Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (15): 2933-2947.doi: 10.3864/j.issn.0578-1752.2025.15.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Identification and Characterization of Retrotransposon Tos17 in the Genomes of Indica Rice

LUO JiaRui1(), WU SanLing2, GUO Fu3, LIU Zhen3, SONG JingHan1,3, TAN YuanYuan1,4, SHU QingYao1,3,*()   

  1. 1 The Advanced Seed Institute, Zhejiang University/State Key Laboratory of Rice Breeding & Biology/Zhejiang Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, Hangzhou 310058
    2 Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University, Hangzhou 310058
    3 Hainan Institute of Zhejiang University, Sanya 572024, Hainan
    4 The New Countryside Development Institute of Zhejiang University, Hangzhou 310058
  • Received:2025-02-08 Accepted:2025-04-01 Online:2025-07-30 Published:2025-07-30
  • Contact: SHU QingYao

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Abstract:

【Objective】 Tos17 is a type of retrotransposon in the rice genome. In the japonica variety Nipponbare, a Tos17 located on chromosome 7 (Tos17Chr.7) can be activated during tissue culture. This study aims to reveal the genomic features of Tos17 of indica varieties in China and determine whether their Tos17 can be activated in tissue culture like in japonica rice, which may affect biotechnological breeding. 【Method】 High-quality genome resequencing data of indica varieties or hybrid parents were retrieved from public databases. An in-house program was developed to identify and analyze Tos17 insertion loci, confirmed by IGV visualization and PCR assays. The varieties were classified through hierarchical clustering and principal component analysis, a phylogenetic tree was constructed based on genome-wide single nucleotide polymorphism (SNP) data, and the association between varietal clusters and Tos17 haplotypes was assessed using Mantel test. Transgenic plants were generated by Agrobacterium -mediated transformation of mature embryo-derived callus of indica varieties, and the changes of Tos17 copy number were analyzed in 125 T0 transgenic plants.【Result】 23 distinct Tos17 insertion loci were identified in 1 511 indica varieties using the Tos17-finder, a program developed specifically for Tos17 identification. All varieties had a Tos17 on Chr.10 (Tos17Chr.10) identical to the one in japonica rice Nipponbare, and there were two high-frequency Tos17 copies on Chr.2, i.e., Tos17Chr.2-1 (79.0%) and Tos17Chr.2-2 (83.7%), but only 85 (5.6%) varieties carried the Tos17Chr.7 common to japonica rice. There were 4.0 Tos17 copies per variety on average, and while 11 varieties had up to 8 Tos17 copies, 35 only had a single Tos17, i.e., Tos17Chr.10. Twelve Tos17 insertions were located within or 2 kb up- or down-stream of annotated genes, with the remaining 11 in intergenic regions. Phylogenetic analysis based on SNPs classified the 1 511 varieties into three subpopulations, each showing partial correlation with specific Tos17 haplotypes. No new Tos17 insertions were detected in the 125 T0 transgenic seedlings of 5 indica varieties. A molecular marker capable of accurately distinguishing Tos17Chr.7 from other Tos17s was developed. 【Conclusion】 The genomic features of Tos17 in indica rice varieties differ from those in japonica rice variety Nipponbare. The developed molecular marker can be used to determine readily whether the test materials carry the activatable Tos17Chr.7.

Key words: Tos17, indica rice, whole-genome resequencing, tissue culture, transposon genomics, transposition activity

Table 1

Genome resequencing information of 1 511 indica rice varieties"

数据来源Data source 品种类型Type of rice variety 品种数量No. of varieties 参考文献Reference
PRJNA656900* 两系法核基因雄性不育系Two-line genic male sterile line 82 [16]
两系法恢复系Two-line restorer 70
三系法恢复系Three-line restorer 249
三系法雄性不育保持系Three-line male sterility maintainer 15
常规品种Conventional variety 271
细胞质雄性不育Cytoplasmic male sterile rice 200
种质资源Germplasm accession 131
PRJNA880974** 籼稻引进品种XIC (Xian Introduced Cultivars) 4 [17]
籼稻地方品种XL (Xian Landraces) 34
籼稻主栽品种XMC (Xian Major Cultivars) 169
CRA000167*** 种质资源Germplasm 286 [18]

Fig. 1

A diagram of bioinformatic analysis pipeline for the identification of Tos17 in rice genomes A: Workflow of Tos17-finder for Tos17 identification using genome sequencing data; B: Chimeric reads (green: genomic, red: Tos17 sequences) containing the Tos17 sequence that can be mapped to Nipponbare genome in Chr.10 (Chr.10: 15 415 374-15 419 577 bp); C: Chimeric reads containing the Tos17 sequence that cannot be mapped to Nipponbare genome in Chr.10 (Chr.10: 15 415 374-15 419 577 bp), the non-Tos17 sequence parts of these reads can be mapped to other chromosomes (dark)"

Fig. 2

Schematic illustration of Tos17 insertion sites on the genome A: The location of Tos17Chr.7 in different genomes; B: The location of Tos17Chr.10 in different genomes; C: The location of Tos17Chr.2-1 in different genomes. Nipponbare is the reference genome for japonica rice, R608 is a variety sample, and R498 is the reference genome for indica rice"

Fig. 3

Visualization of two Tos17 insertion sites in rice genomes by IGV A: Around the Tos17 insertion site on chromosome 7 of Nipponbare, there were no chimeric reads with the LTR sequences at both termini of Tos17 in indica R498, while there were in japonica Xiushui 134; B: Such chimeric reads were detected around the Tos17 insertion site on chromosome 10 in both R498 and Xiushui134. The aligned reference genome is Nipponbare (RGAP v7)"

Fig. 4

Alignment by IGV of sequencing reads of R498 with the sequence of Tos17 on Chr.7 (Tos17Chr.7) of Nipponbare All reads were broken at the 568th nucleotide, indicating the presence of an insertion or deletion at this site"

Fig. 5

Retrotransposon Tos17 in indica rice varieties and their genomic distribution A: Tos17 insertions in indica rice, their genomic location and number of varieties; B: A schematic view of Tos17 insertions in various chromosomes. Each purple circle is at scale and represents the number of accessions that carry the corresponding Tos17 insertions; C: Distribution of rice accessions with different number of Tos17 insertions"

Fig. 6

The indica rice varieties with 8 Tos17 copies Each Tos17 is represented by a differentially colored square"

Fig. 7

Distribution of Tos17 insertions in indica rice genomes A: The distribution of 23 Tos17 insertions in indica rice genome; B: Name and functional annotations (when available) of genes with a Tos17 insertion in their gene body"

Fig. 8

Analysis of the co-evolutionary pattern of Tos17 insertion polymorphism and genomic genetic distance in indica rice population consisting of 1 511 varieties A: Dendrogram based on hierarchical clustering analysis; B: PCA scatter plot; C: The phylogenetic tree constructed using the neighbor-joining method, consisting of 3 clades, i.e., 1(Navy), 2 (Green) and 3 (brown yellow); D: Analysis of correlation between genomic evolution and Tos17 insertion distribution"

Table 2

Statistics of the whole genome-sequencing data of T0 transformed plants of 5 indica rice varieties"

材料
Material		 Q20
(%)		 Q30
(%)		 定位读序数
Mapped reads (bp)		 定位率
Mapped rate (%)		 基因组覆盖率
Genome coverage (%)		 测序深度
Sequencing depth (×)
DR950 96.2(95.2-98.2) 89.6(86.9-95.1) 78663682 (74182194-80276648) 96.4(96.3-96.6) 87.2(86.8-87.5) 27.4(25.9-28.0)
GM03 98.6(98.1-99.0) 89.6(86.9-95.1) 78381573(56149050-80293264) 95.6(95.4-96.3) 86.6(86.4-86.8) 27.3(19.6-28.0)
R17 95.8(95.5-96.1) 88.2(87.5-89.0) 79684843(77671050-80297370) 96.1(96.1-96.1) 87.2(87.2-87.1) 27.8(27.1-28.0)
翠玉B
Cuiyu B		 96.2(95.7-96.9) 89.4(88.1-91.4) 74326250(46121258-80299402) 96.4(96.3-96.4) 86.9(86.2-87.4) 25.9(16.1-28.0)
R7954 97.6(96.1-98.7) 92.6(89.7-95.0) 78062693(27615630-80309870) 96.1(95.5-97.1) 88.0(87.1-88.7) 27.2(9.6-28.0)

Fig. 9

The Tos17 copies identified in 125 T0 transformed plants of indica rice"

Fig. 10

Molecular marker of differentiating two types of retrotransposons Tos17 in rice A: Diagrams of the two types of Tos17 and position information of PCR primers; B: PCR amplicons of Tos17 in indica and japonica rice"

Table 3

Pedigree analysis of 46 indica rice varieties carrying Tos17Chr.7"

亲本之一为粳稻
One of the parents is japonica rice		 祖辈亲本之一为粳稻
One of the ancestral parents is japonica rice		 曾祖辈亲本之一为粳稻
One of the great-grandparents is japonica rice
Q6A、旱恢3号、昌恢871a+b、培矮64Sb、泰R1086c、T136c、成恢448d、新安Se
Q6A, Hanhui 3, Changhui 871a+b, Peiai 64Sb, Tai R1086c, T136c, Chenghui 448d, Xinan Se		 神农2A、万8A、福龙S2、R608、R618、宜恢3003、广占63-4Sa、佳丰68Sa、绿102Sa、绿敏Sa、天源6Sa、宣69Sa、N111Sb、Y58Sb、C815Sb+c、R6547c、扬恢336c、成恢425d、成恢727d、新二Se、软华Af
Shennong 2A, Wan 8A, Fulong S2, R608, R618, Yihui 3003, Guangzhan 63-4Sa, Jiafeng 68Sa, Lü 102Sa, Lümin Sa, Tianyuan 6Sa, Xuan 69Sa, N111Sb, Y58Sb, C815Sb+c, R6547c, Yanghui 336c, Chenghui 425d, Chenghui 727d, Xiner Se, Ruanhua Af		 R9194、鄂早17、华1037S、黔恢1388、03Sa、广茉Sa、华1201Sa、新华Sa、早恢1号a、隆科638Sb、深08Sb、德Sb、望Sb、创5Sb+c、扬恢542c、广茉HSe 、广8Bf
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