Method for screening molecular markers relevant to few-root duckweed leaf growth

A technology of molecular markers and leaves, applied in biochemical equipment and methods, determination/inspection of microorganisms, etc., to achieve the effect of improving the efficiency of biological treatment and the detection speed.

Active Publication Date: 2017-08-01

ZHEJIANG OCEAN UNIV

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AI-Extracted Technical Summary

Problems solved by technology

The detection method of the invention has the advantages of rapidity, accuracy, sensitivity, etc., and can intuitively detect the genotypes of different individuals of the rusty spot, so as to quickly obtain the polymorphism map of the genetic variation of the functional ...

Method used

Plant clone carries out indoor cultivation 50~70d in light incubator, culture condition is 22.5~23.5 ℃, humidity 73~83%, light intensity is 1800~2200lux, light and dark time 14~16:10~8h. Under the above-mentioned culture conditions, the photosynthesis of P. spp. was strong and the growth rate was fast.

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Abstract

The invention discloses a method for screening molecular markers relevant to few-root duckweed leaf growth. The method comprises the steps of clone body culture, genome DNA (deoxyribonucleic acid) extraction, microsatellite primer design, PCR (polymerase chain reaction) amplification and data statistics and analysis. The method has the beneficial effects that the DNA sequences of a great amount of few-root duckweed marked by the microsatellite primers can be fast obtained; the microsatellite mark detection speed is high; the polymorphic map of the few-root duckweed Sp10, Sp14, Sp42, Sp47, Sp51, Sp52 and Sp53 genetic marker loca showing the high-degree genetic variation can be fast obtained. The leaf length and the leaf width of the few-root duckweed are obviously relevant to Sp10, Sp14, Sp42, Sp47, Sp51, Sp52 and Sp53 (P of the Sp51 site is lower than 0.05, and P of other sites is smaller than 0.001). The influence of the genotype differences on the duckweed growth and pollutant purification capability can be known; the wastewater biological treatment efficiency is improved.

Application Domain

Microbiological testing/measurement

Technology Topic

Genetic markerPollutant +10

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Examples

Experimental program(3)
Effect test(1)

Example Embodiment

[0015] Example 1:

[0016] The methods for screening the molecular markers related to leaf growth of the leaf growth of Lapus arvensis include clonal culture, genomic DNA extraction, design of microsatellite primers, PCR amplification, and data statistics and analysis.

[0017] The nutrient solution composition and concentration of plant clone culture are: nitrogen 0.3~0.4g/L, phosphate anhydride 0.1~0.15 g/L, potassium oxide 0.16~0.23 g/L, magnesium oxide 0.01~0.015 g/L and sulfur 0.012 ~0.02 g/L. The above-mentioned nutrient solution can comprehensively provide the macronutrients and trace elements required for the growth of Papaver serrata, the speed of asexual reproduction is fast, the probability of gene mutation is low, a large number of genetically identical plants can be obtained, and the sample amount is sufficient.

[0018] Plant clones were cultured indoors in a light incubator for 50-70 days, culture conditions were 22.5-23.5℃, humidity 73-83%, light intensity 1800-2200 lux, light and dark time 14-16:10-8h. Under the above-mentioned culture conditions, the photosynthesis is strong and the growth rate is fast.

[0019] The microsatellite primers are: Sp 10: F: CCATCTGTCGTCCTTTTTCCC; R: CGCCCCATCACTTATTTCGTA; Sp14: F: GTCCATCCTTCTCAGCACAAT; R: CCGTACAAGATCTAAGCCTTT; Sp42: F: TGAGAGATCAGGCTGGAGCAGTG: GGTAGGAAAGCTGGAGCGATG: GGTA: F: GCCTGAAAGCCA: GTGAG: GGTAGGCTAGGCTAG: F: GCCTAGCTAGGAG: GGGTAG: GCCTAGCTGGAGCTAAGG: GCCTAGCTGGAGCTAAGGGAGAGATCAGGCTGGAGCAGGGAGAGGAGAGATCAGGCTGGAGCAGTGGGGTAAGGCCTG CTCGCACATCAGTTCACAGGA; R: TCAGACATCTGGCGCAGTAGA; Sp52: F: GTCCTCCCTTTGATTGCTCGTC; R: AAGCATCATGGGCTCTTCAGG; Sp53: F: AGGACGACGACCTCTACTGCC; R: TACGAGTTCTGCGGACCATCA.

[0020] The PCR amplification system is 18~22ul, including 3.4~4.2U Taq DNA polymerase (Sangon), 1.6~2.2ul 10×PCRbuffer, 0.7~0.9mM dNTP, upstream and downstream primers each 0.3mM, 50~100ng DNA template, ddH 2 O make up to the final volume of 20ul; perform amplification on the Bio-Rad PCR Amplifier, the amplification program is 92~96℃ pre-denaturation 4.5~5.6min; 93~96℃ denaturation 28~33s, 52~56℃ renaturation 33~37s, 70~75℃ extension for 36~43s, a total of 33~38 cycles; finally 70~75℃ extension for 2.5~3.4min. The above-mentioned PCR amplification can quickly obtain a large amount of DNA sequences of Papaver serrata labeled with microsatellite primers.

[0021] Data statistics and analysis are the statistical description of the number of leaves/plants, leaf length and leaf width of the cloned P. amurense. One-way analysis of variance and least significant difference method or Dunnett T3 non-parametric multiple comparison of the growth characteristics of the two genotypes The difference between the two; using the general linear model process to carry out the least square analysis of the correlation between the leaf number/plant, leaf length and leaf width traits and the microsatellite locus, and multiple comparisons of the genotypes at the same locus. The leaf length and leaf width of Papilla sylvestris were significantly correlated with Sp10, Sp14, Sp42, Sp47, Sp51, Sp52 and Sp53 (except for Sp51 site P <0.05, the rest P <0.001).

Example Embodiment

[0022] Example 2:

[0023] The methods for screening the molecular markers related to leaf growth of the leaf growth of Lapus arvensis include clonal culture, genomic DNA extraction, design of microsatellite primers, PCR amplification, and data statistics and analysis.

[0024] The nutrient solution composition and concentration of plant clone culture are: nitrogen 0.32g/L, phosphate anhydride 0.12 g/L, potassium oxide 0.19g/L, magnesium oxide 0.012 g/L and sulfur 0.016g/L. The above-mentioned nutrient solution can comprehensively provide the macronutrients and trace elements required for the growth of Papaver serrata, the speed of asexual reproduction is fast, the probability of gene mutation is low, a large number of genetically identical plants can be obtained, and the sample amount is sufficient.

[0025] The plant clones were cultured indoors in a light incubator for 60 days under the conditions of 23°C, humidity 78%, light intensity of 2000 lux, and light and dark time of 16: 8h. Under the above-mentioned culture conditions, the photosynthesis is strong and the growth rate is fast.

[0026] The microsatellite primers are: Sp 10: F: CCATCTGTCGTCCTTTTTCCC; R: CGCCCCATCACTTATTTCGTA; Sp14: F: GTCCATCCTTCTCAGCACAAT; R: CCGTACAAGATCTAAGCCTTT; Sp42: F: TGAGAGATCAGGCTGGAGCAGTG: GGTAGGAAAGCTGGAGCGATG: GGTA: F: GCCTGAAAGCCA: GTGAG: GGTAGGCTAGGCTAG: F: GCCTAGCTAGGAG: GGGTAG: GCCTAGCTGGAGCTAAGG: GCCTAGCTGGAGCTAAGGGAGAGATCAGGCTGGAGCAGGGAGAGGAGAGATCAGGCTGGAGCAGTGGGGTAAGGCCTG CTCGCACATCAGTTCACAGGA; R: TCAGACATCTGGCGCAGTAGA; Sp52: F: GTCCTCCCTTTGATTGCTCGTC; R: AAGCATCATGGGCTCTTCAGG; Sp53: F: AGGACGACGACCTCTACTGCC; R: TACGAGTTCTGCGGACCATCA.

[0027] The PCR amplification system is 20ul, including 4U Taq DNA polymerase (Sangon), 2ul10×PCRbuffer, 0.8mMdNTP, upstream and downstream primers each 0.3mM, 50-100ng DNA template, ddH 2 O make up to the final volume of 20ul. Amplify on the Bio-Rad PCR Amplifier. The amplification program is 94°C pre-denaturation for 5 min; 94°C denaturation for 30s, 54°C renaturation for 35s, 72°C extension for 40s, a total of 35 cycles; final 72°C extension for 3min . The PCR products were detected by capillary electrophoresis on the 3730XL sequencer. The above-mentioned PCR amplification can quickly obtain a large amount of DNA sequences of Papaver serrata labeled with microsatellite primers.

[0028] Data statistics and analysis are the statistical description of the number of leaves/plants, leaf length and leaf width of the cloned P. amurense. One-way analysis of variance and least significant difference method or Dunnett T3 non-parametric multiple comparison of the growth characteristics of the two genotypes The difference between the two; using the general linear model process to carry out the least square analysis of the correlation between the leaf number/plant, leaf length and leaf width traits and the microsatellite locus, and multiple comparisons of the genotypes at the same locus. The leaf length and leaf width of Papilla sylvestris were significantly correlated with Sp10, Sp14, Sp42, Sp47, Sp51, Sp52 and Sp53 (except for Sp51 site P <0.05, the rest P <0.001).

Example Embodiment

[0029] Example 3:

[0030] The methods for screening the molecular markers related to leaf growth of the leaf growth of Lapus arvensis include clonal culture, genomic DNA extraction, design of microsatellite primers, PCR amplification, and data statistics and analysis.

[0031] The nutrient solution components and their concentrations for plant clone culture are: nitrogen 0.32g/L, phosphate anhydride 0.12g/L, potassium oxide 0.2g/L, magnesium oxide 0.013g/L and sulfur 0.017g/L. The above-mentioned nutrient solution can comprehensively provide the macronutrients and trace elements required for the growth of Papaver serrata, the speed of asexual reproduction is fast, the probability of gene mutation is low, a large number of genetically identical plants can be obtained, and the sample amount is sufficient.

[0032] Count the number of leaves for each plant (leaf connected) in different culture lines, and use vernier calipers to measure the leaf length (the longest leaf length), the leaf width (the shortest leaf length) and the length of each root.

[0033] The microsatellite primers are: Sp 10: F: CCATCTGTCGTCCTTTTTCCC; R: CGCCCCATCACTTATTTCGTA; Sp14: F: GTCCATCCTTCTCAGCACAAT; R: CCGTACAAGATCTAAGCCTTT; Sp42: F: TGAGAGATCAGGCTGGAGCAGTG: GGTAGGAAAGCTGGAGCGATG: GGTA: F: GCCTGAAAGCCA: GTGAG: GGTAGGCTAGGCTAG: F: GCCTAGCTAGGAG: GGGTAG: GCCTAGCTGGAGCTAAGG: GCCTAGCTGGAGCTAAGGGAGAGATCAGGCTGGAGCAGGGAGAGGAGAGATCAGGCTGGAGCAGTGGGGTAAGGCCTG CTCGCACATCAGTTCACAGGA; R: TCAGACATCTGGCGCAGTAGA; Sp52: F: GTCCTCCCTTTGATTGCTCGTC; R: AAGCATCATGGGCTCTTCAGG; Sp53: F: AGGACGACGACCTCTACTGCC; R: TACGAGTTCTGCGGACCATCA.

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Classification and recommendation of technical efficacy words

detection speed
Improve bioremediation efficiency

Method for screening molecular markers relevant to few-root duckweed leaf growth

A technology of molecular markers and leaves, applied in biochemical equipment and methods, determination/inspection of microorganisms, etc., to achieve the effect of improving the efficiency of biological treatment and the detection speed.

AI-Extracted Technical Summary

Problems solved by technology

Method used

Abstract

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Examples

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Description & Claims & Application Information

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