189份桃种质肉质性状形成相关位点基因型鉴定及组合分析

doi:10.3864/j.issn.0578-1752.2023.12.011

摘要/Abstract

摘要：

【目的】通过分子标记与生物信息学手段，鉴定189份桃种质与肉质形成相关的F-M基因座、YUC11启动子区转座子插入、NAC72编码区9 bp插入的基因型及3个位点组合情况，为深入研究桃果实肉质形成机制及育种亲本选配提供理论基础。【方法】通过PCR扩增、竞争性等位基因特异PCR（kompetitive allele specific PCR，KASP）、高分辨率溶解曲线（high resolution melting analysis，HRM）技术，对F-M基因座倍型、YUC11转座子插入、NAC72 9 bp插入进行检测；同时结合重测序数据利用生物信息学方法进一步验证，从而准确鉴定189份桃种质肉质性状形成相关位点基因型及组合情况。【结果】F-M基因座编码多聚半乳糖醛酸酶（endo-PG）的PGM与PGF是调控桃果实肉质形成的两个重要基因。189份桃种质中，PGM与PGF分别有159份（84%）、99份（52%）。F-M基因座共发现H0、H1、H2、H3 4种单倍型，占比分别为33%、39%、4%、24%。在F-M基因座，不溶质粘核以H3H3、H2H3组合为主，溶质粘核以H0H0、H0H1组合为主，溶质离核以H1H1、H0H1组合为主。发现18份含有硬质基因型的种质，在YUC11启动子区含有转座子纯合插入。HRM结果表明，NAC72编码区9 bp插入纯合（早熟）有45份种质、插入杂合（中熟）有71份种质、无插入（晚熟）有73份种质，中熟与晚熟种质占比77%；其中6份种质基因型与表型差别较大。不溶质粘核、溶质粘核、溶质离核3种常见肉质类型，在3个位点最多的基因型组合分别是mmffHdHdI、MMffHdHdI、MMFFHdHdL。【结论】通过分子试验与生物信息学相结合进一步证实了F-M基因座存在4种单倍型；发现18份含有硬质基因型的种质；开发了一种用于鉴定桃成熟期的分子标记；鉴定出不同种质在肉质相关位点的基因型组合形式。

关键词: 桃, 肉质, 基因型鉴定, 基因型组合

Abstract:

【Objective】 The molecular markers and bioinformatics were used to identify the genotypes and combinations of the F-M locus, transposon insertion in YUC11 promoter region, and 9 bp insert in NAC72 coding region by 189 peach germplasms, in order to provide the theoretical foundation for the mechanism of peach flesh formation and the selection of breeding parents.【Method】PCR amplification, KASP, and HRM were used to identify genotypes. Results of loci associated with the formation of flesh trait were further validated using bioinformatics by resequencing data in 189 peach germplasms.【Result】Two genes encoding endopolygalacturonase in F-M locus, designated PGM and PGF, are associated with peach texture. Through primer amplification, 159 (84%) PGM and 99 (52%) PGF were detected in 189 peach germplasms. Four haplotypes (H0, H1, H2, and H3) were found in F-M locus, while H0 and H1 were the major genotypes. The haplotype combination of non-melting flesh, melting flesh, free-stone melting flesh were H3H3 and H2H3, H0H0 and H0H1, H1H1 and H0H1, respectively. In addition, it was found that 18 germplasms contain homozygous transposon insertion. HRM results demonstrated that 45 germplasms were homozygous insertion (early ripening), 71 germplasms were heterozygous insertion (middle ripening), and 73 germplasms were no insertion (late ripening). But six germplasm genotypes were inconsistent with the phenotypes. The most frequent genotypic combinations at the three loci of non-melting flesh, melting flesh, and free-stone melting flesh were mmffHdHdI, MMffHdHdI, MMFFHdHdL, respectively.【Conclusion】The existence of four haplotypes at the F-M locus further was confirmed, 18 germplasms containing stony hard genotype were identified, a molecular marker for the identification of maturity date was developed, and genotype combinations of different germplasms at flesh-related loci were identified.

Key words: peach, flesh texture, genotype identification, genotype combinations

王朝晖, 李勇, 曹珂, 朱更瑞, 方伟超, 陈昌文, 王新卫, 吴金龙, 王力荣. 189份桃种质肉质性状形成相关位点基因型鉴定及组合分析[J]. 中国农业科学, 2023, 56(12): 2367-2379.

WANG ZhaoHui, LI Yong, CAO Ke, ZHU GengRui, FANG WeiChao, CHEN ChangWen, WANG XinWei, WU JinLong, WANG LiRong. Genotype Identification and Combination Analysis of Loci Related to the Peach Flesh Texture Trait via 189 Peach Accessions[J]. Scientia Agricultura Sinica, 2023, 56(12): 2367-2379.

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参考文献 48

[1]	王力荣. 中国桃品种改良历史回顾与展望. 果树学报, 2021, 38(12): 2178-2195.
	WANG L R. History and prospect of peach breeding in China. Journal of Fruit Science, 2021, 38(12): 2178-2195. (in Chinese)
[2]	LI Y, CAO K, LI N, ZHU G R, FANG W C, CHEN C W, WANG X W, GUO J, WANG Q, DING T Y, WANG J, GUAN L, WANG J X, LIU K Z, GUO W W, ARÚS P, HUANG S W, FEI Z, WANG L R. Genomic analyses provide insights into peach local adaptation and responses to climate change. Genome Research, 2021, 31(4): 592-606. doi: 10.1101/gr.261032.120 pmid: 33687945
[3]	孟君仁. 桃慢溶质性状调控机制初步研究[D]. 北京: 中国农业科学院, 2021.
	MENG J R. Preliminary study on regulation mechanism of slow melting flesh characteristics in peach[D]. Beijing: Chinese Academy of Agricultural Sciences, 2021. (in Chinese)
[4]	牛良, 曾文芳, 潘磊, 孟君仁, 鲁振华, 崔国朝, 王志强. 硬质桃研究现状及展望. 果树学报, 2020, 37(8): 1227-1235.
	NIU L, ZENG W F, PAN L, MENG J R, LU Z H, CUI G C, WANG Z Q. Research status and perspective for stony-hard peach. Journal of Fruit Science, 2020, 37(8): 1227-1235. (in Chinese)
[5]	曾文芳, 王志强, 牛良, 潘磊, 丁义峰, 鲁振华, 崔国朝. 桃果实肉质研究进展. 果树学报, 2017, 34(11): 1475-1482.
	ZENG W F, WANG Z Q, NIU L, PAN L, DING Y F, LU Z H, CUI G C. Research process on peach fruit flesh texture. Journal of Fruit Science, 2017, 34(11): 1475-1482. (in Chinese)
[6]	BAILEY J S, FRENCH A P. The inheritance of certain fruit and foliage characteristics in the peach[R]. Massachusetts Agricultur- al Experimental Station Bulletin,452,University of Massachu- setts, 1949.
[7]	LESTER D R, SHERMAN W B, ATWELL B J. Endopolygalacturonase and the melting flesh (M) locus in peach. Journal of the American Society for Horticultural Science, 1996, 121(2): 231-235. doi: 10.21273/JASHS.121.2.231
[8]	DIRLEWANGER E, COSSON P, BOUDEHRI K, RENAUD C, CAPDEVILLE G, TAUZIN Y, LAIGRET F, MOING A. Development of a second-generation genetic linkage map for peach[Prunus persica (L.) Batsch] and characterization of morphological traits affecting flower and fruit. Tree Genetics & Genomes, 2006, 3(1): 1-13.
[9]	GU C, WANG L, WANG W, ZHOU H, MA B Q, ZHENG H Y, FANG T, OGUTU C, VIMOLMANGKANG S, HAN Y P. Copy number variation of a gene cluster encoding endopolygalacturonase mediates flesh texture and stone adhesion in peach. Journal of Experimental Botany, 2016, 67(6): 1993-2005. doi: 10.1093/jxb/erw021 pmid: 26850878
[10]	NAKANO R, KAWAI T, FUKAMATSU Y, AKITA K, WATANABE S, ASANO T, TAKATA D, SATO M, FUKUDA F, USHIJIMA K. Postharvest properties of ultra-late maturing peach cultivars and their attributions to melting flesh (M) locus: Re-evaluation of M locus in association with flesh texture. Frontiers in Plant Science, 2020, 11: 554158. doi: 10.3389/fpls.2020.554158
[11]	YOSHIDA M. Genetical studies on the fruit quality of peach varieties. 3. Texture and keeping quality[R]. Bulletin of the Fruit Tree Research Station. Series A. Hiratsuka, 1976.
[12]	HAJI T, YAEGAKI H, YAMAGUCHI M. Inheritance and expression of fruit texture melting, non-melting and stony hard in peach. Scientia Horticulturae, 2005, 105(2): 241-248. doi: 10.1016/j.scienta.2005.01.017
[13]	PAN L, ZENG W F, NIU L, LU Z H, LIU H, CUI G C, ZHU Y Q, CHU J F, LI W P, FANG W C, 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, 66(22): 7031-7044. doi: 10.1093/jxb/erv400
[14]	曾文芳, 丁义峰, 潘磊, 王小贝, 牛良, 鲁振华, 崔国朝, 王志强. 桃硬质性状可能源于PpYUC11基因启动子区域CACTA型转座子的插入. 果树学报, 2017, 34(10): 1239-1248.
	ZENG W F, DING Y F, PAN L, WANG X B, NIU L, LU Z H, CUI G C, WANG Z Q. A CACTA transposable element in a PpYUC11 gene promoter is associated with the stony hard phenotype in peach. Journal of Fruit Science, 2017, 34(10): 1239-1248. (in Chinese)
[15]	TATSUKI M, SOENO K, SHIMADA Y, SAWAMURA Y, SUESADA Y, YAEGAKI H, SATO A, KAKEI Y, NAKAMURA A, BAI S L, MORIGUCHI T, NAKAJIMA N. Insertion of a transposon-like sequence in the 5’-flanking region of the YUCCA gene causes the stony hard phenotype. The Plant Journal, 2018, 96(4): 815-827. doi: 10.1111/tpj.14070
[16]	孟君仁, 曾文芳, 邓丽, 潘磊, 鲁振华, 崔国朝, 王志强, 牛良. 桃若干重要性状的KASP分子标记开发与应用. 中国农业科学, 2021, 54(15): 3295-3307. doi: 10.3864/j.issn.0578-1752.2021.15.013
	MENG J R, ZENG W F, DENG L, PAN L, LU Z H, CUI G C, WANG Z Q, NIU L. Development and application of KASP molecular markers of some important traits for peach. Scientia Agricultura Sinica, 2021, 54(15): 3295-3307. (in Chinese) doi: 10.3864/j.issn.0578-1752.2021.15.013
[17]	孟君仁, 牛良, 邓丽, 潘磊, 鲁振华, 崔国朝, 王志强, 曾文芳. 控制桃粘/离核PG基因的BAC克隆筛选与序列分析. 中国农业科学, 2021, 54(20): 4396-4404. doi: 10.3864/j.issn.0578-1752.2021.20.013. doi: 10.3864/j.issn.0578-1752.2021.20.013
	MENG J R, NIU L, DENG L, PAN L, LU Z H, CUI G C, WANG Z Q, ZENG W F. Screening and sequence analysis of BAC clone contained PG gene controlling clingstone/freestone characteristic of peach. Scientia Agricultura Sinica, 2021, 54(20): 4396-4404. doi: 10.3864/j.issn.0578-1752.2021.20.013. (in Chinese) doi: 10.3864/j.issn.0578-1752.2021.20.013
[18]	PIRONA R, EDUARDO I, PACHECO I, DA SILVA LINGE C, MICULAN M, VERDE I, TARTARINI S, DONDINI L, PEA G, BASSI D, ROSSINI L. Fine mapping and identification of a candidate gene for a major locus controlling maturity date in peach. BMC Plant Biology, 2013, 13(1): 166. doi: 10.1186/1471-2229-13-166
[19]	余银梅. 桃果实成熟期调控基因PpNAC56、PpNAC72的表达分析及功能初探[D]. 郑州: 河南农业大学, 2018.
	YU Y M. Expression analysis and functional exploration of PpNAC56 and PpNAC72 related to peach fruit maturity date[D]. Zhengzhou: Henan Agricultural University, 2018. (in Chinese)
[20]	何雪娇, 郑涛, 苏金强, 陈振东. 改良CTAB法提取野牡丹科7种植物DNA. 热带农业科学, 2011, 31(10): 73-77.
	HE X J, ZHENG T, SU J Q, CHEN Z D. DNA extraction of 7 species plants of Melastomaceae using modified CTAB method. Chinese Journal of Tropical Agriculture, 2011, 31(10): 73-77. (in Chinese)
[21]	殷豪, 王彩虹, 田义轲, 李节法, 王然, 戴洪义. 利用高分辨率熔解曲线(HRM)分析梨微卫星标记. 园艺学报, 2011, 38(8): 1601-1606.
	YIN H, WANG C H, TIAN Y K, LI J F, WANG R, DAI H Y. Differentiation of microsatellites in pear by high resolution melting (HRM) analysis. Acta Horticulturae Sinica, 2011, 38(8): 1601-1606. (in Chinese)
[22]	WINGETT S W, ANDREWS S. FastQ Screen: A tool for multi-genome mapping and quality control. F1000Research, 2018, 7: 1338.
[23]	LI H, DURBIN R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 2009, 25(14): 1754-1760. doi: 10.1093/bioinformatics/btp324 pmid: 19451168
[24]	LI H, HANDSAKER R E, WYSOKER A, FENNELL T, RUAN J, HOMER N, MARTH G, ABECASIS G, DURBIN R. The Sequence Alignment/Map format and SAMtools. Bioinformatics, 2009, 25(16): 2078-2079. doi: 10.1093/bioinformatics/btp352 pmid: 19505943
[25]	RAUSCH T, ZICHNER T, SCHLATTL A, STÜTZ A M, BENES V, KORBEL J O. DELLY: Structural variant discovery by integrated paired-end and split-read analysis. Bioinformatics, 2012, 28(18): i333-i339. doi: 10.1093/bioinformatics/bts378
[26]	THORVALDSDÓTTIR H, ROBINSON J T, MESIROV J P. Integrative Genomics Viewer (IGV): High-performance genomics data visualization and exploration. Briefings in Bioinformatics, 2013, 14(2): 178-192. doi: 10.1093/bib/bbs017 pmid: 22517427
[27]	CIRILLI M, GIOVANNINI D, CIACCIULLI A, CHIOZZOTTO R, GATTOLIN S, ROSSINI L, LIVERANI A, BASSI D. Integrative genomics approaches validate PpYUC11-like as candidate gene for the stony hard trait in peach (P. persica L. Batsch). BMC Plant Biology, 2018, 18(1): 1-12. doi: 10.1186/s12870-017-1213-1
[28]	韩晴. 桃果肉质地和粘离核性状两个关键PpPG基因的表达和调控关系分析[D]. 北京: 中国农业科学院, 2019.
	HAN Q. Expression and regulation relationship analysis of two key PpPG genes related to peach flesh texture and adhension traits[D]. Beijing: Chinese Academy of Agricultural Sciences, 2019. (in Chinese)
[29]	SHI Y N, LI B J, SU G Q, ZHANG M X, GRIERSON D, CHEN K S. Transcriptional regulation of fleshy fruit texture. Journal of Integrative Plant Biology, 2022, 64(9): 1649-1672. doi: 10.1111/jipb.13316
[30]	QIAN M, ZHANG Y K, YAN X Y, HAN M Y, LI J J, LI F, LI F R, ZHANG D, ZHAO C P. Identification and expression analysis of polygalacturonase family members during peach fruit softening. International Journal of Molecular Sciences, 2016, 17(11): 1933. doi: 10.3390/ijms17111933
[31]	ZHANG W W, ZHAO S Q, ZHANG L C, XING Y, JIA W S. Changes in the cell wall during fruit development and ripening in Fragaria vesca. Plant Physiology and Biochemistry, 2020, 154: 54-65. doi: 10.1016/j.plaphy.2020.05.028
[32]	ZHU N, ZHAO C N, WEI Y Q, SUN C D, WU D, CHEN K S. Biosynthetic labeling with 3-O-propargylcaffeyl alcohol reveals in vivo cell-specific patterned lignification in loquat fruits during development and postharvest storage. Horticulture Research, 2021, 8: 61. doi: 10.1038/s41438-021-00497-z
[33]	王巍. 生长素应答基因PpIAA5-ARF8对桃果实成熟软化的调控作用[D]. 北京: 北京农学院, 2021.
	WANG W. Auxin-responsive gene PpIAA-ARF8 on peach fruit ripening and softing[D]. Beijing: Beijing University of Agriculture, 2021. (in Chinese)
[34]	KUMAR R, KHURANA A, SHARMA A K. Role of plant hormones and their interplay in development and ripening of fleshy fruits. Journal of Experimental Botany, 2014, 65(16): 4561-4575. doi: 10.1093/jxb/eru277 pmid: 25028558
[35]	LIU Y D, TANG M F, LIU M C, SU D D, CHEN J, GAO Y S, BOUZAYEN M, LI Z G. The molecular regulation of ethylene in fruit ripening. Small Methods, 2020, 4(8): 1900485. doi: 10.1002/smtd.v4.8
[36]	ZHOU L L, TANG R K, LI X J, TIAN S P, LI B B, QIN G Z. N6-methyladenosine RNA modification regulates strawberry fruit ripening in an ABA-dependent manner. Genome Biology, 2021, 22(1): 1-32. doi: 10.1186/s13059-020-02207-9
[37]	BAI Q, HUANG Y, SHEN Y Y. The physiological and molecular mechanism of abscisic acid in regulation of fleshy fruit ripening. Frontiers in Plant Science, 2021, 11: 619953. doi: 10.3389/fpls.2020.619953
[38]	ZHU F, JADHAV S S, TOHGE T, SALEM M A, LEE J M, GIOVANNONI J J, CHENG Y J, ALSEEKH S, FERNIE A R. A comparative transcriptomics and eQTL approach identifies SlWD40 as a tomato fruit ripening regulator. Plant Physiology, 2022, 190(1): 250-266. doi: 10.1093/plphys/kiac200
[39]	WANG J F, TIAN S W, YU Y T, REN Y, GUO S G, ZHANG J, LI M Y, ZHANG H Y, GONG G Y, WANG M, XU Y. Natural variation in the NAC transcription factor NONRIPENING contributes to melon fruit ripening. Journal of Integrative Plant Biology, 2022, 64(7): 1448-1461. doi: 10.1111/jipb.13278
[40]	GUO Z H, ZHANG Y J, YAO J L, XIE Z H, ZHANG Y Y, ZHANG S L, GU C. The NAM/ATAF1/2/CUC₂ transcription factor PpNAC.A 59 enhances PpERF.A 16 expression to promote ethylene biosynthesis during peach fruit ripening. Horticulture Research, 2021, 8: 209. doi: 10.1038/s41438-021-00644-6
[41]	王小贝. 桃ERFs和PpIAA1协同调控桃成熟软化的分子机制研究[D]. 武汉: 华中农业大学, 2019.
	WANG X B. Study on the molecular mechanism of synergistic regulation of peach ripening and softening by ERFs and PpIAA1[D]. Wuhan: Huazhong Agricultural University, 2019. (in Chinese)
[42]	李金金. PpSEP1(SEPALLATA)在不同质地桃果实成熟软化过程中的调控机制[D]. 杨凌: 西北农林科技大学, 2016.
	LI J J. Regulatory mechanism of the PpSEP1 (SEPALLATA) gene during fruit ripening and softening in melting and non-melting peachs[D]. Yangling: Northwest A & F University, 2016. (in Chinese)
[43]	李芳. NAP家族成员克隆及其在桃果实发育与成熟期间的表达特性研究[D]. 杨凌: 西北农林科技大学, 2016.
	LI F. Cloning and expression characterization of NAP family members during development and ripening of peach[D]. Yangling: Northwest A & F University, 2016. (in Chinese)
[44]	HUANG B W, HU G J, WANG K K, FRASSE P, MAZA E, DJARI A, DENG W, PIRRELLO J, BURLAT V, PONS C, GRANELL A, LI Z G, VAN DER REST B, BOUZAYEN M. Interaction of two MADS-box genes leads to growth phenotype divergence of all-flesh type of tomatoes. Nature Communications, 2021, 12: 6892. doi: 10.1038/s41467-021-27117-7 pmid: 34824241
[45]	MIGICOVSKY Z, YEATS T H, WATTS S, SONG J, FORNEY C F, BURGHER-MACLELLAN K, SOMERS D J, GONG Y H, ZHANG Z Q, VREBALOV J, VAN VELZEN R, GIOVANNONI J G, ROSE J K C, MYLES S. Apple ripening is controlled by a NAC transcription factor. Frontiers in Genetics, 2021, 12: 671300. doi: 10.3389/fgene.2021.671300
[46]	郭健. 基于基因组结构变异发掘桃重要农艺性状关键基因[D]. 武汉: 华中农业大学, 2021.
	GUO J. Identification of causal genes responsible for key agronomic traits based on genome structural variations in peach[D]. Wuhan: Huazhong Agricultural University, 2021. (in Chinese)
[47]	LI J J, LI F, QIAN M, HAN M Y, LIU H K, ZHANG D, MA J J, ZHAO C P. Characteristics and regulatory pathway of the PrupeSEP1 SEPALLATA gene during ripening and softening in peach fruits. Plant Science, 2017, 257: 63-73. doi: 10.1016/j.plantsci.2017.01.004
[48]	WANG W H, WANG Y Y, CHEN T, QIN G Z, TIAN S P. Current insights into posttranscriptional regulation of fleshy fruit ripening. Plant Physiology, 2022, 190(22): 921-930. doi: 10.1093/plphys/kiac353

名称 Code	引物序列 Primer sequence	退火温度 Annealing temperature (℃)	片段长度 Size (bp)
PGM-F	ACTAACCCAACCAATTACTCAAACC	56	3066
PGM-R	TGTCCTTAGTACTGGAGGGCAAAC
PGF-F	AGGAGAAATTGGAGACGCCG	58	3353
PGF-R	CGTCTTCCGGACATAATCTTACA
YUC-F	TAAAGCCGCCCAAAAATAAA	58	600
YUC-R1	TGGGAAGGAAGAAAATAGTCACA
YUC-R2	ATTTTCAACTTTCCCGAGCA	58	448/3015
PGH0-F	TACGACTACAAGCCAAGTCGG	56	591
PGH0-R	ACTCCAGTTGCGCTGAAAGA
PGH2-F	CATATTGAAGAACCAGCCA	56	1364
PGH2-R	TGGTATATTGCATCGACGTG
PGH3-F	AAAGATAGGAAGGGGGAAGG	56	524
PGH3-R	GTTTGCACTGGGGGTTTTAGA

表型 Phenotype				基因型出现个数 Number of genotype occurrences
溶质粘核		溶质离核	不溶质粘核	1	2	3	4
MMffHdHdI	MMFFHdHdL	MMFFHdHdL	mmffHdHdL	12	2	22	11
MMFFHdHdE	MmffHdhdI	MMFfHdhdI	mmffHdHdE	9	1	8	5
MMFfHdHdI	MmffhdhdE	MMFfHdHI	mmffHdHdI	9	1	7	5
MMffHdHdL	MMFFHdhdE	MMFfHdHdL	MmffHdHdE	8	1	5	3
MMFfHdHdE	MMFFHdhdL	MmFfHdHdL	MMffHdHdI	4	1	5	3
MmFfHdhdE	MMFfHdhdI	MMFFhdhdI	MMffHdHdL	4	1	4	2
MmffHdHdL	MmFfHdHdI	MMffHdHdL	mmffhdhdE	4	1	3	2
MmffHdHdL	MmFfHdHdL	MmFfHdhdE	MmffHdHdI	4	1	2	1
MMffhdhdI	MMffHdHdE	MmFfHdhdI	MmffHdHdL	3	1	2	1
MmffHdHdI	MMffHdhdI	MMFFHdHdI	MmffHdhdI	3	1	2	1
MmFfHdHdE	MMffHdhdI	MMFfhdhdE	MMffHdhdI	3	1	1	1
MMFFHdHdI	MMffhdhdL	MmFfHdHdE	MmFfHdHdI	2	1	1	1
MMffHdhdL		MMFFhdhdL	MMFfHdHdI	2		1	1
MMffhdhdE		MMFFhdhdE		2		1
MmffHdHdE		MmFfhdhdE		2		1
mmffHdHdE		MMFfHdhdE		2		1