Enteromorpha gender specific molecular marker and application thereof

A technology of molecular marker and prolifera, applied in the field of marine algae molecular genetics, can solve the problems of lack of reliable determination methods for sex identification of prolifera, lack of accumulation of genetic research, and difficulties in genetic research of prolifera and hybrid breeding.

Active Publication Date: 2021-03-19
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In summary, because Enteromorpha has the same type of life history type with alternation of generations and diverse reproductive methods, especially the lack of necessary genetic research accumulation, the sex identification of Enteromorpha lacks a reliable method, especially when dealing with a large number of When collecting samples, it is impossible to efficiently and accurately identify the breeding parents and offspring, which has caused great difficulties for the genetics research and hybrid breeding of Enteromorpha.

Method used

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  • Enteromorpha gender specific molecular marker and application thereof
  • Enteromorpha gender specific molecular marker and application thereof
  • Enteromorpha gender specific molecular marker and application thereof

Examples

Experimental program
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Embodiment 1

[0023] Example 1: Screening and Design of Sex-Specific Molecular Markers in Enteromorpha Gametophytes

[0024]Based on the original unannotated sequence (NC_036137.1) of the Ulva prolifera genome in NCBI, by comparing and analyzing the resequencing data of the female and male gametophyte genomes of Ulva prolifera, it is speculated that the sequence of sex difference may be located in scaffold 107 (SDUY01000439. 1) with scaffold 115 (SDUY01000200.1). Based on bioinformatics analysis, specific amplification primers were designed respectively. Among them, using the female gametophyte genomic DNA as a template, using the primer pair Up1-F (5'-AGATGTATGATTCCTGTGGCTTC-3') and Up1-R (5'-GCAGCAATAACCAGTATCTTGTC-3') to amplify, the approximately A 1460bp nucleotide fragment; using the male gametophytic genomic DNA as a template, using the primer pair Up2-F (5'-AGTGAATCAGTACGAAACACCTC-3') and Up2- (5'-AGTTCCCAGCTCATACAAATACC-3') to amplify, the male gametophyte containing An approxima...

Embodiment 2

[0064] Example 2: Using sex-specific molecular markers to detect ploidy and sex in each generation in the typical life history of Enteromorpha

[0065] One individual plant of the identified female and male gametophytes of Enteromorpha enteromorpha was selected as the parent, and the formation and release of germ cells were induced by the cutting method. The specific steps include: cut the algae into 1-2 mm segments with a sterile blade, place them in a petri dish, add VSE culture solution, transfer to a light incubator, set the culture temperature at 20°C, and the light-dark cycle 14h / 10h (L:D), light intensity about 100μmol photons m -2 ·s -1 Reproductive induction was performed and germ cells were seen to be released from the algal segment after about 2-3 days. Collect germ cells from female and male gametophytes respectively, mix them in equal amounts in a new petri dish, add VSE culture medium (prepared with sterilized seawater, containing NaNO 3 0.5mmol / L;Na 2 HPO ...

Embodiment 3

[0068] Example 3: Using sex-specific molecular markers to detect ploidy and sex in the atypical life cycle of Enteromorpha—gametophytic parthenogenesis

[0069] The reproductive mode of Enteromorpha is very diverse. In addition to the typical life history, there are also many atypical life histories. The most basic genetic law, including the change characteristics of algae ploidy, has yet to be elucidated. The study found that Enteromorpha algae (speculated to be gametocytes) capable of producing double flagella and positive phototaxis germ cells can also germinate alone to form new algae without cell fusion. However, for this process, there has never been a result of molecular identification to prove that the specific aspects include: 1) whether the parents are haploid gametophytes; 2) whether there is no ploidy change in the atypical life history process, and the offspring are still the same sex 3) Whether both male and female gametophytes can complete this life cycle, or on...

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Abstract

The invention belongs to the field of seaweed molecular genetics, and relates to an enteromorpha gender specific molecular marker and application thereof. The enteromorpha gender specific molecular marker is characterized in that one enteromorpha female gametophyte DNA specific molecular marker and one enteromorpha male gametophyte DNA specific molecular marker are provided, wherein the nucleotidesequences of the enteromorpha female gametophyte DNA specific molecular marker and the enteromorpha male gametophyte DNA specific molecular marker are respectively SEQ ID NO. 3 and SEQ ID NO. 4, theSEQ ID NO. 3 is obtained by amplifying a primer sequence SEQ ID NO. 1, and the SEQ ID NO. 4 is obtained by amplifying a primer sequence SEQ ID NO. 2. The specific molecular marker is completed by detecting the specific fragment through PCR (polymerase chain reaction), and the process comprises the following steps of extracting to-be-detected enteromorpha genomic DNA, performing PCR amplification,and performing agarose gel electrophoresis detection on an amplification product. The specific primer is obtained by referring to existing high-throughput data result in a laboratory, combining genomeDNA extraction, target fragment amplification and recovery, cloning by using pGEM-T vector and escherichia coli competent strain TOP10 and the like. By means of the specific molecular marker, ploidyof a large number of enteromorpha algae and gender of gametophytes can be rapidly, simply and accurately identified, and an important molecular tool is provided for enteromorpha life history researchand variety breeding.

Description

technical field [0001] The invention belongs to the field of seaweed molecular genetics, and relates to a gender-specific molecular marker of Enteromorpha and its application. Background technique [0002] Ulva prolifera belongs to Chlorophyta, Chlorophyceae, Ulvales, Ulvaceae, Ulva, is a common large green algae . Enteromorpha is rich in dietary fiber, essential amino acids, trace elements and minerals. It has high nutritional value and has edible and medicinal value. Its artificial breeding has reached a certain scale. As an emerging large economic algae, it is urgent to carry out systematic and in-depth research on its basic genetic characteristics such as life history type, reproductive mode, and algae sex, so as to lay a theoretical and methodological foundation for the hybrid breeding of Enteromorpha. [0003] There is a typical isotype alternation in the life history of Enteromorpha, which consists of haploid gametophytic generations and diploid sporophytic generati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6879C12N15/11
CPCC12Q1/6895C12Q1/6879
Inventor 姜鹏刘倩纯赵瑾吴春辉
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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