A high-density genetic map developed by specific-locus amplified fragment (SLAF) sequencing and identification of a locus controlling anthocyanin pigmentation in stalk of Zicaitai (Brassica rapa L. ssp. chinensis var. purpurea)

- A high-density genetic map developed by specific-locus amplified fragment (SLAF) sequencing and identification of a locus controlling anthocyanin pigmentation in stalk of Zicaitai ( Brassica rapa L.
- The aim of this study is to construct a high density genetic map using the specific length amplified fragment sequencing (SLAF-seq) technique to explore genetic basis for anthocyanin pigmentation traits via quantitative trait loci (QTL) mapping..
- Bioinformatics analysis of the major locus identified 62 protein-coding genes.
- However, there were several transcription factors like helix-loop-helix (bHLH) bHLH, MYB in the locus.
- An insertion and deletion (InDel) marker developed from deletion/insertion in the promoter region of bHLH49 showed significant correlation with the stalk color trait in the F2 population..
- 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- Recently, a genetic map of 120 Chinese cabbage F2 individuals of 711 bins representing 3985 single nucleotide polymorphism (SNP) markers was constructed using restriction site-associated DNA se- quencing (RAD-seq) [5].
- Multiple markers developed by sequencing facilitate high-density genetic map con- struction and QTL analysis.
- successfully used for high-density linkage map construction in diverse species such as common carp [15], sesame [16], soybean [17, 18], mei [19], and cucumber [20, 21].
- Thus, it is necessary to construct high density genetic map for Zicaitai to perform agronomic traits mapping..
- The biosynthetic pathway of the purple pigment antho- cyanin is a conserved network in many plant species and is well characterized [24].
- In the current study, genetic mapping of the purple stalk trait was performed using F2 progeny of two B..
- The SLAF-seq technique was used for construc- tion of a high-density genetic map containing 10,940 SNP markers distributed into 10 linkage groups (LGs), and spanning 1030.04 cM.
- Stalk and stem color in the F2 population followed a continuous distribution that indicates that this trait might be quantitative and the alleles conferring purple stalks might only derive from the maternal parent..
- In the maternal line (Xianghongtai reads representing 80,935 SLAFs with average depth of 39.4 were generated (Table 1).
- In the paternal line (Yinong 50D reads representing 87,833 SLAFs with average coverage of 40.75 were generated (Table 1).
- In the F2 population reads representing 77,079 SLAFs with average depth of 9.89 were generated (Table 1)..
- Finally, 10,112 of the 13,756 polymorphic SLAFs with aa×bb segregation pattern were selected for linkage map construction as Xianghongtai 01 and Yinong 50D are homozygous lines with geno- types of aa and bb (Fig.
- Characteristics and evaluation of the high-density genetic map.
- The integrity of the mapped markers ranged.
- from 99.15 to 100% which indicated high quality of the map (Table 2).
- The genetic lengths of the 10 LGs ranged from 73.12 (LG8) to 140.03 cM (LG5).
- The locations of all markers in the genetic map were presented in Additional file 1..
- The double crossover should be at a low rate for high-density genetic map construction [38].
- In this study, the percentage of double crossover of each LG is less than 0.04%, which is in a permis- sible limit for high-density genetic map construction (Table 2).
- all 4253 markers on the final map were mapped onto 10 chromosomes in the genome (Fig.
- The Spearman values of the 10 LGs ranged from 0.92 to 1.00 (Additional file 2)..
- Most of the SLAF markers on the genetic map were in the same order as those in the corresponding chromosomes of the B.
- Based on the high-density Table 1 Summary of SLAF depths.
- genetic map and phenotypic characterization, QTL mapping of the stalk color trait were performed using Highmap with the Kosambi mapping function.
- Among all loci, qBrps2 had the highest effect on purple color, explaining 61.2% of the phenotypic variation and mapping on to a region between Marker2181741 and Marker2262922, which spanned a genetic distance of ~ 0.33 cM and a physical distance of ~ 0.38 Mb (Table 3).
- The sum of PVE values of the five QTLs was 77%, indicating that the mapped QTLs explained most of the variance..
- Based on the B.
- [1], 62 predicted protein-coding genes were identified in the small physical interval of qBrps2.
- Table 2 Basic information of the B.
- rapa genetic map.
- Linked InDel marker development and identification To confirm that linkage of qBrps2 with trait segregation, an InDel marker located in the promoter region was designed targeting a 7 bp deletion in locus 1771 in the genome sequence of Bra004348 (Fig.
- The InDel marker was polymorphic between the parents and were heterozygous in the F1, suggesting this marker might be associated with the purple stalk trait (Fig.
- The effect of envir- onment on the trait or the influence of the presence of other QTLs could contribute to misidentification of.
- This InDel marker was further tested for genetic differentiation of the cultivars based on the stalk color..
- Here the honglu hybrids with green stalk and F1 hybrid culti- var with light purple color at the bottom of the stalk showed the same genotype as of F1 (Fig.
- These findings confirmed the associ- ation of this InDel marker with the phenotypic variation of the stalk color of different cultivars.
- 4 LOD scores along the 10 chromosomes for variation of the purple stalk trait.
- The horizontal ordinate is the order of markers in the linkage group.
- Curves in the plot indicate the genetic coordinate and LOD score (top) of the detected QTLs.
- The box inset shows qBrps2 and the zoom-in view of the peak on Chr07 and the imaginary line indicates the LOD for genome wide significances for the purple stalk trait.
- We identified a total of 26,557 polymorphic SLAFs, and finally selected 4253 polymorphic markers with 75% of progeny for construct- ing the genetic map.
- The selection criterion is applicable for high-density genetic map construction (Table 2).
- The linear analysis of this genetic map and reference genome showed that the SLAF markers in most LGs were well ordered and the marker quality fulfilled the require- ments for genetic map construction.
- Compared with another reported high density map of B.
- However, we found that F2 individuals with 2nd color grade purple stalks have green leaves and the purple color is focused on the stalk and stem in one of the parent (Zicaitai).
- The identification of re- sponsible genes by genetic mapping can facilitate under- standing of the molecular mechanism of anthocyanin biosynthesis..
- The physical region of the chr07 QTL was from to Mb in internal length)..
- The major locus on chr07 from to kb in internal length) was included in the reported minor QTL on this chromosome [22].
- These results suggest that the high-density genetic map generated in this study can be successfully applied in QTL mapping..
- The small size of the region facilitated Table 4 Annotation of BrPs candidate genes.
- Anthocyanin biosynthesis variation associated with this region may result from transcriptional regulation of structural genes since no salient structural genes were found in the locus.
- The transcript levels of the genes are normalized to actin transcript level and expressed relative to PPIS which is set to 1.
- In tomato, bHLH TF gene SlGL3 is also not participating in the putative MBW pro- tein complex in tomato as is the case in Arabidopsis [51].
- The difference of major QTLs in the leaves and stalk regions suggests that bHLH TFs activity can be of tissue specific during anthocyanin biosynthesis in Zicaitai.
- Moreover, the members of the MBW complex,.
- In the present study, we found that purple stalk color was not linked with change of leaf color, suggesting the two traits could be mainly regulated by two different genes which might be tis- sue specific.
- In the present study, BrbHLH49 on chr07 seems to be the best candidate gene for purple stalk color since the regulation of anthocyanin biosyn- thesis in Xianghongtai 01 is likely due to the high ex- pression of this gene.
- The environmental stimuli involved in the regulation of anthocyanin biosynthesis has not yet been found.
- 6 Frequency distribution of the 150 F2 individuals based on the genotype of the InDel marker.
- These results implicate that the BrbHLH49 was the most interesting candidate gene for increased anthocyanin biosynthesis in the stalk of Xianghongtai 01.
- Further analysis to identify candidate gene function and the genes responsible for anthocyanin biosynthesis needed to clarify the genetic mechanism underling anthocyanin accumulation in the stalk of Zicaitai..
- In this study, we used SLAF-seq method to construct a high-genetic map with the most markers for B.
- Three-week old seedlings of the inbred lines and individ- uals were planted in the experimental field of the Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, China in November of 2016.
- Seedlings were planted in the ridges of hilled rows (30 cm in height.
- Only the SLAF markers with the pattern of aa×bb were used for genetic map construction since F2 individuals were derived from two fully homozygous parental lines..
- Linkage map construction and QTL mapping of stalk color.
- All high quality SLAF markers were allocated to one of the ten LGs based on the location on the chromosome.
- The function of the predicted genes was determined using the Swissprot database and BLASTX..
- To examine the expression of the 8 genes, a PCR mix was prepared with 1 μ L cDNA samples as templates in the qRT-PCR assay in the presence of a SYBR Green PCR Master Mix (Invitrogen, USA) and gene-specific primers..
- The relative levels of the amplified mRNA were evaluated according to Livak and Schmittgen according to the 2 − ΔΔ Ct method using actin gene for normalization [62].
- An InDel marker located in at chr07 was developed based on the deletions/insertions in the can- didate gene of Bra004348 .
- (PDF 91 kb) Additional file 3: Annotation of the genes in the major QTL locus..
- This study was supported by the projects of the Guangdong Science and Technology Foundation (2015B the Guangzhou Science and Technology Foundation the projects of the Guangdong Science and Technology Foundation (2018A030313770).
- The listed markers in the genetic map in our study are available in the Additional file 1.The sequences of markers and the raw datasets analyzed in the study are not publicly available but can be available, on reasonable request from the corresponding author..
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