Development of Functional Molecular Markers for Tomato Fruit Weight Gene and Population Genotyping Analysis

doi:10.13560/j.cnki.biotech.bull.1985.2025-0101

Abstract

Abstract:

Objective This study is aimed to develop and apply molecular markers to identify and analyze the patterns of genetic variation in Fruit weight 3.2 (Fw3.2) and Fruit weight 11.3 (Fw11.3), two pivotal genes involved in the regulation of tomato (Solanum lycopersicum) fruit weight, at different domestication stages. It is expected to provide effective tools for the precise identification and utilization of tomato genetic resources, as well as the rapid domestication of wild germplasm. Method Fragment length polymorphism molecular markers were designed specifically based on the copy number variation (CNV) of Fw3.2 and the 1.4 kb large fragment insertion/deletion (Indel) variation at the 3′ end of Fw11.3. These markers were used to conduct genotype analysis on a collection of 259 tomato germplasms (including 79 Solanum pimpinellifolium (PIM) accessions, 95 Solanum lycopersicum var. cerasiforme (CER) accessions and 85 big-fruited S. lycopersicum accessions (BIG)), determining variation of Fw3.2 and Fw11.3 and their distribution within different tomato populations. Result By the molecular markers developed in this study, the CNVs (fw3.2^WT and fw3.2^dup ) of the Fw3.2 gene and the large fragment Indel variants of the Fw11.3 gene (fw11.3-WT and fw11.3-D) were successfully identified. Within PIM population, the frequencies of the large-fruit alleles fw3.2^dup and fw11.3-D were 0 and 2.53%, respectively. In the CER population, these allele frequencies increased to 10.53% for fw3.2^dup and 8.42% for fw11.3-D. Notably, in the BIG population, the prevalence of fw3.2^dup and fw11.3-D alleles rose significantly to 69.41% and 92.94%, respectively. Conclusion Both fw3.2^dup and fw11.3-D alleles significantly increase tomato fruit weight. Furthermore, these alleles lead to a synergistic effect that further enhances fruit weight in tomatoes. During the domestication and genetic improvement of tomatoes, the frequency of these large-fruit alleles has progressively increased, mirroring the evolutionary trajectory toward larger fruit size.

Key words: tomato, fruit weight, molecular marker, Fw3.2, Fw11.3, copy number variation, insertion/deletion variation, domestication

HOU Ya-tao, LI Ying-hui, DENG Lei, LI Chang-bao, LI Chuan-you, SUN Chuan-long. Development of Functional Molecular Markers for Tomato Fruit Weight Gene and Population Genotyping Analysis[J]. Biotechnology Bulletin, 2025, 41(4): 98-105.

Figures/Tables 7

Table 1 Primer sequences for molecular markers of the Fw3.2 and Fw11.3 gene

引物名称 Primer name	引物序列 Primer sequence （5′‒3′）
Fw3.2-pol-F	GTATAAGACTGTTCACTACG
Fw3.2-pol-R1	CGAATCAGCAGTATACACTG
Fw3.2-pol-R2	GTGGATGTGATGCATCAACT
Fw11.3-polF	TCACCGTCATCAATCAAAAT
Fw11.3-polR1	CAATTAATTACTATCGAAACGC
Fw11.3-polR2	CATATGCATTTAGTGAGGTTG

Fig. 1 Schematic diagram of the Fw3.2 (A) and Fw11.3 (B) gene structures and primer locationsfw3.2WT refers to the single-copy allele of the Fw3.2 gene, while fw3.2dup refers to the duplicated allele of the Fw3.2 gene. fw11.3-WT refers to the wild-type allele ofthe Fw11.3 gene, and fw11.3-D indicates the allele with a large fragment deletion in the 3′ region of the Fw11.3 gene. The same below

Fig. 2 Polymorphic primer detection for Fw3.2 (A) and Fw11.3 (B) in different tomato materialsM: Marker. LA1589 and LA1375 are S. pimpinellifolium (PIM) accessions, while Brandywine and M82 are big-fruited S. lycopersicum (BIG) accessions

Fig. 3 Allele frequency analysis of Fw3.2 (A) and Fw11.3 (B)A: PIM: S. pimpinellifolium. CER: S. lycopersicum var. cerasiforme. BIG: Big-fruited S. lycopersicum. The same below

Table 2 Genotype analysis of tomato accessions collected from different geographic origins

地理来源 Geographic origin	类别 Category	fw3.2^WT	fw3.2^dup	fw11.3-WT	fw11.3-D
美洲 the Americas	PIM	76	0	74	2
	CER	83	10	85	8
	BIG	16	3	5	14
亚洲 Asia	BIG	2	48	1	49

Fig. 4 Haplotype analysis of Fw3.2 and Fw11.3 in different tomato populationsfw3.2WTfw11.3-WT: Haplotype 1; fw3.2WTfw11.3-D: haplotype 2; fw3.2dupfw11.3-WT: haplotype 3; fw3.2dup fw11.3-D: haplotype 4. The same below

Fig. 5 Phenotypic analysis of fruit weight in different tomato populationsA: Representative images of PIM, CER and BIG varieties. Bar = 1 cm. B: Fruit weight of PIM, CER, and BIG tomatoes. C‒D: Phenotypic variation in fruit weight among distinct Fw3.2 genotypes within CER (C) and BIG (D) populations. E‒F: Phenotypic variation in fruit weight among distinct Fw11.3 genotypes within CER (E) and BIG (F) populations. G: Phenotypic variation in fruit weight among distinct Fw3.2 and Fw11.3 haplotypes within the BIG. Significance of differences was tested using one-way ANOVA for panels B and G, and two-tailed t-tests for panels C‒F. Different letters indicate significant differences (P < 0.05)

References 30

1	Chaudhary J, Alisha A, Bhatt V, et al. Mutation breeding in tomato: advances, applicability and challenges [J]. Plants, 2019, 8(5): 128.
2	杜敏敏, 周明, 邓磊, 等. 番茄分子育种现状与展望——从基因克隆到品种改良 [J]. 园艺学报, 2017, 44(3): 581-600.
	Du MM, Zhou M, Deng L, et al. Current status and prospects on tomato molecular breeding—from gene cloning to cultivar improvement [J]. Acta Hortic Sin, 2017, 44(3): 581-600.
3	Yang JW, Liu Y, Liang B, et al. Genomic basis of selective breeding from the closest wild relative of large-fruited tomato [J]. Hortic Res, 2023, 10(8): uhad142.
4	Pereira L, Zhang L, Sapkota M, et al. Unraveling the genetics of tomato fruit weight during crop domestication and diversification [J]. Theor Appl Genet, 2021, 134(10): 3363-3378.
5	Frary A, Nesbitt TC, Grandillo S, et al. fw2.2: a quantitative trait locus key to the evolution of tomato fruit size [J]. Science, 2000, 289(5476): 85-88.
6	Beauchet A, Bollier N, Grison M, et al. The cell number regulator FW2.2 protein regulates cell-to-cell communication in tomato by modulating callose deposition at plasmodesmata [J]. Plant Physiol, 2024, 196(2): 883-901.
7	Cong B, Barrero LS, Tanksley SD. Regulatory change in YABBY-like transcription factor led to evolution of extreme fruit size during tomato domestication [J]. Nat Genet, 2008, 40(6): 800-804.
8	Alonge M, Wang XG, Benoit M, et al. Major impacts of widespread structural variation on gene expression and crop improvement in tomato [J]. Cell, 2020, 182(1): 145-161.e23.
9	Huang ZJ, van der Knaap E. Tomato fruit weight 11.3 maps close to fasciated on the bottom of chromosome 11 [J]. Theor Appl Genet, 2011, 123(3): 465-474.
10	Mu Q, Huang ZJ, Chakrabarti M, et al. Fruit weight is controlled by cell size regulator encoding a novel protein that is expressed in maturing tomato fruits [J]. PLoS Genet, 2017, 13(8): e1006930.
11	Rodríguez GR, Muños S, Anderson C, et al. Distribution of SUN, OVATE, LC, and FAS in the tomato germplasm and the relationship to fruit shape diversity [J]. Plant Physiol, 2011, 156(1): 275-285.
12	Lippman Z, Tanksley SD. Dissecting the genetic pathway to extreme fruit size in tomato using a cross between the small-fruited wild species Lycopersicon pimpinellifolium and L. esculentum var. Giant Heirloom [J]. Genetics, 2001, 158(1): 413-422.
13	Tanksley SD. The genetic, developmental, and molecular bases of fruit size and shape variation in tomato [J]. Plant Cell, 2004, 16(): S181-S189.
14	Xu C, Liberatore KL, MacAlister CA, et al. A cascade of arabinosyltransferases controls shoot meristem size in tomato [J]. Nat Genet, 2015, 47(7): 784-792.
15	Yuste-Lisbona FJ, Fernández-Lozano A, Pineda B, et al. ENO regulates tomato fruit size through the floral meristem development network [J]. Proc Natl Acad Sci USA, 2020, 117(14): 8187-8195.
16	孟思达, 韩磊磊, 相恒佐, 等. 番茄心室数的调控机制研究进展 [J]. 园艺学报, 2024, 51(7): 1649-1664.
	Meng SD, Han LL, Xiang HZ, et al. Research progress on the mechanism of regulating the number of tomato locules [J]. Acta Hortic Sin, 2024, 51(7): 1649-1664.
17	Lin T, Zhu GT, Zhang JH, et al. Genomic analyses provide insights into the history of tomato breeding [J]. Nat Genet, 2014, 46(11): 1220-1226.
18	Razifard H, Ramos A, Della Valle AL, et al. Genomic evidence for complex domestication history of the cultivated tomato in Latin America [J]. Mol Biol Evol, 2020, 37(4): 1118-1132.
19	Jamann TM, Balint-Kurti PJ, Holland JB. QTL mapping using high-throughput sequencing [J]. Methods Mol Biol, 2015, 1284: 257-285.
20	Li ZQ, Xu YH. Bulk segregation analysis in the NGS era: a review of its teenage years [J]. Plant J, 2022, 109(6): 1355-1374.
21	Gao L, Gonda I, Sun HH, et al. The tomato pan-genome uncovers new genes and a rare allele regulating fruit flavor [J]. Nat Genet, 2019, 51(6): 1044-1051.
22	Li N, He Q, Wang J, et al. Super-pangenome analyses highlight genomic diversity and structural variation across wild and cultivated tomato species [J]. Nat Genet, 2023, 55(5): 852-860.
23	Consortium TG. The tomato genome sequence provides insights into fleshy fruit evolution [J]. Nature, 2012, 485(7400): 635-641.
24	Zhou Y, Zhang ZY, Bao ZG, et al. Graph pangenome captures missing heritability and empowers tomato breeding [J]. Nature, 2022, 606(7914): 527-534.
25	Wang Y, Sun CL, Ye ZB, et al. The genomic route to tomato breeding: Past, present, and future [J]. Plant Physiol, 2024, 195(4): 2500-2514.
26	Han HY, Li XH, Li TZ, et al. Chromosome-level genome assembly of Solanum pimpinellifolium [J]. Sci Data, 2024, 11(1): 577.
27	王娟, 王柏柯, 李宁, 等. 基因编辑技术在番茄育种中的应用进展 [J]. 植物生理学报, 2020, 56(12): 2606-2616.
	Wang J, Wang BK, Li N, et al. Advances in the application of genome editing in tomato breeding [J]. Plant Physiol J, 2020, 56(12): 2606-2616.
28	Zsögön A, Čermák T, Naves ER, et al. De novo domestication of wild tomato using genome editing [J]. Nat Biotechnol, 2018. DOI: 10.1038/nbt.4272 .
29	Li TD, Yang XP, Yu Y, et al. Domestication of wild tomato is accelerated by genome editing [J]. Nat Biotechnol, 2018. DOI: 10.1038/nbt.4273 .
30	刘慧, 黄婷, 陶建平, 等. 番茄生物钟基因SlLNK1、SlEID1和SlELF3光周期响应分析 [J]. 园艺学报, 2023, 50(10): 2079-2090.
	Liu H, Huang T, Tao JP, et al. Analysis of circadian clock genes SlLNK1, SlEID1, and SlELF3 response to photoperiod in tomato [J]. Acta Hortic Sin, 2023, 50(10): 2079-2090.

[1]	LIU Jie, WANG Fei, TAO Ting, ZHANG Yu-jing, CHEN Hao-ting, ZHANG Rui-xing, SHI Yu, ZHANG Yi. Overexpression of SlWRKY41 Improves the Tolerance of Tomato Seedlings to Drought [J]. Biotechnology Bulletin, 2025, 41(2): 107-118.
[2]	CHEN Hui-ting, XIA Yi-nan, YAN Wei, ZHANG Meng-fei, CHEN Zhou-mei, HOU Li-xia, LIU Xin. Effect of Exogenous Chlorogenic Acid Treatment on Fruit Quality of Facility Tomato [J]. Biotechnology Bulletin, 2025, 41(2): 119-126.
[3]	SONG Ying-pei, WANG Can, ZHOU Hui-wen, KONG Ke-ke, XU Meng-ge, WANG Rui-kai. Analysis of Soybean Pod Dehiscence Habit Based on Whole Genome Association Analysis and Genetic Diversity [J]. Biotechnology Bulletin, 2025, 41(2): 97-106.
[4]	LIU Wen-zhi, HE Dan, LI Peng, FU Ying-lin, ZHANG Yi-xin, WEN Hua-jie, YU Wen-qing. Paenibacillus polymyxa New Strain X-11 and Its Growth-promoting Effects on Tomato and Rice [J]. Biotechnology Bulletin, 2024, 40(9): 249-259.
[5]	RAO Yong-bin, ZHANG Jun-li. Biological Characteristics, Domestication and Cultivation of Wild Gymnopilus subpurpuratus [J]. Biotechnology Bulletin, 2024, 40(4): 264-270.
[6]	ZHAO Yao, WEN Lang, LUO Shao-dan, LI Zi-xing, LIU Chao-chao. Identification of HMA Gene Family and Cadmium Transport Function of SlHMA1 in Tomato [J]. Biotechnology Bulletin, 2024, 40(2): 212-222.
[7]	WANG Nan, LIAO Yong-qin, SHI Zhu-feng, SHEN Yun-xin, YANG Tong-yu, FENG Lu-yao, YI Xiao-peng, TANG Jia-cai, CHEN Qi-bin, YANG Pei-wen. Identification of Three Strains of Bacillus from the Forest Soil of Wuliang Mountain and Mining of Their Bioactivities [J]. Biotechnology Bulletin, 2024, 40(2): 277-288.
[8]	LI Qing, SHI Yu-he, ZHU Jue, LI Xiao-ling, HOU Chao-wen, TONG Qiao-zhen. Genetic Diversity Analysis and DNA Fingerprint Construction of Atractylodes macrocephala Germplasm Resources Based on SCoT Molecular Markers [J]. Biotechnology Bulletin, 2024, 40(11): 142-151.
[9]	SUN Mei-hua, SUN Hui-xian, TIAN Lin-lin, MIAO Yan-xiu, HOU Lei-ping, QI Ming-fang, LI Tian-lai. Functional Identification of YABBY2b Gene and Expression Analysis of Downstream Genes in Tomato [J]. Biotechnology Bulletin, 2024, 40(11): 162-168.
[10]	XIAO Liang, WU Zheng-dan, LU Liu-ying, SHI Ping-li, SHANG Xiao-hong, CAO Sheng, ZENG Wen-dan, YAN Hua-bing. Research Progress of Important Traits Genes in Cassava [J]. Biotechnology Bulletin, 2023, 39(6): 31-48.
[11]	SHI Jian-lei, ZAI Wen-shan, SU Shi-wen, FU Cun-nian, XIONG Zi-li. Identification and Expression Analysis of miRNA Related to Bacterial Wilt Resistance in Tomato [J]. Biotechnology Bulletin, 2023, 39(5): 233-242.
[12]	CHE Yong-mei, GUO Yan-ping, LIU Guang-chao, YE Qing, LI Ya-hua, ZHAO Fang-gui, LIU Xin. Isolation and Identification of Bacterial Strain C8 and B4 and Their Halotolerant Growth-promoting Effects and Mechanisms [J]. Biotechnology Bulletin, 2023, 39(5): 276-285.
[13]	HU Ming-yue, YANG Yu, GUO Yang-dong, ZHANG Xi-chun. Functional Analysis of SlMYB96 Gene in Tomato Under Cold Stress [J]. Biotechnology Bulletin, 2023, 39(4): 236-245.
[14]	SHEN Yun-xin, SHI Zhu-feng, ZHOU Xu-dong, LI Ming-gang, ZHANG Qing, FENG Lu-yao, CHEN Qi-bin, YANG Pei-wen. Isolation, Identification and Bio-activity of Three Bacillus Strains with Biocontrol Function [J]. Biotechnology Bulletin, 2023, 39(3): 267-277.
[15]	XIE Yang, ZHOU Guo-yan, SU Hang, XING Yu-meng, YAN Li-ying. Transcriptome Analysis of Cucumber Seeds Early and Late Germination Under PEG Drought Simulation [J]. Biotechnology Bulletin, 2023, 39(12): 148-157.