基于SNP标记的桃矮化基因精细定位

doi:10.3864/j.issn.0578-1752.2017.18.013

摘要/Abstract

摘要： 【目的】矮化型桃树体矮小、节间短，是盆栽观赏和砧木育种的重要遗传资源。明确矮化性状形成的遗传机制并对桃矮化基因进行精细定位，是建立目标性状分子辅助选种体系和遗传改良的前提，可为有目标的选育矮化观赏桃和砧木品种奠定基础。【方法】以‘05-2-144’（‘97矮’×‘鸳鸯垂枝’桃）套袋自交获得的395个后代单株构建的分离群体为材料。参考桃基因组信息并基于Sanger技术开发的SNP标记对亲本和后代单株进行分析，在扩大群体单株中进行连锁关系分析，确定连锁的SNP标记，初步定位目标基因。在定位区域内基于二代测序技术开发更多的基因型和表型一致的SNP标记，对后代单株进行基因分型，完成目标性状的精细定位。然后在精细定位区域内开发SNP标记，即杂交群体双亲均为Aa杂合基因型，对‘10-7’×‘96-5-1’杂交后代89个单株进行分子鉴定，以验证基因定位结果的准确性。【结果】通过对桃单株‘05-2-144’自交后代实生苗表型鉴定表明，普通型和矮化型单株数分别为300株和95株，性状分离比例接近3﹕1（P值为0.67；χ2为0.19），符合孟德尔遗传规律，桃矮化性状受隐性单基因控制。用于分子鉴定的单株来源于‘10-7’（普通型）×‘96-5-1’（普通型）杂交组合，共获得89个后代单株，其中普通型66株；矮化型23株（P值为0.854；χ2为0.034）。基于Sanger测序技术开发了SNP标记，在桃基因组数据库Pp06上25 230 425 bp和27 191 090 bp处获得了连锁的SNP标记，且目标基因位于这两个标记的右侧，初步获得了连锁的SNP分子标记。在此基础上，对亲本进行66.89X深度测序，继续开发符合Aa杂合基因型的SNP标记位点。根据参考基因组和物理距离区间共设计了15对SNP引物，其中12对引物与重测序结果中SNP的类型一致，3对引物与重测序结果中SNP的类型不一致，连锁标记的基因分型成功率为80.0%。通过基于SNP基因分型分析，最终完成了目标性状的精细定位，位点位于Pp06的28 712 165 bp（引物为JXSNP-5）和28 899 661 bp（引物为JXHRM-SNP-3）之间，遗传距离分别为0.38 cM和0.13 cM，物理距离约为277 kb，精细定位区域内有54个已知转录本。在定位区域内桃基因组Pp06的28 108 436 bp处和29 247 763 bp处开发SNP标记用于杂交后代表型的鉴定，结果表明所有后代单株基因型和表型鉴定结果完全一致，鉴定准确率为100%。【结论】本研究精细定位了桃矮化基因，物理距离约为277 kb，为基因克隆、亲本早期筛选以选育矮化观赏桃和砧木品种等奠定了基础。

关键词: 桃, 矮化基因, SNP, 精细定位

Abstract: 【Objective】 Dwarfing peach, due to the small trees, shorter internodes, is an important genetic resources in ornamental and rootstock breeding program. The fine mapping of peach dwarfing genes and explication of the genetic mechanism will provide a basis for the establishment of a molecular-assisted selection system and desired variety improvement, which could be used in breeding dwarf ornamental peach and rootstock varieties. 【Method】 Based on the results of genetic analysis in this study, 395 individuals generated from a self-pollinated population of 05-2-144 were selected for fine mapping the dwarfing gene in peach (Prunus persica (L.) Batsch). By referencing the peach genome sequence, SNP markers were developed in the parents and progenies to generate markers linked to the locus and used to map the gene based on Sanger sequencing. Subsequently, the parent was resequenced to generate SNP markers in the mapping region and acquire heterozyous SNPs for fine mapping. Within the fine mapping region, SNP primers were designed to verify the phenotype of 89 individuals generated from the F₁ segregation population of 10-7×96-5-1.【Result】The segregating population of 05-2-144 was generated to assess the genetic characteristics, resulting in observed 3:1 (300 standard type and 95 dwarfing type individuals) Mendelian ratio fitting with the expected ratio for a monogenic recessive genetic control (P=0.67, χ²=0.19). Moreover, the ratio of standard type to dwarf type corresponded to the expected 3:1 (66 standard type and 23 dwarfing type individuals) segregation (P=0.854, χ²=0.034) for molecular detection. Based on Sanger sequencing results, the linked SNP markers were identified in the position 25 230 425 bp and 27 191 090 bp on Pp06 of peach genome and the locus were on the right side of these two markers. For fine mapping, the parent of this segregation population was resequenced with 66.89X depth and identified the heterozygous SNP to develop SNP markers. Totally, fifteen SNP primers were designed and 12 SNPs (80.0%) were consistent with resequencing data. The dwarfing locus of this type was narrowed on Pp06 between the position 28 712 165 bp and 28 899 661 bp with genetic distances of 0.38 cM and 0.13 cM. The physical region of fine mapping was 277 kb containing 54 known transcripts. Within 28 108 436bp and 29 247 763 bp on Pp06, SNP markers were developed and detected the progeny phenotype of a segregation population of 10-7×96-5-1 with 100% accuracy, constituted of 89 individuals. 【Conclusion】 The dwarfing gene was fine mapped on Pp06 within a physical distance 277 kb. The results of this study will be helpful to clone dwarfing gene and select parents for breeding dwarfing ornamental peach and rootstock varieties.

Key words: peach, dwarfing gene, SNP, fine mapping

鲁振华，牛良，张南南，姚家龙，崔国朝，曾文芳，潘磊，王志强. 基于SNP标记的桃矮化基因精细定位[J]. 中国农业科学, 2017, 50(18): 3572-3580.

LU ZhenHua, NIU Liang, ZHANG NanNan, YAO JiaLong, CUI GuoChao, ZENG WenFang, PAN Lei, WANG ZhiQiang. Fine Mapping of Dwarfing Gene for Peach Based on SNP Markers[J]. Scientia Agricultura Sinica, 2017, 50(18): 3572-3580.

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参考文献

[1] BASSI D, DIMA A, SCORZA R. Tree structure and pruning response of six peach growth forms. Journal of the American Society for Horticultural Science, 1994, 119(3): 378-382.

[2] LAMMERTS W E. The breeding of ornamental edible peaches for mild climates.I. inheritance of tree and flower characters. American Journal of Botany, 1945, 32: 53-61.

[3] SCORZA R. Characterization of four distinct peach tree growth types. Journal of the American Society for Horticultural Science, 1984, 109(4): 455-457.

[4] MOORE J N, ROM R C, BROWN S A, KLINGAMAN G L. ‘Bonfire’ dwarf peach, ‘Leprechaun’ dwarf nectarine, and ‘Crimson Cascade’ and ‘Pink Cascade’ weeping peaches. Hortscience, 1993, 28(8): 854.

[5] 宗学普, 张贵荣, 王志强, 刘淑娥. 矮化型油桃新品种-矮丽红. 落叶果树, 1997(2): 26.

ZONG X P, ZHANG G R, WANG Z Q, LIU S E. Dwarf nectarine variety - ‘Ailihong’. Deciduous Fruits, 1997(2): 26. (in Chinese)

[6] YAMAMOTO T, SHIMADA T, IMAI T, YAEGAKI H, HAJI T, MATSUTA N, YAMAGUCHI M, HAYASHI T. Characterization of morphological traits based on a genetic linkage map in peach. Breeding Science, 2001, 51: 271-278.

[7] HOLLENDER C A, HADIARTOT, SRINIVASAN C, SCORZA R, DARDICK C. A brachytic dwar?sm trait (dw) in peach trees is caused by anonsense mutation within the gibberellic acid receptor PpeGID1c. New Phytologist, 2015, 210(1): 227-239.

[8] DOYLE J J, DOYLE J L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 1987, 19: 11-15.

[9] ROBINSON J T, THORVALDSDÓTTIR H, WINCKLER W, GUTTMAN M, LANDER E S, GETZ G, MESIROV J P. Integrative genomics viewer. Nature Biotechnology, 2011, 29: 24-26.

[10] 鲁振华, 牛良, 张南南, 崔国朝, 潘磊, 曾文芳, 王志强. 基于HRM获得与桃Tssd紧密连锁的SNP标记. 中国农业科学, 2017, 50(8): 1505-1513.

LU Z H, NIU L, ZHANG N N, CUI G C, PAN L, ZENG W F, WANG Z Q. SNP marker tightly linked to Tssd for peach using high resolution melting analysis. Scientia Agricultura Sinica, 2017, 50(8): 1505-1513. (in Chinese)

[11] VAN OOIJEN J W, VOORRIPS R E. JionMap version 3.0: Software for the calculation of genetic linkage maps. Plant Research International, Wageningen, The Netherlands, 2001.

[12] BALL A, STAPLEY J, DAWSON D, BIRKHEAD T, BURKE T, SLATE J. A comparison of SNPs and microsatellites as linkage mapping markers: lessons from the zebra finch (Taeniopygia guttata). BMC Genomics, 2010, 11(1): 218.

[13] MARTÍNEZ-GARCÍA P J, PARFITT D E, OGUNDIWIN E A, FASS J, CHAN H M, AHMAD R, LURIE S, DANDEKAR A, GRADZIEL T M, CRISOSTO C H. High density SNP mapping and QTL analysis for fruit quality characteristics in peach (Prunus persica L.). Tree Genetics and Genomes, 2013, 9: 19-36.

[14] TAKAGI H, ABE A, YOSHIDA K, KOSUGI S, NATSUME S, MITSUOKA C, UEMURA A, UTSUSHI H, TAMIRU M, TAKUNO S, INNAN H, CANO L M, KAMOUN S, TERAUCHI R. QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. The Plant Journal, 2013, 74: 174-183.

[15] ABE A, KOSUGI S, YOSHIDA K, NATSUME S, TAKAGI H, KANZAKI H, MATSUMURA H, YOSHIDA K, MITSUOKA C, TAMIRU M, INNAN H, CANO L, KAMOUN S, TERAUCHI R. Genome sequencing reveals agronomically important loci in rice using MutMap. Nature Biotechnology, 2012, 30(2): 174-179.

[16] 李娜, 王吉明, 尚建立, 李楠楠, 徐永阳, 马双武. 西瓜枯萎病生理小种1抗性QTL精细定位与InDel标记开发. 中国农业科学, 2017, 50(1): 131-141.

LI N, WANG J M, SHANG J L, LI N N, XU Y Y, MA S W. Fine-mapping of QTL and development of InDel markers for Fusarium oxysporum race 1 resistance in watermelon. Scientia Agricultura Sinica, 2017, 50(1): 131-141. (in Chinese)

[17] LU Z H, NIU L, CHAGNÉ D, CUI GG C, PAN L, FOSTER T, ZHANG R P, ZENG W F, WANG Z Q. Fine mapping of the temperature-sensitive semi-dwarf (Tssd) locus regulating the internode length in peach (Prunus persica). Molecular Breeding, 2016, 36: 20. DOI: 10.1007/s11032-016-0442-6.

[18] SHEN Z J, CONFOLENT C, LAMBERT P, POËSSEL J-L, QUILOT-TURION B, YU M L, MA R J, PASCAL T. Characterization and genetic mapping of a new blood-flesh trait controlled by the single dominant locus DBF in peach. Tree Genetics and Genomes, 2013, 9: 1435-1446.

[19] ADAMI M, FRANCESCHI P D, BRANDI F, LIVERANI A, GIOVANNINI D, ROSATI C, DONDINI L, TARTARINI S. Identifying a carotenoid cleavage dioxygenase (ccd4) gene controlling yellow/white fruit flesh color of peach. Plant Molecular Biology Reporter, 2013, 31(5): 1166-1175.

[20] MA J J, LI J, ZHAO J B, ZHOU H, REN F, WANG L, GU C, LIAO L, HAN Y P. Inactivation of a gene encoding carotenoid cleavage dioxygenase (CCD4) leads to carotenoid-based yellow coloration of fruit flesh and leaf midvein in Peach. Plant Molecular Biology Reporter, 2014, 32(4): 246-257.

[21] BRETÓ M P, CANTÍN C M, IGLESIAS I, ARÚS P, EDUARDO I. Mapping a major gene for red skin color suppression (highlighter) in peach. Euphytica, 2017, 213: 14. DOI: 10.1007/s10681-016-1812-1.

[22] PAN L, ZENG W F, NIU L, LU Z H, WANG X B, LIU H, CUI G C, ZHU Y Q, CHU J F , LI W P, FANGＷC, CAI Z G, LI G H, WANG Z Q. PpYUC11, a strong candidate gene for the stony hard phenotype in peach (Prunus persica L. Batsch), participates in IAA biosynthesis during fruit ripening. Journal of Experimental Botany, 2015. Doi: 10.1093/jxb/erv400.

[23] DARDICK C, CALLAHAN A, HORN R, RUIZ KB, ZHEBENTYAYEVA T, HOLLENDER C, WHITAKER M, ABBOTT A, SCORZA R. PpeTAC1 promotes the horizontal growth of branches in peach trees and is a member of a functionally conserved gene family found in diverse plants species. The Plant Journal, 2013, 75: 618-630.

[24] OTTO D, PETERSEN R, BRAUKSIEPE B, BRAUN P, SCHMIDT E R. The columnar mutation (‘Co gene’) of apple (Malus × domestica) is associated with an integration of a Gypsy-like retrotransposon. Molecular Breeding, 2013, 33(4): 863-880.

[25] MORIYA S, OKADA K, HAJI T, YAMAMOTO T, ABE K. Fine mapping of Co, a gene controlling columnar growth habit located on apple (Malus × domestica Borkh.) linkage group 10. Plant Breeding, 2012, 131(5): 641-647.

[26] OGNJANOV V, VUJANIC-VARGA D, GASIC K. Breeding columnar apples in Novi Sad. Acta Horticulturae, 1999, 484: 207-209.

[27] MORIYA S, IWANAMI H, KOTODA N, TAKAHASHI S, YAMAMOTO T, ABE K. Development of a marker-assisted selection system for columnar growth habit in apple breeding. Journal of the Japanese Society for Horticultural Science, 2009, 78: 279-287.

[28] VERDE I, BASSIL N, SCALABRIN S, GILMORE B, LAWLEY C T. Correction: Development and evaluation of a 9K SNP array for peach by internationally coordinated SNP detection and validation in breeding germplasm. PLoS ONE, 2012, 7(6): e35668. DOI: 10.1371/ journal.pone.0035668.

[29] CANTÍNA C M, CRISOSTOB C H, OGUNDIWINB E A, GRADZIELB T, TORRENTSC J, MORENOA M A, GOGORCENA Y. Chilling injury susceptibility in an intra-speci?c peach [Prunus persica (L.) Batsch] progeny. Postharvest Biology and Technology, 2010, 58: 79-87.

[30] VERDE I, QUARTA R, CEDROLA C, DETTORI M T. QTL analysis of agronomic traits in a BC1 peach population. Acta Horticulturae, 2002, 592: 291-297.

[31] 张妤艳, 俞明亮, 马瑞娟, 蔡志翔, 沈志军, 许建兰. 与桃树性高/矮性状相关的SRAP标记. 果树学报, 2011, 28(4): 586-590.

ZHANG Y Y, YU M L, MA R J, CAI Z X, SHEN Z J, XU J L. Identification of SRAP markers linked to peach gene with the character of tall normal/brachytic dwarf. Journal of Fruit Science, 2011, 28 (4): 586-590. (in Chinese)