Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (4): 802-810.doi: 10.3864/j.issn.0578-1752.2020.04.012

• HORTICULTURE • Previous Articles     Next Articles

Fine Mapping and Candidate Gene Analysis of a Short Internodes Gene Cmdm1 in Melon (Cucumis melo L.)

Jian MA1,CongCong LI2,JianShe WANG1()   

  1. 1 Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences/Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Ministry of Agriculture and Rural Affairs, Beijing 100097
    2 Beijing Agro-Biotechnology Research Center, Beijing Academy of Agricultural and Forestry Sciences/Beijing Key Laboratory of Agricultural Genetic Resources and Biotechnology, Beijing 100097
  • Received:2019-08-07 Accepted:2019-09-16 Online:2020-02-16 Published:2020-03-09
  • Contact: JianShe WANG E-mail:wangjianshe@nercv.org

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Abstract:

【Objective】In this study, the Z8 was a mutant with short internode in melon, and its gene fine mapping and candidate gene analysis were conducted to provide the theoretical guidance for the improvement of melon architecture. 【Method】Agronomic traits, such as internode number, main stem length, main stem internodes length and lateral branch length were measured in the Z8 and wild-type B15. The Z8 was crossed with B15, and the F2 and BC1F1 generations were used for genetic analysis. The recessive individuals in F2 were used to locate the gene by the map-based cloning strategy, and the candidate gene in the mapping region was determined by gene sequencing. 【Result】The Z8 mutant was dwarf because the internodes were significantly shorter compared with the wild-type B15. In addition, the flower buds of Z8 mutant were clustered at the top, which showed significantly compact inflorescences. Genetic test indicated that the mutant phenotype segregated as a single recessive gene locus named Cmdm1, which had been mapped to a 56 kb interval on the short arm of chromosome 7, flanked by markers c7-112 and s2 and co-segregated with marker dm-1. The MELO3C016916 gene was homologous to Arabidopsis ERECTA gene, which was one of the four annotated open reading frames (ORFs) in the 56 kb region. Sequence analysis of MELO3C016916 gene in the Z8 mutant indicated that a single nucleotide substitution (T 1995→G 1995) in its 25 th exon caused premature termination of the encoded protein, thus placing MELO3C016916 as a candidate gene for Cmdm1.【Conclusion】The short internode trait of Z8 was controlled by a single recessive gene named Cmdm1, which was finally mapped to a 56 kb interval between markers c7-112 and s2 on chromosome 7, and the MELO3C016916 gene was presumed to be the candidate gene.

Key words: melon (Cucumis melo L.), short internodes, gene mapping, Cmdm1, ERECTA

Table 1

PCR primer sequences used in this study"

引物名称 Primer 正向引物 Forward primer (5'-3') 反向引物 Reverse primer (5'-3')
c7-3 AATTCCCTTACCCTTGACAGCA GTTGAACCGACTCGACCGAC
c7-13 CAGGCAGGCTACGCTACATT CGCTCTGACGTTATGAAGTTGT
c7-70 GGAGAAAATGACGCTCTTAATTGAT CCCCATCTCCATTACAATGTTTACT
c7-94 ATGCATGTTAAGTTGTCCTACGG CCCACCACTATGGCATGTGTAA
c7-101 ACATGTCAGAGCAACAGAATTGA AAGCTCCATTCGAGGATTCATT
c7-103 ACCTCGTTCATGCCAAAGATG ACCTTTGACTCCAAAAGAAGGA
c7-112 GTGTTCGACCAACCAATTTTCAC GGTGTTAGTGTGGATTTGGGA
c7-119 AAAACCTTCAAATCATGGACTGTT ACAGTTAAAATAGCATGCAAGAAGT
c7-120 AATTTTTAGATCCACCGCTGCT CATACTTCTCCCCAATCTTCCC
c7-130 GTGTCATGTCGAAACACCGTC CTTCGTGGTACGTCGGGAAA
c7-132 AAAGGTGTAATTCACATTCATCCAA ACTTTGTTGGAACATAGAGTTGGT
c7-167 ATTCCACGACTGCCCATCAG TGCTGTCTGCAACATTAAAGGG
c7-200 TCGTTTACCGACTACATTGAGC TGTCCAGATTTTAAAGTTGATTCGT
c7-206 CAATACATGGCTCTAAAGACAAACT AGAGTGTTTCACTCGCATCCT
dm-1 CAACTTCAAAATCTTCTCTCCTTGT TAAACATGCAGCAGGAAGGAAA
s2 GGCTGTTGCTTGGACTTTGG AGCATGACCGGCTCATCAAA
MELO3C016916 CCAGCCATGAACACTGCAAAA CCATGTTGCTGCTTGCCTAC
MELO3C016917 CTCTCCTTGTTGTCACTGTTCAAA ATCTGCTACCCTCAGCTTTCC
MELO3C016918 CTTCTGGATCTCGCTAGGGTTT TGGTGTGGCACCTGTGTTT
MELO3C016919 TTTGTAGGCTGGCTTTGAGGA ACTCCCCAAAAGAGAAGTCCCA
q3C016916 TCGTCCGAACAACACAATACCT CTCATAAACGTGAAGGGCCA
CmActin TTCCCTCAGCACTTTCCAACA TCCCTTTCCACCTTTTTGCTTC

Fig. 1

Phenotypes of wild-type B15 and Z8 mutant A: Cotyledon stage (Scale bars, 5 cm); B: Vine elongation stage (Scale bars, 20 cm); C: The top inflorescence of Z8 (Scale bars, 5 cm)"

Table 2

Comparison of agronomic traits between the wild-type B15 and Z8 mutant (mean±SD, n=10)"

材料名称
Material		 主蔓节数
Node number (No.)		 主蔓长度
Main stem length (cm)		 主蔓平均节间长度
Main stem internodes length (cm)		 侧枝长度
Lateral branch length (cm)
B15 43±9.8 363.3±77.7 8.5±0.1 61.6±10.4
Z8 47.3±8.1 181±13.9** 3.2±0.2** 64.7±15.3

Table 3

Genetic analysis of the Z8 mutant"

品种或组合
Generation		 正常单株
Normal individuals		 短蔓单株
Short internodes individuals		 分离比
Expected ratio		 χ20.05 值
χ20.05
B15 (P1) 20 0
Z8 (P2) 0 20
F1 (P1 × P2) 25 0
F2 (F1)? 175 55 3﹕1 0.40
BC1F1 (F1 × P2) 15 16 1﹕1 0.39

Fig. 2

Physical maps of the Cmdm1 locus A: the Cmdm1 gene was located on the short arm of chromosome 7 between markers c7-3 and c7-206; B: the Cmdm1 gene was preliminarily delimited to the c7-101-c7-132 interval using 210 short internodes F2 individuals; the numerals below the corresponding markers indicate the numbers of recombinants (Rec) identified among F2 plants; C: fine genetic mapping of the Cmdm1 gene based on 780 short internodes F2 individuals; D: physical map of the Cmdm1 region based on the reference sequence and arrows indicated four annotated ORFs in this region"

Fig. 3

Gene structure, predicated function domains and gene expression levels of MELO3C016916 gene A: Gene structure of MELO3C016916 and T1995 to G1995 mutation site was identified in the 25th exon generating a premature termination codon; B: The predicted function domains of MELO3C016916 protein in B15 and Z8, respectively"

Fig. 4

Relative expression analysis of MELO3C016916 in B15 and Z8"

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