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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (8): 1948-1956.doi: 10.3724/SP.J.1006.2022.12049

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

Fine mapping and cloning of a seed shattering gene SH8 in rice (Oryza sativa L.)

WANG Mu-Mu1(), HE Yan-Fan2, ZHENG Yong-Sheng1, WANG Hui1, WANG Li-Yuan1, WANG Dong-Jian1, ZHANG Han1, LI Ru-Yu1,*()   

  1. 1Crop research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China
    2Shandong Seed Administration Station, Jinan 250100, Shandong, China
  • Received:2021-07-22 Accepted:2021-11-29 Online:2022-08-12 Published:2021-12-21
  • Contact: LI Ru-Yu E-mail:opmcy@163.com;li_ruyu@sina.com
  • Supported by:
    National Natural Science Foundation of China(31801340)

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Abstract:

Seed shattering is an important trait for seed dispersal and reproduction of offspring in wild rice. It is a vital guarantee for seed dispersal and offspring reproduction of wild rice, but easy grain dropping will cause yield loss in domesticated rice production. Although some genes related to seed shattering have been cloned in rice, these genes were not responsible for all phenotype variation of rice seed shattering. In this study, a recombinant inbred line (E6-5) was selected from a cross between an indica variety Pei-kuh and an Asian common wild rice W1944 and hybridized with Pei-kuh to construct the isolated population. E6-5 harbored W1944 introgression segment on chromosome 8, but it had not carried SH4 gene and exhibited a very easy seed shattering phenotype. By analyzing an F2 segregation population constructed using E6-5 and Pei-kuh, we narrowed down the SHATTERING8 (SH8, qSHT-8) in the 26.4 kb interval. There were four protein-coding genes in this region. The comparison of PEI-Kuh and E6-5 sequencing revealed that LOC_Os08g41950 contained three single nucleotide polymorphisms (SNPs) in coding region. Meanwhile, qRT-PCR revealed that LOC_Os08g41950 was expressed in the junction of flower and pedicel, where the abscission layer was formed and shed off. We considered LOC_Os08g41950 as the candidate gene for SH8. To verify whether LOC_Os08g41950 was responsible for rice seed shattering, we constructed an overexpression vector using LOC_Os08g41950 encoding sequence from E6-5 and transformed it into Pei-huh. Compared with Pei-kuh, the shattering ability of transgenic plants was significantly enhanced. In addition, consistent with the BTS testing results, transgenic plants had smoother fracture surface of pedicel compared with that of Pei-kuh in scanning electron microscopy photos. These results would facilitate the study of rice domestication and seed shattering regulation mechanism.

Key words: O. rufipogon, seed shattering, abscission layer, gene cloning, SH8 gene

Table S1

Primers used in this study (restriction sites are indicated in lowercase letters)"

引物名称
Name		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')		 用途
Application
FNP-SH4 ACTACCGCAAGGGGAACTG TGGGGAGGTTGCAGTCCTT 功能位点检测FNP detection
RM23469 CAACAGAAGCCATTAGGCATTAGC ACCGAAGTGATCAACAACCTACC 基因定位Gene mapping
RM23489 AGCAGGAGGAGGAGAAGGGAAGG CGAGCCACTGCTTCTTCTGTGC 基因定位Gene mapping
RM447 ACGGGCTTCTTCTCCTTCTCTCC TCCCTTGTGCTGTCTCCTCTCC 基因定位Gene mapping
RM23532 TATTGCCATGAATGCTGGGAAGG GGCATCGCAATGACAAACATCC 基因定位Gene mapping
RM23586 TTCTGTGGATAGCTGGAACATGC CCAGATACATCATCAGGAAGGAAGG 基因定位Gene mapping
M1(CAPS)a TGAATTGACAAAACGGACCA GGACCTGTTCTTGGGTTGAA 基因定位Gene mapping
M2(IN/DEL) AACATGATGGTGCCAAGTGA AGGATGGAGATGGTGAGTGG 基因定位Gene mapping
ACTIN TACAGTGTCTGGATTGGAGGAT TCTTGGCTTAGCATTCTTGGGT 实时定量PCR RT-qPCR
qPCR-950 ATTCATCCCTGAAGCACGTC GGCTGACGTTCATAGCCAAT 实时定量PCR RT-qPCR
qPCR-960 CTTCCACCTCCAGCATTGAT ATCGCTTCCTCCAAACCTCT 实时定量PCR RT-qPCR
950QC-1 AACATGATGGTGCCAAGTGA ACAGGACAGATCCAATCGAA 测序 Sequencing
950QC-2 GCTGAAGAGGATCGAGAACA CAGCGCTATGACATCGAAAA 测序 Sequencing
950QC-3 CTGAAGGCAAGGGTTGAAAA GTTGCTTTCCTCCAGCTACA 测序 Sequencing
950QC-4 TGCAGATATCATGGAATGGA TGCGACAGTGAATCTGTCAT 测序 Sequencing
960QC-1 TATCCGCGTACGGCTTAATG AATGTGGTGGTGAGGGAATC 测序 Sequencing
960QC-2 TCCGAGTGCTCCTAGTGGAT AGAAAGGTGGGAAGGGAAAA 测序 Sequencing
960QC-3 GAGGCGAAACACCAAATCTC TACCCCTAGCCTCTGCTTGA 测序 Sequencing
引物名称
Name		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')		 用途
Application
960QC-4 TATCTCCACCATCCCCACTC GGCGTGCCAGTTAGTTTGAT 测序 Sequencing
960QC-5 GTTCGGTCCGATCTATCAAGA CTCACACAACCACCATCCTG 测序 Sequencing
960QC-6 ATGGCGACAACCTACTGACC GAACCAGGCAAAGGGTTTCT 测序 Sequencing
pSH8-0E AAAggatcc- ATGGGGAGGGGTCGGGTGGA AAAactagt- TCATGGTAGCCATGGGGGCAT 载体构建Vector construction
HYG AAGTTCGACAGCGTCTCCGAC TCTACACAGCCATCGGTCCAG 转基因检测Transgenic detection
CDS-1 ATGGGGAGGGGTCGGGTGGA CGGCTAGCTTTCAATTGCTC 转基因检测Transgenic detection

Fig. 1

Phenotypic characterization of E6-5 and Pei-kuh A: breaking tensile strength (BTS) required to pull grain away from pedicels; B and C: fluorescence images of longitudinal sections of abscission layer, AL means abscission layer; D and F: scanning electron microscopy photos of pedicel junction after detachment of seeds. Bar = 50 μm in B-E. ** indicates significant difference at P < 0.01 by Student's t-test."

Fig. 2

Map-based cloning of SH8 gene N is the number of mapping populations."

Table 1

Gene annotation of Nipponbare in mapping region"

基因名称
Locus name		 基因注释
Gene annotation
LOC_Os08g41950 OsMADS7-MADS-box family gene with MIKCc type-box, expressed
LOC_Os08g41960 OsMADS37-MADS-box family gene with MIKCc* type-box, expressed
LOC_Os08g41970 Conserved hypothetical protein
LOC_Os08g41980 Aminotransferase, putative, expressed

Fig. 3

Analysis of candidate MADS-box genes A: expression pattern of candidate MADS-box genes at heading stage in E6-5; B: relative expression of Os08g41950 in FP and F at heading stage; C: coding sequence alignment of Os08g41950 between E6-5 and Pei-kuh, SNPs are highlighted in red blocks, bases causing amino acid change are underlined. FP: the junction of flower and pedicel; F: flower."

Fig. 4

Detection of transgenic plants A: detection of transgenic plants by primer HYG; B: detection of transgenic plants by SH8 gene specific primer CDS-1. "

Fig. 5

Phenotypic characterization of transgenic plants A: relative expression of SH8 in abscission layer at heading stage; B: breaking tensile strength (BTS) required to pull grain away from pedicels; C and D: scanning electron microscopy photos of pedicel junction after detachment of seeds. Bar = 50 μm in C and D. ** indicates significant difference at P < 0.01 by Student's t-test."

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