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Identification and fine mapping of a stay-green gene (Brnye1) in pakchoi (Brassica campestris L. ssp. chinensis)

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Using bulked segregant analysis combined with next-generation sequencing, we delimited the Brnye1 gene responsible for the stay-green trait of nye in pakchoi. Sequence analysis identified Bra019346 as the candidate gene.

Abstract

“Stay-green” refers to a plant trait whereby leaves remain green during senescence. This trait is useful in the cultivation of pakchoi (Brassica campestris L. ssp. chinensis), which is marketed as a green leaf product. This study aimed to identify the gene responsible for the stay-green trait in pakchoi. We identified a stay-green mutant in pakchoi, which we termed “nye”. Genetic analysis revealed that the stay-green trait is controlled by a single recessive gene, Brnye1. Using the BSA-seq method, a 3.0-Mb candidate region was mapped on chromosome A03, which helped us localize Brnye1 to an 81.01-kb interval between SSR markers SSRWN27 and SSRWN30 via linkage analysis in an F2 population. We identified 12 genes in this region, 11 of which were annotated based on the Brassica rapa annotation database, and one was a functionally unknown gene. An orthologous gene of the Arabidopsis gene AtNYE1, Bra019346, was identified as the potential candidate for Brnye1. Sequence analysis revealed a 40-bp insertion in the second exon of Bra019346 in nye, which generated the TAA stop codon. A candidate gene-specific Indel marker in 1561 F2 individuals showed perfect cosegregation with Brnye1 in the nye mutant. These results provide a foundation for uncovering the molecular mechanism of the stay-green trait in pakchoi.

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References

  • Abe A, Kosugi S, Yoshida K, Natsume S, Takagi H, Kanzaki H, Matsumura H, Yoshida K, Mitsuoka C, Tamiru M (2012) Genome sequencing reveals agronomically important loci in rice using MutMap. Nat Biotechnol 30:174–178

    Article  CAS  PubMed  Google Scholar 

  • Alós E, Roca M, Iglesias DJ, Mínguez-Mosquera MI, Damasceno CM, Thannhauser TW, Rose JK, Talón M, Cercós M (2008) An evaluation of the basis and consequences of a stay-green mutation in the navel negra Citrus mutant using transcriptomic and proteomic profiling and metabolite analysis. Plant Physiol 147:1300–1315

    Article  PubMed  PubMed Central  Google Scholar 

  • Armstead I, Donnison I, Harper J, Hörtensteiner S, James C, Mani J, Moffet M, Ougham H, Roberts L, Thomas A, Weeden N, Thomas H, King I (2006) From crop to model to crop: identifying the genetic basis of the stay green mutation in the Lolium/Festuca, forage and amenity grasses. New Phytol 172(4):592–597

    Article  PubMed  Google Scholar 

  • Aubry S, Mani J, Hörtensteiner S (2008) Stay-green protein, defective in Mendel’s green cotyledon mutant, acts independent and upstream of pheophorbide a oxygenase in the chlorophyll catabolic pathway. Plant Mol Biol 67:243–256

    Article  CAS  PubMed  Google Scholar 

  • Bachmann A, Fernandezlopez J, Ginsburg S, Thomas H, Bouwkamp JC, Solomos T, Matile P (1994) Stay green genotypes of Phaseolus vulgaris L.: chloroplast proteins and chlorophyll catabolites during foliar senescence. New Phytol 126(4):593–600

    Article  CAS  Google Scholar 

  • Barry CS, Giovannoni JJ (2008) Amino acid substitutions in homologs of the STAY GREEN protein are responsible for the green-flesh and chlorophyll retainer mutations of tomato and pepper. Plant Physiol 147(1):179–187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Behm-Ansmant I, Izaurralde E (2006) Quality control of gene expression: a stepwise assembly pathway for the surveillance complex that triggers nonsense-mediated mRNA decay. Genes Dev 20:391–398

    Article  CAS  PubMed  Google Scholar 

  • Borovsky Y, Paran I (2008) Chlorophyll degradation during pepper fruit ripening in the chlorophyll retainer mutation is impaired at the homolog of the senescence-inducible stay green gene. Theor Appl Genet 117:235–240

    Article  CAS  PubMed  Google Scholar 

  • Brogna S, Wen J (2009) Nonsense-mediated mRNA decay (NMD) mechanisms. Nat Struct Mol Biol 16:107–113

    Article  CAS  PubMed  Google Scholar 

  • Canfield MR, Guiamét JJ, Noodén LD (1995) Alteration of soybean seedling development in darkness and light by the stay-green mutation cytG and Gd1d2. Ann Bot (Lond) 75:143–150

    Article  Google Scholar 

  • Cha KW, Lee YJ, Koh HJ, Lee BM, Nam YW, Paek NC (2002) Isolation, characterization and mapping of stay green mutant in rice. Theor Appl Genet 104(4):526–532

    Article  CAS  PubMed  Google Scholar 

  • Chang YF, Imam JS, Wilkinson MF (2007) The nonsense-mediated decay RNA surveillance pathway. Annu Rev Biochem 76:51–74

    Article  CAS  PubMed  Google Scholar 

  • Doma MK, Parker R (2007) RNA quality control in eukaryotes. Cell 131(4):660–668

    Article  CAS  PubMed  Google Scholar 

  • Fang C, Li C, Li W, Wang Z, Zhou Z, Shen Y, Wu M, Wu Y, Li G, Kong LA, Liu C, Jackson SA, Tian Z (2014) Concerted evolution of D1 and D2 to regulate chlorophyll degradation in soybean. Plant J 77(5):700–712

    Article  CAS  PubMed  Google Scholar 

  • Gong YH, Zhang J, Gao JY, Lu JY, Wang JR (2005) Slow export of photoassimilate from stay-green leaves during late grain-filling stage in hybrid winter wheat (Triticum aestivum L.). J Agron Crop Sci 191:292–299

    Article  CAS  Google Scholar 

  • Horie Y, Ito H, Kusaba M, Tanaka R, Tanaka A (2009) Participation of chlorophyll b reductase in the initial step of the degradation of light-harvesting chlorophyll a/b-protein complexes in Arabidopsis. J Biol Chem 284:17449–17456

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hörtensteiner S (2009) Stay green regulates chlorophyll and chlorophyll-binding protein degradation during senescence. Trends Plant Sci 14(3):155–162

    Article  PubMed  Google Scholar 

  • Islam MS, Zeng L, Thyssen GN, Delhom CD, Kim HJ, Li P, Fang DD (2016) Mapping by sequencing in cotton (gossypium hirsutum) line MD52ne identified candidate genes for fiber strength and its related quality attributes. Theor Appl Genet 129(6):1071–1086

    Article  CAS  PubMed  Google Scholar 

  • Jiang H, Li M, Liang N, Yan H, Wei Y, Xu X, Liu J, Xu Z, Chen F, Wu G (2007) Molecular cloning and function analysis of the stay green gene in rice. Plant J. 52(2):197–209

    Article  CAS  PubMed  Google Scholar 

  • Jibran R, Sullivan KL, Crowhurst R, Erridge ZA, Chagné D, McLachlan AR, Brummell DA, Dijkwel PP, Hunter DA (2015) Staying green postharvest: how three mutations in the Arabidopsis chlorophyll b reductase gene NYC1 delay degreening by distinct mechanisms. J Exp Bot 66:6849–6862

    Article  CAS  PubMed  Google Scholar 

  • Kosambi DD (1944) The estimation of map distance from recombination values. Ann Eugen 12:172–175

    Article  Google Scholar 

  • Kusaba M, Ito H, Morita R, Iida S, Sato Y, Fujimoto M, Kawasakie S, Tanakab R, Hirochikae H, Nishimurac M, Tanakab A (2007) Rice NON-YELLOW COLORING1 is involved in light-harvesting complex ii and grana degradation during leaf senescence. Plant Cell 19(4):1362–1375

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kusaba M, Tanaka A, Tanaka R (2013) Stay green plants: what do they tell us about the molecular mechanism of leaf senescence? Photosynth Res 117(1):221–234

    Article  CAS  PubMed  Google Scholar 

  • Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25:1754–1760

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−△△Ct method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Matile P (2000) Biochemistry of indian summer: physiology of autumnal leaf coloration. Exp Gerontol 35(2):145–158

    Article  CAS  PubMed  Google Scholar 

  • Moore MJ (2005) From birth to death: the complex lives of eukaryotic mRNAs. Science 309:1514–1518

    Article  CAS  PubMed  Google Scholar 

  • Morita R, Sato Y, Masuda Y, Nishimura M, Kusaba M (2009) Defect in non-yellow coloring 3, an alpha/beta hydrolase-fold family protein, causes a stay green phenotype during leaf senescence in rice. Plant J 59:940–952

    Article  CAS  PubMed  Google Scholar 

  • Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8(19):4321–4325

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nagane T, Tanaka A, Tanaka R (2010) Involvement of AtNAP1 in the regulation of chlorophyll degradation in Arabidopsis thaliana. Planta 231:939–949

    Article  CAS  PubMed  Google Scholar 

  • Park S, Yu J, Park J, Li J, Yoo S, Lee N, Lee S, Jeong S, Seo H, Koh H, Jeon J, Park Y, Paek N (2007) The senescence-induced stay green protein regulates chlorophyll degradation. Plant Cell 19(5):1649–1664

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pruzinská A, Tanner G, Anders I, Roca M, Hörtensteiner S (2003) Chlorophyll degradation: pheophorbide a oxygenase is a rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene. Proc Natl Acad Sci 100(25):15259–15264

    Article  PubMed  PubMed Central  Google Scholar 

  • Pruzinská A, Anders I, Aubry S, Schenk N, Tapernoux-Lüthi E, Müller T, Kräutler B, Hörtensteiner S (2007) In vivo participation of red chlorophyll catabolite reductase in chlorophyll breakdown. Plant Cell 19(1):369–387

    Article  PubMed  PubMed Central  Google Scholar 

  • Ren G, An K, Liao Y, Zhou X, Cao Y, Zhao H, Ge X, Kuai B (2007) Identification of novel chloroplast protein AtNYE1 regulating chlorophyll degradation during leaf senescence in Arabidopsis. Plant Physiol 144(3):1429–1441

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rivero RM, Kojima M, Gepstein A, Sakakibara H, Mittler R, Gepstein S, Blumwald E (2007) Delayed leaf senescence induces extreme drought tolerance in a flowering plant. Proc Natl Acad Sci USA 104:19631–19636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rong H, Tang Y, Zhang H, Wu P, Chen Y, Li M, Wu G (2013) The Stay green Rice like (SGRL) gene regulates chlorophyll degradation in rice. J Plant Physiol 170(15):1367–1373

    Article  CAS  PubMed  Google Scholar 

  • Sakuraba Y, Schelbert S, Park S-Y, Han S-H, Lee B-D, Andrès CB, Kessler F, Hörtensteiner S, Paek N-C (2012) Stay-green and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis. Plant Cell 24:507–518

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sakuraba Y, Park S, Kim Y, Wang S, Yoo S, Hörtensteiner S, Paek N (2014a) Arabidopsis STAY GREEN2 is a negative regulator of chlorophyll degradation during leaf senescence. Mol Plant 7(8):1288–1302

    Article  CAS  PubMed  Google Scholar 

  • Sakuraba Y, Kim D, Kim YS, Hörtensteiner S, Paek NC (2014b) Arabidopsis STAYGREEN-LIKE (SGRL) promotes abiotic stress-induced leaf yellowing during vegetative growth. FEBS Lett 588(21):3830–3837

    Article  CAS  PubMed  Google Scholar 

  • Sato Y, Morita R, Nishimura M, Yamaguchi H, Kusaba M (2007) Mendel’s green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway. Proc Natl Acad Sci 104(35):14169–14174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schelbert S, Aubry S, Burla B, Agne B, Kessler F, Krupinska K, Hörtensteiner S (2009) Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll degradation during leaf senescence in Arabidopsis. Plant Cell 21(3):767–785

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Singh VK, Mangalam AK, Dwivedi S, Naik S (1998) Primer premier: program for design of degenerate primers from a protein sequence. Biotechniques 24:318–319

    CAS  PubMed  Google Scholar 

  • Spano G, Shewry PR (2003) Physiological characterization of ‘stay green’ mutants in durum wheat. J Exp Bot 54(386):1415–1420

    Article  CAS  PubMed  Google Scholar 

  • Takagi H, Abe A, Yoshida K, Kosugi S, Natsume S, Mitsuoka C, Uemura A, Utsushi H, Tamiru M, Takuno S, Innan H, Cano LM, Kamoun S, Terauchi R (2013) QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of dna from two bulked populations. Plant J 74:174–183

    Article  CAS  PubMed  Google Scholar 

  • Tanaka R, Hirashima M, Satoh S, Tanaka A (2003) The Arabidopsis accelerated cell death gene ACD1 is involved in oxygenation of pheophorbide a: inhibition of the pheophorbide a oxygenase activity does not lead to the ‘‘stay green’’ phenotype in Arabidopsis. Plant Cell Physiol 44:1266–1274

    Article  CAS  PubMed  Google Scholar 

  • Thomas H (1987) Sid: a Mendelian locus controlling thylakoid membrane disassembly in senescing leaves of Festuca pratensis. Theor Appl Genet 73(4):551–555

    Article  CAS  PubMed  Google Scholar 

  • Thomas H, Howarth CJ (2000) Five ways to stay green. J Exp Bot 51:329–337

    Article  CAS  PubMed  Google Scholar 

  • Tian F, Gong J, Zhang J, Zhang M, Wang G, Li A, Wang W (2013) Enhanced stability of thylakoid membrane proteins and antioxidant competence contribute to drought stress resistance in the tasg1 wheat stay-green mutant. J Exp Bot 64(6):1509–1520

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Van Ooijen J (2006) Joinmap 4.0 Software for the calculation of genetic linkage maps in experimental populations. Wageningen, Kyazma BV

    Google Scholar 

  • Vicentini F, Hortensteiner S, Schellenberg M, Thomas H, Matile P (1995) Chlorophyll breakdown in senescent leaves: identification of the biochemical lesion in a stay-green genotype of Festuca pratensis Huds. New Phytol 129(2):247–252

    Article  CAS  Google Scholar 

  • Wang XW, Wang HZ, Wang J, Sun RF, Wu J, Liu SY, Bai YQ, Mun JH, Bancroft I, Cheng F, Huang S, Li X, Hua W, Wang J, Wang X, Freeling M, Pires JC, Paterson AH, Chalhoub B, Wang B, Hayward A, Sharpe AG, Park BS, Weisshaar B, Liu B, Li B, Liu B, Tong C, Song C, Duran C, Peng C, Geng C, Koh C, Lin C, Edwards D, Mu D, Shen D, Soumpourou E, Li F, Fraser F, Conant G, Lassalle G, King GJ, Bonnema G, Tang H, Wang H, Belcram H, Zhou H, Hirakawa H, Abe H, Guo H, Wang H, Jin H, Parkin IA, Batley J, Kim JS, Just J, Li J, Xu J, Deng J, Kim JA, Li J, Yu J, Meng J, Wang J, Min J, Poulain J, Wang J, Hatakeyama K, Wu K, Wang L, Fang L, Trick M, Links MG, Zhao M, Jin M, Ramchiary N, Drou N, Berkman PJ, Cai Q, Huang Q, Li R, Tabata S, Cheng S, Zhang S, Zhang S, Huang S, Sato S, Sun S, Kwon SJ, Choi SR, Lee TH, Fan W, Zhao X, Tan X, Xu X, Wang Y, Qiu Y, Yin Y, Li Y, Du Y, Liao Y, Lim Y, Narusaka Y, Wang YP, Wang ZY, Li ZY, Wang ZW, Xiong ZY, Zhang ZH (2011) The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 43:1035–1039

    Article  CAS  PubMed  Google Scholar 

  • Wang X, Wu J, Liang J, Cheng F, Wang X (2015) Brassica database (BRAD) version 2.0: integrating and mining Brassicaceae species genomic resources. Database. https://doi.org/10.1093/database/bav093

    Google Scholar 

  • Win KT, Vegas J, Zhang C, Song K, Lee S (2017) QTL mapping for downy mildew resistance in cucumber via bulked segregant analysis using next-generation sequencing and conventional methods. Theor Appl Genet 130(1):199–211

    Article  CAS  PubMed  Google Scholar 

  • Wu S, Li Z, Yang L, Xie Z, Chen J, Zhang W, Liu T, Gao S, Gao J, Zhu Y, Xin J, Ren G, Kuai (2015) NON-YELLOWING2 (NYE2), a close paralog of NYE1, plays a positive role in chlorophyll degradation in Arabidopsis. Mol Plant 9(4):624–627

    Article  PubMed  Google Scholar 

  • Yoo SC, Cho SH, Zhang H, Paik HC, Lee CH, Li J, Yoo JH, Lee BW, Koh HJ, Seo HS, Paek NC (2007) Quantitative trait loci associated with functional stay green SNU-SG1 in rice. Mol Cells 24:83–94

    CAS  PubMed  Google Scholar 

  • Zheng HJ, Wu AZ, Zheng CC, Wang YF, Cai R, Shen XF, Xu RR, Liu P, Kong LJ, Dong ST (2009) QTL mapping of maize (Zea mays) stay green traits and their relationship to yield. Plant Breed 128:54–62

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by Grants from the National Natural Science Foundation of China (no. 31601749). We would like to thank Editage for English language editing. We also would like to thank Oebiotech for whole-genome resequencing.

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Authors and Affiliations

  1. Liaoning Key Laboratory of Genetics and Breeding for Cruciferous Vegetable Crops, Shenyang Agricultural University, Shenyang, 110866, China

    Nan Wang, Zhiyong Liu & Hui Feng

  2. Department of Horticulture, Shenyang Agricultural University, Shenyang, 110866, China

    Yun Zhang, Chengyu Li & Hui Feng

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  1. Nan Wang
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  2. Zhiyong Liu
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  4. Chengyu Li
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Correspondence to Hui Feng.

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Communicated by Maria Laura Federico.

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122_2017_3028_MOESM1_ESM.tif

Supplementary Fig. S1 Gel image of confirmation polymorphism of tightly linked SSR markers between nye and ‘FT’. Note: P1: ‘FT’; P2: nye. (TIFF 2304 kb)

122_2017_3028_MOESM2_ESM.tif

Supplementary Fig. S2 Gel images of markers tightly linked to Brnye1. a: Recombinant individuals with SSRWN26 in the mapping population; b: Recombinant individuals with SSRWN25 in the mapping population; c: Recombinant individuals with SSRWN30 in the mapping population; d: Recombinant individuals with SSRWN27 in the mapping population. Note: M: Marker; P1: ‘FT’; P2: nye; *: recombinant individuals. (TIFF 14851 kb)

122_2017_3028_MOESM3_ESM.pdf

Supplementary Fig. S3 Putative ORF of the unknown gene Bra019336 in the 81.01-kb region identified by Softberry. (PDF 21 kb)

Supplementary Fig. S4 Sequence alignment of Bra019336 in nye and ‘13A510’. (TIFF 10160 kb)

122_2017_3028_MOESM5_ESM.tif

Supplementary Fig. S5 Gel images of polymorphisms of the full length (a), RT-PCR (b), and 3′RACE of Bra019346 (c) between ‘13A510’ and nye; and gel images of the full length of Bra019336 (d) and the promoter of Bra019346 (e) between ‘13A510’ and nye. (TIFF 24768 kb)

Supplementary Fig. S6 Sequence alignment of the CDS (a) in ‘13A510’ and nye (TIFF 21176 kb)

Supplementary Fig. S6 Partial 3′RACE of Bra019346 (b) in ‘13A510’ and nye (TIFF 4511 kb)

122_2017_3028_MOESM8_ESM.tif

Supplementary Fig. S7 Gel images of the indel marker of the bands cosegregated with the mutant nye. Note: M: Marker; P1: ‘FT’; P2: nye. (TIFF 1406 kb)

Supplementary Fig. S8 Sequence alignment of the promoter of Bra019346 in ‘13A510’ and nye. (TIFF 53618 kb)

Supplementary Fig. S9 Protein conservative domains analysis of Bra019346. (TIFF 602 kb)

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Wang, N., Liu, Z., Zhang, Y. et al. Identification and fine mapping of a stay-green gene (Brnye1) in pakchoi (Brassica campestris L. ssp. chinensis). Theor Appl Genet 131, 673–684 (2018). https://doi.org/10.1007/s00122-017-3028-8

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