) applied BSR-Seq to locate the genes controlling male sterility on chromosome A05 and white petal on chromosome A02. Here, the mutant pylm and Chinese cabbage DH 'FT' lines were chosen as parents to construct the F 2 separation population. The phenotypes of the wild type and mutant individuals signi cantly differed. Thus, extreme mixed pools could be accurately and conveniently created for BSR-Seq. Release of Brassica rapa genomic data enhanced the reliability of these populations in BSR-Seq applications. Five candidate regions related to theyellow leaf phenotypein pylm were identi ed on chromosomes A07 and A09. Molecular markers were developed according to the locations of the candidate regions. The etiolation genes py1 andpy2were separately mapped with different F 3:4 families. The ndings con rmed the feasibility of BSR-Seq for mapping two recessive nuclear genes. They also showed that BSR-Seq simpli es molecular marker development and screening in traditional mapping methods and greatly improves their e ciency.
New molecular markers were developed near the target regions based on preliminary py1 mapping by BSR-Seq. The py1 was mapped between the markers Indelzk125 and SSRzk36 on A09 chromosome over a 258.3-kb localization interval containing 34 predicted genes. No new polymorphic SSR or Indel markers were available to limit the localization interval. The gene expression patterns determined by BSR-Seq disclosed only one differentially expressed gene (BraA09004189) within the py1 mapping region. A gene annotation referenced from the Brassica database indicated that BraA09004189 encodes heme oxygenase-1 (HO1). This enzyme plays a vital role in phytochrome chromophore metabolism, the photoresponse mechanism, adventitious root formation, and oxidative damage mitigation (Davis et al. 1999;Ortiz de Montellano and Wilks 2001;Quail 2002;Emborg et al. 2006). HO1 stabilizes and maintains the heme content by transforming heme into BV-IXα (Terry et al. 1993). As embranchments of tetrapyrrole biosynthesis, Chl and heme biosynthesis share a common metabolic pathway from ALA to Proto IX. Excessive heme accumulation caused by abnormal heme metabolism leads to feedback inhibition of Chl biosynthesis (Weller et al. 1996). Therefore, a decrease in HO1 activity may in uence Chl biosynthesis. The hy1 mutant of Arabidopsis and the yellow-green leaf 2 mutant of rice presented with the reduced-Chl phenotype because of free heme inhibition resulting from HO1 mutations (Davis et al. 1999;Chen et al. 2013). HO1 defects strongly affected thylakoid development in rice . Davis et al. (1999) found that the abnormally elongated hypocotyl phenotype of mutant Arabidopsis seedlings may be associated with HO1 defects. Thus, py1 may encode HO and a mutation thereof may in uence Chl biosynthesis and leaf color. In this study, qRT-PCR demonstrated that BraA09004189 was downregulated in pylm. This nding was consistent with those obtained by BSR-Seq. The SNP BraA09004189 was detected between pylm and 'CK-51'. A candidate gene-speci c SNP marker in 1,520 F 3:4 yellow-colored individuals co-segregated with py1. Thus, BraA09004189 corresponds to the yellow leaf locus py1 in pylm.
It was already known that certain Chl de ciency traits are controlled by two recessive nuclear genes. However, there was a lack of appropriate mapping populations or reliable molecule markers. Therefore, they were either approximately mapped without de nite locations Wu et al. 2014) or only one of the pair could be localized (Zhao et al. 2014). In previous studies, little progress was made in the simultaneous ne mapping or accurate prediction of the candidate genes. Here, we used the same mapping strategy as that for py1 to accomplish ne mapping for py2. The linkage analysis disclosed that py2 was mapped between SSR11 and SSR15 on chromosome A07. The mapping interval was narrowed to 4.4 kb by the SNV11 marker linked to py2. Sequence analysis of the ve genes in the py2 localization interval showed that only BraA07001774 expression signi cantly differed between pylm and 'CK-51'. For pylm, BraA07001774 had a SNP missense mutation on the rst exon such that the wild type had an Asp residue whereas pylm had an Asn. The qRT-PCR revealed that BraA07001774 was downregulated in the mutant relative to the wild type. Gene annotation in the Brassica database indicated that BraA09004189 encodes embryo defective 1187 (emb 1187) and a phosphotransferase. In Arabidopsis, the emb (embryo defective) genes are essential for seed development (Despres et al. 2001). EMB genes encode various proteins. Thirty percent of them are active in the plastids (Shen et al. 2013). Most emb mutations result in albinism or etiolated seeds and embryos which are secondary effects of mutations in chloroplast biogenesis and function (Li and Thomas 1998). The albino mutants (pds1, pds2) and hypocotyl elongation phenotypes in Arabidopsis may be related to mutations in EMB genesPatton et al. 1991;Franzmann et al. 1995). Thus, BraA07001774 is a candidate gene for py2. Here, we successfully achieved ne mapping and predictions for the two candidate etiolation genes py1 and py2. These discoveries may help elucidate the molecular mechanisms underlying the trait controlled by two duplicate recessive nuclear genes. In future studies, functional validation will be conducted to clarify the functions of these candidate genes. In this manner, the molecular mechanism of gene interactions may be better understood.

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Availability of data and materials
All data are fully available without restriction.  Tables   Table 1 Genetic   ED5 distributions on chromosomes. Each color on the X-axis represents different chromosomes of Brassica rapa. Y-axis represents ED5 for each differential SNP locus. Horizontal line is the threshold of the top 1% ED5 Figure 4 Genetic and physical py1 maps and candidate gene analysis. a: Chromosome A09 linkage map was constructed with 1,520 pylm individuals from the No. 1 F3:4 family. The py1 was preliminarily mapped between SSRzk17 and SSRzk28; b: Fine mapping of py1. The py1 was restricted to the region between Indelzk125 and SSRzk36. Number of recombinants between the markers and py1 is shown below the genetic map. The mapping distance above the linkage map is in centimorgan (cM) units; c: Candidate py1 region and the annotated genes in the Brassica database. The py1 locus was narrowed to a 258.3-kb region comprising 34 predicted genes. The numbers 1-34 refer to the candidate genes. The arrows indicate the direction of gene expression. Detailed information on the 34 genes is presented in Table S6 Page 24/29 Genetic and physical maps of py2 and candidate gene analysis. a: Linkage map of chromosome A07 was constructed with 1,860 individuals bearing the pylm phenotype in the No. 3 F3:4 family. The py2 was preliminarily mapped between SSR133 and SSR103; b: Fine mapping of py2. The py2 was restricted to the region between SSR11 and SSR15. The number of recombinants between the markers and py2 is shown under the genetic map. The distance above the linkage map is in centimorgan (cM) units; c: Candidate py2 region and annotated genes in the Brassica database. The py2 locus was narrowed to a 4.4-kb region containing the ve predicted genes BraA07001773-BraA07001777. Arrows indicate the direction of gene expression. Detailed information on these genes is presented in Table S9 Page 27/29