Construction of a high-density bin-map and identification of fruit quality-related quantitative trait loci and functional genes in pear

Abstract

Pear (Pyrus spp.) is one of the most common fruit crops grown in temperate regions worldwide. Genetic enhancement of fruit quality is a fundamental goal of pear breeding programs. The genetic control of pear fruit quality traits is highly quantitative, and development of high-density genetic maps can facilitate fine-mapping of quantitative trait loci (QTLs) and gene identification. Bin-mapping is a powerful method of constructing high-resolution genetic maps from large-scale genotyping datasets. We performed whole-genome sequencing of pear cultivars ‘Niitaka’ and ‘Hongxiangsu’ and their 176 F₁ progeny to identify genome-wide single-nucleotide polymorphism (SNP) markers for constructing a high-density bin-map of pear. This analysis yielded a total of 1.93 million SNPs and a genetic bin-map of 3190 markers spanning 1358.5 cM, with an average adjacent interval of 0.43 cM. This bin-map, along with other high-density genetic maps in pear, improved the reference genome assembly from 75.5 to 83.7% by re-anchoring the scaffolds. A quantitative genetic analysis identified 148 QTLs for 18 fruit-related traits; among them, QTLs for stone cell content, several key monosaccharides, and fruit pulp acids were identified for the first time in pear. A gene expression analysis of six pear cultivars identified 399 candidates in the identified QTL regions, which showed expression specific to fruit developmental stages in pear. Finally, we confirmed the function of PbrtMT1, a tonoplast monosaccharide transporter-related gene responsible for the enhancement of fructose accumulation in pear fruit on linkage group 16, in a transient transformation experiment. This study provides genomic and genetic resources as well as potential candidate genes for fruit quality improvement in pear.

References

[1] Lombard PB, Westwood MN . Pear rootstocks. In: Rom RC, Carlson RF, eds. Rootstocks for Fruit Crops.John Wiley: New York, 1987, 145-83.

[2] Wu J, Li L, Li M et al. High-density genetic linkage map construction and identification of fruit-related QTLs in pear using SNP and SSR markers. J Exp Bot. 2014; 65: 5771-81.

[3] Khan MA, Zhao YF, Korban SS . Identification of genetic loci associated with fire blight resistance in Malus through combined use of QTL and association mapping. Physiol Plant. 2013; 148: 344-53.

[4] Kumar S, Garrick DJ, Bink MC et al. Novel genomic approaches unravel genetic architecture of complex traits in apple. BMC Genomics. 2013; 14: 393.

[5] Iketani H, Abe K, Yamamoto T et al. Mapping of disease-related genes in Japanese pear using a molecular linkage map with RAPD markers. Ikushugaku Zasshi. 2001; 51: 179-84.

[6] Yamamoto T, Kimura T, Shoda M et al. Genetic linkage maps constructed by using an interspecific cross between Japanese and European pears. Theor Appl Genet. 2002; 106: 9-18.

[7] Dondini L, Pierantoni L, Gaiotti F et al. Identifying QTLs for fire-blight resistance via a European pear (Pyrus communis L.) genetic linkage map . Mol Breed. 2005; 14: 407-18.

[8] Yamamoto T, Kimura T, Terakami S et al. Integrated reference genetic linkage maps of pear based on SSR and AFLP markers. Ikushugaku Zasshi. 2007; 57: 321-9.

[9] Terakami S, Kimura T, Nishitani C et al. Genetic linkage map of the Japanese pear ‘Housui’ identifying three homozygous genomic regions. Hortic J. 2009; 78: 417-24.

[10] Zhang R, Wu J, Li X et al. An AFLP, SRAP, and SSR genetic linkage map and identification of QTLs for fruit traits in pear (Pyrus L.) . Plant Mol Biol Rep. 2013; 31: 678-87.

[11] Wu J, Wang Z, Shi Z et al. The genome of the pear (Pyrus bretschneideri Rehd.) . Genome Res. 2013; 23: 396-408.

[12] Linsmith G, Rombauts S, Montanari S et al. Pseudo-chromosome-length genome assembly of a double haploid ‘Bartlett’ pear (Pyrus communis L.) . Gigascience. 2019; 8, giz138.

[13] Yao G, Ming M, Allan AC et al. Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis. Plant J. 2017; 92: 437-51.

[14] Vision TJ, Brown DG, Shmoys DB et al. Selective mapping: a strategy for optimizing the construction of high-density linkage maps. Genetics. 2000; 155: 407-20.

[15] Chen Z, Wang B, Dong X et al. An ultra-high density bin-map for rapid QTL mapping for tassel and ear architecture in a large F2 maize population. Genomics. 2014; 15: 433.

[16] Zhou Z, Zhang C, Zhou Y et al. Genetic dissection of maize plant architecture with an ultra-high density bin map based on recombinant inbred lines. Genomics. 2016; 17: 178.

[17] Pereira L, Ruggieri V, Pérez S et al. QTL mapping of melon fruit quality traits using a high-density GBS-based genetic map. Plant Biol. 2018; 18: 324.

[18] Hu Z, Deng G, Mou H et al. A re-sequencing-based ultra-dense genetic map reveals a gummy stem blight resistance-associated gene in Cucumis melo . DNA Res. 2018; 25: 1-10.

[19] Di Pierro EA, Gianfranceschi L, Di Guardo M et al. A high-density, multi-parental SNP genetic map on apple validates a new mapping approach for outcrossing species. Hortic Res. 2016; 3: 16057.

[20] Howad W, Yamamoto T, Dirlewanger E et al. Mapping with a few plants: using selective mapping for microsatellite saturation of the Prunus reference map. Genetics. 2005; 171: 1305-9.

[21] Sargent DJ, Cipriani G, Vilanova S et al. The development of a bin mapping population and the selective mapping of 103 markers in the diploid Fragaria reference map. Can J Genet Cytol. 2008; 51: 120-7.

[22] Li X, Singh J, Qin M et al. Development of an integrated 200K SNP genotyping array and application for genetic mapping, genome assembly improvement and genome wide association studies in pear (Pyrus) . Plant Biotechnol J. 2019; 17: 1582-94.

[23] Wu Y, Close TJ, Lonardi S . Accurate construction of consensus genetic maps via integer linear programming. IEEE/ACM Trans Comput Biol Bioinform. 2011; 8: 292-3.

[24] Shapiro SS, Wilk MB . An analysis of variance test for normality (complete samples). Biometrika. 1965; 52: 591-611.

[25] Xue C, Yao J, Qin M et al. PbrmiR397a regulates lignification during stone cell development in pear fruit. Plant Biotechnol J. 2019; 17: 103-17.

[26] Cheng R, Cheng Y, Lü J et al. The gene PbTMT4 from pear (Pyrus bretschneideri) mediates vacuolar sugar transport and strongly affects sugar accumulation in fruit . Physiol Plant. 2018; 164: 307-19.

[27] Li J, Qin M, Qiao X et al. A new insight into the evolution and functional divergence of SWEET transporters in Chinese white pear (Pyrus bretschneideri) . Plant Cell Physiol. 2017; 58: 839-50.

[28] Li JM, Zheng DM, Li LT et al. Genome-wide function, evolutionary characterization and expression analysis of sugar transporter family genes in pear (Pyrus bretschneideri Rehd) . Plant Cell Physiol. 2015; 56: 1721-37.

[29] Van Ooijen J . MapQTL^® 6. Software for the mapping of quantitative trait loci in experimental populations of diploid species . Wageningen, Netherlands: Kyazma BV, 2009.

[30] Ooijen J. Multipoint maximum likelihood mapping in a full-sib family of an outbreeding species. Genet Res. 2011; 93: 343-9.

[31] Li W, Zhang J, Mou Y et al. Integration of Solexa sequences on an ultradense genetic map in Brassica rapa L. BMC Genomics 2011; 12: 249.

[32] Ma X, Fu Y, Zhao X et al. Genomic structure analysis of a set of Oryza nivara introgression lines and identification of yield-associated QTLs using whole-genome resequencing. Sci Rep. 2016; 6: 27425.

[33] Yao GF, Yang ZJ, Zhang SL et al. Characteristics of components and contents of organic acid in pear fruits from different cultivated species. Acta Hortic Sin. 2014; 41: 755-64.

[34] Kenis K, Keulemans J, Davey MW . Identification and stability of QTLs for fruit quality traits in apple. Tree Genet Genomes. 2008; 4: 647-61.

[35] Zhang Q, Ma B, Li H et al. Identification, characterization, and utilization of genome-wide simple sequence repeats to identify a QTL for acidity in apple. Genomics. 2012; 13: 537.

[36] Rymenants M, van de Weg E, Auwerkerken A et al. Detection of QTL for apple fruit acidity and sweetness using sensorial evaluation in multiple pedigreed full-sib families. Tree Genet Genomes. 2020; 16: 71.

[37] Fanizza G, Lamaj F, Costantini L et al. QTL analysis for fruit yield components in table grapes (Vitis vinifera) . Theor Appl Genet. 2005; 111: 658-64.

[38] Jung M, Roth M, Aranzana MJ et al. The apple REFPOP-a reference population for genomics-assisted breeding in apple. Hortic Res. 2020; 7: 189.

[39] Muranty H, Troggio M, Sadok IB et al. Accuracy and responses of genomic selection on key traits in apple breeding. Hortic Res. 2015; 2: 15060.

[40] Roth M, Muranty H, Di Guardo M et al. Genomic prediction of fruit texture and training population optimization towards the application of genomic selection in apple. Hortic Res. 2020; 7: 148.

[41] Martin-Cabrejas MA, Waldron KW, Selvendran RR et al. Ripening-related changes in the cell walls of Spanish pear (Pyrus communis) . Physiol Plant. 1994; 91: 671-9.

[42] Berthet S, Demont-Caulet N, Pollet B et al. Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems. Plant Cell. 2011; 23: 1124-37.

[43] Wang Y, Bouchabke-Coussa O, Lebris P et al. LACCASE5 is required for lignification of the Brachypodium distachyon culm. Plant Physiol. 2015; 168: 192-204.

[44] Zhao Q, Nakashima J, Chen F et al. LACCASE is necessary and nonredundant with PEROXIDASE for lignin polymerization during vascular development in Arabidopsis . Plant Cell. 2013; 25: 3976-87.

[45] Fornalé S, Shi X, Chai C et al. ZmMYB31 directly represses maize lignin genes and redirects the phenylpropanoid metabolic flux. Plant J. 2010; 64: 633-44.

[46] Borevitz JO, Xia Y, Blount J et al. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell. 2000; 12: 2383-93.

[47] Zhang J, Ge H, Zang C et al. EjODO1, a MYB transcription factor, regulating lignin biosynthesis in developing loquat (Eriobotrya japonica) fruit . Front Plant Sci. 2016; 7: 1360.

[48] Zeng JK, Li X, Zhang J et al. Regulation of loquat fruit low temperature response and lignification involves interaction of heat shock factors and genes associated with lignin biosynthesis. Plant Cell Environ. 2016; 39: 1780-9.

[49] Schulz A, Beyhl D, Marten I et al. Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2. Plant J. 2011; 68: 129-36.

[50] Wormit A, Trentmann O, Feifer I et al. Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport. Plant Cell. 2007; 18: 3476-90.

[51] Tao S, Khanizadeh S, Zhang H et al. Anatomy ultrastructure and lignin distribution of stone cells in two Pyrus species . Plant Sci. 2009; 176: 413-9.

[52] Yao G, Zhang S, Cao Y et al. Characteristics of components and contents of soluble sugar in pear fruits from different species. Sci Agric Sin. 2010; 43: 4229-37.

[53] Liu L, Chen C, Zhu Y et al. Maternal inheritance has impact on organic acid content in progeny of pear (Pyrus spp.) fruit . Euphytica. 2016; 209: 305-21.

[54] Bolger AM, Lohse M, Usadel B . Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30: 2114-20.

[55] Li H, Durbin R . Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25: 1754-60.

[56] McKenna A, Hanna M, Banks E et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20: 1297-303.

[57] Li H, Handsaker B, Wysoker A et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009; 25: 2078-9.

[58] Grattapaglia D. Pseudo-testcross mapping strategy using RAPD markers. In: Micheli MRAB, ed. Fingerprinting Methods Based on Arbitrarily Primed PCR.Springer: Berlin, 1997, 201-18.

[59] Broman KW, Wu H, Sen S et al. R/qtl: QTL mapping in experimental crosses. Bioinformatics. 2003; 19: 889-90.

[60] Tang H, Zhang X, Miao C et al. ALLMAPS: robust scaffold ordering based on multiple maps. Genome Biol. 2015; 16: 3.

[61] Marçais G, Delcher AL, Phillippy AM et al. MUMmer4: a fast and versatile genome alignment system. PLoS Comput Biol. 2018; 14: e1005944.

[62] Zhao J, Xu Y, Li H et al. A SNP-based high-density genetic map of leaf and fruit related quantitative trait loci in wolfberry (Lycium Linn.) . Front Plant Sci. 2019; 10: 977.

[63] Lander ES, Botstein D . Mapping mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics. 1989; 121: 185-99.

[64] Zhang MY, Xue C, Xu L et al. Distinct transcriptome profiles reveal gene expression patterns during fruit development and maturation in five main cultivated species of pear (Pyrus L.) . Sci Rep. 2016; 6: 28130.

[65] Li J, Yang Z, Zhang S et al. Change of organic acid contents and related enzyme activities in different pear cultivars. Acta Botan Boreali-Occiden Sin. 2013; 33: 2024-30.

[66] Wang D, Yang Z, Sun J et al. Difference in soluble sugar accumulation and related enzyme activity of fruits among different pear cultivars. J Fruit Sci. 2014; 31: 30-8.

[67] Xiao-Yang L, Gui-Zhen G, Hong-Xia LI et al. The studies on the changing laws of the fruit growth, development and the stone cell forming of Pyrus bretschneider cv. Dangshan Supear. J Huaibei Coal Industry Teachers College. 2006; 1: 49-53.

[68] Curtis MD, Grossniklaus U . A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 2003; 133: 462-9.

[69] Zhou H, Lin-Wang K, Wang H et al. Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. Plant J. 2015; 82: 105-21.

[70] Holzapfel B, Wickert L . Die quantitative real-time-PCR (qRT-PCR). Methoden und Anwendungsgebiete. Biol Unserer Zeit. 2007; 37: 120-6.