‘溧阳白芹’雄性不育材料的选育及线粒体候选基因的鉴定

Breeding of male sterile materials and identification of mitochondria candidate gene in ‘Liyangbaiqin’

摘要

[目的]本文旨在创制和选育‘溧阳白芹’雄性不育材料，打破‘溧阳白芹’杂交品种的培育限制，以培育‘溧阳白芹’新材料。[方法]用甲基磺酸乙酯(EMS)诱变‘溧阳白芹’的种子，通过筛选、形态鉴定、育性研究、线粒体基因组测序、基因克隆等方法对‘溧阳白芹’雄性不育材料进行分析。[结果]以‘溧阳白芹’种子为诱变材料，从0.6mol·L^-1 EMS、浸泡2h的组合中，筛选出1株花丝花药萎缩，花药中未有花粉粒的雄性不育材料(命名为LYSQBU001)。线粒体基因组测序结果表明，LYSQBU001线粒体基因组为单环结构(NCBI:PQ790628)，长度为384078bp，GC含量为45.03%，含35个unique蛋白质编码基因，22个tRNA基因，3个rRNA基因；有79个SSR，其中四聚体数量最高(32个，40.5%)；含有20个串联复制和437对重复序列；丙氨酸(Ala)对密码子GCU有较高的使用，而对密码子GCG使用最低。对比其他伞形科植物，LYSQBU001线粒体atp6基因在5′端发生缺失，克隆测序表明LYSQBU001与可育的‘溧阳白芹’(LYSQKU001)在5′端缺失462bp片段，导致LYSQBU001仅为744bp(LYSQKU001中长度为1206bp)，缺失片段为跨膜结构。进化分析表明，LYSQBU001中atp6蛋白序列与不育的胡萝卜、茴香同属于一个分支，而LYSQKU001中atp6蛋白序列与可育的胡萝卜和茴香属于同一个分支。[结论]线粒体atp6基因突变是‘溧阳白芹’LYSQBU001雄性不育产生的原因，可用于‘溧阳白芹’新品种培育。

Abstract

[Objectives] The paper aimed to create and select male sterile materials of ‘Liyangbaiqin’ [Oenanthe javanica (Blume) DC], to break the limitations of cultivating hybrid varieties of ‘Liyangbaiqin’, and thereby breed new variety of ‘Liyangbaiqin’. [Methods] Using ‘Liyangbaiqin’ seeds as mutagenic materials, ethyl methanesulfonate (EMS) was used for mutagenesis of ‘Liyangbaiqin’ seeds, and the male sterile materials of ‘Liyangbaiqin’ were analyzed by screening, morphological identification, fertility research, mitochondrial genome sequencing, gene cloning, and other methods. [Results] One male sterile material of ‘Liyangbaiqin’ (named: LYSQBU001) without pollen grains in the anthers was selected from the combination of EMS with a concentration of 0.6 mol·L^-1 and soaking for 2 h. The results showed that the mitochondrial genome of LYSQBU001 was a single loop structure (NCBI: PQ790628), with a length of 384078 bp and the GC content of 45.03%. It contained 35 unique protein coding genes, 22 tRNA genes, and 3 rRNA genes. There were 79 SSR, and the highest number was the tetramers (32, 40.5%). It contained 20 tandem replicates and 437 pairs of repetitive sequences. Alanine (Ala) had a higher usage of codon GCU, while the lowest usage of codon GCG. Compared with other Apiaceae plants, the mitochondrial atp6 gene had a fragment missing at the 5′ end. Cloning and sequencing showed that a 462 bp fragment was missing at the 5′ end compared to the fertile ‘Liyangbaiqin’ (LYSQKU001), resulting in the length of LYSQBU001 being only 744 bp (LYSQKU001 was 1206 bp), and the missing fragment was the transmembrane segment. Evolutionary analysis showed that the atp6 protein sequence in LYSQBU001 belonged to the same branch with sterile carrots and fennel, while the atp6 protein sequence in LYSQKU001 belonged to the same branch with fertile carrots and fennel. [Conclusions] The mitochondrial atp6 gene mutation was the cause of male infertility in ‘Liyangbaiqin’ LYSQBU001, and could be used for the cultivation of new varieties of ‘Liyangbaiqin’.

关键词

溧阳白芹 / 雄性不育 / atp6 / 线粒体 / 选育

Key words

Liyangbaiqin / male sterile / atp6 / mitochondrion / breeding

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基金资助

贵州省科学技术厅项目(黔科合服企〔2022〕014,黔科合服企〔2024〕003-1,黔科合服企〔2024〕003-2)

贵州省农业科学院项目(黔农科博士基金〔2024〕03号,黔农科种质资源〔2023〕17号)

贵州省高原特色蔬菜产业体系(GZMARS)

南京农业大学三亚研究院引导资金项目(NAUSY-MS33)

贵州省山地农业关键核心技术攻关项目(GZNYGJHGX-2023013)