甜瓜短蔓基因Cmdm1的精细定位及候选基因分析

doi:10.3864/j.issn.0578-1752.2020.04.012

摘要/Abstract

摘要：

【目的】对甜瓜短蔓突变体Z8进行短蔓基因的精细定位并确定候选基因,为甜瓜株型的分子改良奠定基础。【方法】考察短蔓突变体Z8和野生型B15的主蔓节数、主蔓长度、主蔓节间长度以及侧枝长度等农艺性状。配制Z8/B15杂交组合并进行遗传分析,利用F₂群体中的短蔓单株进行基因精细定位。通过对定位区间内注释基因编码区进行测序以确定候选基因。【结果】与野生型B15相比,突变体Z8节间显著变短导致植株矮化,顶端花序紧凑簇生,遗传分析表明其短蔓性状由一对隐性核基因Cmdm1控制。采用基因图位克隆策略,利用780个F₂短蔓单株最终将该基因精细定位于第7染色体短臂标记c7-112和s2之间约56 kb的区间内,并与标记dm-1共分离,区间内共包含4个注释基因。经测序鉴定,发现Z8中与拟南芥ERECTA同源的MELO3C016916 ATG下游第1 995位碱基由T突变为G而产生终止密码子,导致蛋白翻译提前终止,致使后面激酶结构域完全缺失,推测MELO3C016916即为控制蔓长的Cmdm1。【结论】Z8短蔓性状受隐性核基因Cmdm1控制,利用分子标记最终将该基因定位于7号染色体短臂标记c7-112和s2之间约56 kb区间内,推测MELO3C016916为最有可能的候选基因。

关键词: 甜瓜, 短蔓, 基因定位, Cmdm1, ERECTA

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

马建,李丛丛,王建设. 甜瓜短蔓基因Cmdm1的精细定位及候选基因分析[J]. 中国农业科学, 2020, 53(4): 802-810.

Jian MA,CongCong LI,JianShe WANG. Fine Mapping and Candidate Gene Analysis of a Short Internodes Gene Cmdm1 in Melon (Cucumis melo L.)[J]. Scientia Agricultura Sinica, 2020, 53(4): 802-810.

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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

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

品种或组合 Generation	正常单株 Normal individuals	短蔓单株 Short internodes individuals	分离比 Expected ratio	χ²_0.05 值 χ²_0.05
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