CN113234848A - Molecular marker related to poplar stomatal morphology and photosynthetic efficiency and application thereof - Google Patents

Abstract

The invention discloses a molecular marker related to pore shape and photosynthetic efficiency of poplar, wherein the molecular marker is an SNP marker, is positioned at 3140 th base downstream of a genome ZCWCCC 3 gene of poplar, and has C/T polymorphism, wherein the genotype of the molecular marker is a poplar individual of CC, and has larger pore width, pore length-width ratio and higher photosynthetic efficiency; the poplar individual with TT as the genotype of the molecular marker has smaller pore width, pore length-width ratio and lower photosynthetic efficiency. The molecular marker provided by the invention has definite functions and obvious effect, can be directly applied to the molecular marker-assisted breeding of poplar, has environmental adaptability, and is suitable for regional breeding and climatic adaptation breeding. The invention also provides a primer pair for amplifying the molecular marker, application of the molecular marker, a poplar genetic improvement method and a system for predicting the stomatal morphology and the photosynthetic efficiency of poplar, and provides an important basis for the molecular marker-assisted selective breeding of poplar.

Description

Molecular marker related to poplar stomatal morphology and photosynthetic efficiency and application thereof

Technical Field

The invention belongs to the technical field of tree molecular breeding, and particularly relates to a molecular marker, a primer group, a kit and application thereof, wherein the molecular marker is related to the pore morphology and the photosynthetic efficiency of poplar.

Background

The poplar is an industrial material and an ecological tree species which are widely distributed in the world, is the first tree species for the construction of artificial forests in the world, has the advantages of early fast growth, strong adaptability, wide distribution, multiple varieties, easy hybridization, easy improvement of heredity, easy propagation and the like, and is widely applied to intensive cultivation. Photosynthesis directly affects the accumulation of forest biomass and is one of the most important physiological processes that determine forest productivity. Among the factors influencing the photosynthesis of the trees, the air hole form is particularly important and can balance the water transpiration and carbon acquisition inside the leaves as a direct channel for gas exchange between the trees and the outside, so that the genetic improvement on the air hole form of the poplar trees has important theoretical and application values.

The genetic improvement of poplar as perennial tree species in the traditional crossbreeding mode takes a long time, and the development of functional molecular markers for auxiliary breeding is the current main research trend. Single nucleotide polymorphism is known as the latest third generation DNA molecular marker, and has been widely used in the research fields of gene localization, cloning, genetic breeding, genetic diversity and the like. The principle of genome-wide association analysis is linkage disequilibrium, namely that one SNP always exists in the genome-wide range and linkage disequilibrium exists with causative mutation causing the traits, and genome positions influencing the traits can be narrowed down or identified by detecting the SNP.

However, the genetic improvement practice of poplar stomata morphology lacks molecular markers which have definite functions and obvious effects and can be directly applied due to the interference of factors such as genetic background differences, founder effects, multi-gene interaction effects, gene-environment interaction effects, incomplete selection/balanced selection effects, the limitation of the number of genetic markers, linkage disequilibrium and the like of different experimental groups.

In addition, in recent years, related researches show that similar to other quantitative traits of trees, stomata forms have abundant phenotypic variation among different ecotypes, are selected by various biotic and abiotic stresses of local environments, and show obvious phenotypic plasticity along the change of space and environmental gradients. Functional adaptation of the stomatal morphology regulatory pathway can provide valuable candidate alleles for future regional breeding programs and climatic adaptation.

Therefore, there is a need to provide a molecular marker related to stomata morphology and photosynthetic efficiency of poplar, which has a clear function and a remarkable effect, can be directly applied and is suitable for region breeding.

Disclosure of Invention

In order to overcome the problems, the inventor of the present invention has conducted intensive research, integrates a whole genome association analysis and a selective elimination strategy, screens out molecular markers significantly related to stomatal morphology and photosynthetic efficiency of poplar, can be directly applied to molecular marker assisted breeding of poplar, and the obtained molecular markers have environmental suitability and are suitable for regional breeding and climatic adaptation breeding. In addition, a primer pair for amplifying the molecular marker, application of the molecular marker, a poplar genetic improvement method and a system for predicting the stomatal morphology and the photosynthetic efficiency of poplar are provided, so that the stomatal morphology of poplar can be accurately evaluated on a molecular level, on the basis of the stomatal morphology, the tree species with high photosynthetic efficiency can be accurately and efficiently screened out, the breeding period of poplar can be effectively shortened, and the invention is completed.

Specifically, the present invention aims to provide the following:

in a first aspect, a molecular marker related to the stomatal morphology and photosynthetic efficiency of a poplar is provided, and the molecular marker is an SNP marker, is positioned at 3140 th base downstream of a ZCWCC3 gene of a poplar genome, and has C/T polymorphism.

In a second aspect, there is provided a method for obtaining the molecular marker of the first aspect, the method comprising the steps of:

step 1, determining the phenotypic character of a poplar group;

step 2, extracting genome DNA of the poplar group;

and 3, obtaining the molecular marker which is obviously related to the phenotypic character.

In a third aspect, a primer pair for amplifying the molecular marker related to poplar stomatal morphology and photosynthetic efficiency of the first aspect is provided, and the primer pair comprises a primer P1 and a primer P2,

wherein, the primer P1 has the sequence shown in SEQ ID NO: 3, primer P2 has the nucleotide sequence shown as SEQ ID NO: 4.

In a fourth aspect, there is provided a molecular marker according to the first aspect, a molecular marker obtained by the method according to the second aspect, or a primer pair according to the third aspect, wherein the molecular marker is used in any one of the following aspects (1) to (4):

(1) screening or auxiliary screening of a new poplar variety with high air hole width, air hole length-width ratio and photosynthetic efficiency; (2) molecular marker assisted breeding of poplar; (3) preparing and screening or auxiliary screening products of new poplar varieties with high air hole width, air hole length-width ratio and photosynthetic efficiency; (4) preparing a product of poplar molecular marker assisted breeding.

In a fifth aspect, a method for genetic improvement of poplar is provided, the method comprises the steps of subculturing poplar individuals with CC genotype of the molecular marker in the first aspect, and eliminating all other genotype individuals of the molecular marker;

preferably, the method for genetic improvement of poplar comprises the following steps:

step i, extracting genomic DNA of poplar;

step ii, using poplar genome DNA as a template to perform PCR amplification;

step iii, determining the genotype of the molecular marker of the poplar to be detected;

step iv, determining the stomatal morphology and the photosynthetic efficiency of the poplar to be detected according to the genotype detection result;

and v, subculturing and breeding the CC genotype individuals of the molecular markers.

In a sixth aspect, a system for predicting stomatal morphology and photosynthetic efficiency of poplar is provided, the system comprising an amplification subsystem, a genotype identification subsystem and a prediction subsystem connected in sequence, wherein,

the amplification subsystem is used for amplifying the genomic DNA of the poplar to be detected;

a genotype identification subsystem, which is used for determining the genotype of the molecular marker of the first aspect of the poplar to be detected;

and the prediction subsystem is used for predicting the stomatal morphology and the photosynthetic efficiency of the poplar according to the genotype of the molecular marker.

The invention has the advantages that:

(1) the molecular marker related to the pore morphology and the photosynthetic efficiency of the poplar provided by the invention has definite functions and obvious effects, and can be directly applied to molecular marker-assisted breeding of the poplar; (2) the molecular marker related to the pore morphology and the photosynthetic efficiency of the poplar can accurately evaluate the pore morphology of the poplar on a molecular level, and on the basis, the tree species with high photosynthetic efficiency can be accurately and efficiently screened out, so that the breeding period of the poplar can be effectively shortened; (3) the method for obtaining the molecular marker related to the stomatal morphology and the photosynthetic efficiency of the poplar, provided by the invention, integrates and utilizes a whole genome association analysis and selective elimination method, so that the obtained molecular marker has environmental adaptability and is suitable for regional breeding and climatic adaptation breeding.

Drawings

Fig. 1 shows the climate zone specific division criteria in embodiment 1 of the present invention; FIG. 2 is a diagram showing the effect of different genotypes of SNP marker loci according to example 1 on the aspect ratio of stomata of three subpopulations of Populus tomentosa; FIG. 3 is a graph showing the effect of different genotypes of SNP marker sites on the gas pore widths of three subgroups of Populus tomentosa according to example 1 of the present invention; FIG. 4 is a diagram showing the effect of different genotypes of SNP marker loci according to example 1 on the photosynthetic efficiency of three subpopulations of Populus tomentosa.

Detailed Description

The present invention will be described in further detail below with reference to preferred embodiments and examples. The features and advantages of the present invention will become more apparent from the description.

The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

The inventor finds that the adaptive genetic mechanism of perennial trees is analyzed by applying the strategy of selective elimination and genome-wide association analysis, and provides new insight for adaptive genetic variation prediction and local adaptive mechanism under global change.

Therefore, in the first aspect of the invention, the selective elimination and the whole genome association analysis are combined to discover a molecular marker related to the stomata morphology and the photosynthetic efficiency of the poplar, wherein the molecular marker is an SNP marker, is positioned at the 3140 th base downstream of the ZCWCCC 3 gene of the poplar genome and has C/T polymorphism.

In the invention, the version of the poplar genome is the poplar reference genome v3.0(http:// popgenie.org/)。

According to a preferred embodiment of the invention, the molecular marker is located as set forth in SEQ ID NO: 1, the 7897 th nucleotide sequence of the ZCWCC3 gene, and the 201 th to 4757 th nucleotide sequence of the ZCWCC3 gene.

In the present invention, the homologous gene of ZCWCCC 3 gene in Arabidopsis thaliana is AT5G57110, encoding self-inhibitory Ca²⁺ATPases, which are located mainly in the plasma membrane, plasmodesmata, have P-type calcium ion transport activity and are involved in calcium ion transport across the membrane, both in the plasma membrane components and in intracellular membrane-protected organelles.

In a further preferred embodiment, the encoding amino acid sequence of the ZCWCC3 protein is as set forth in SEQ ID NO: 2, respectively.

In a further preferred embodiment, the pore morphology comprises a pore width and a pore aspect ratio, preferably a pore width and a pore aspect ratio.

Preferably, the poplar is populus tomentosa.

The populus tomentosa is a special local tree species in China, is widely distributed, has distribution in Liaoning (south), Hebei, Shandong, Shanxi, Shaanxi and other provinces, takes yellow river territories and downstream as central distribution areas, plays an important role in the production of forestry and the construction of ecological environment in northern China, and is a pioneer tree species for forest cultivation in northern areas.

According to a preferred embodiment of the invention, the genotypes of the molecular markers are CC, TT and CT, wherein,

the molecular marker is a poplar individual with CC genotype and has larger pore width, pore length-width ratio and higher photosynthetic efficiency;

the poplar individual with TT as the genotype of the molecular marker has smaller pore width, pore length-width ratio and lower photosynthetic efficiency.

In the present invention, the CC genotype is homozygous for the above molecular marker C of poplar, the TT genotype is homozygous for the above molecular marker T of poplar, and the CT genotype is heterozygous for the above molecular markers C and T of poplar.

The inventor researches and discovers that the stomata form has abundant phenotypic variation among different ecotypes, and the stomata form is selected by the stress of various organisms and non-organisms in the local environment, and shows obvious phenotypic plasticity along the change of space and environmental gradient. The selected gene may determine the variation of the stomata of the populations in different habitats, so as to cause the difference of the allele frequency of the molecular marker in different areas, thereby maximally improving the photosynthetic capacity of the trees to respond to the change of the environment, the preferential mode causes different alleles to show different differentiation modes after undergoing different selective pressures,

in a further preferred embodiment, the molecular marker is an individual poplar with a genotype of CC, suitable for growth in southern climatic regions;

the molecular marker is a poplar individual with the genotype of TT and is suitable for growing in the northwest climate zone;

the genotype of the molecular marker is a poplar individual with CT, and the molecular marker is suitable for growing in northeast climate zones.

Preferably, the northwest climate zone is located in high-altitude, arid and semi-arid regions, preferably the regions with the east longitude of 102 degrees 61 '-112 degrees 90', the northern latitude of 34 degrees 38 '-39 degrees 23', the annual precipitation of 200-400 mm, and the annual sunshine duration of over 2200 hours; the northeast climate zone is located in the areas of northeast of Shandong and West of Shanxi, preferably the areas with the longitude from east 113 degrees 58 'to 119 degrees 51', the latitude from north 34 degrees 93 'to 41 degrees 73', the annual precipitation amount of 500-900 mm, and the annual sunshine duration of 2800 hours or more; the southern climate zone is located in a low-latitude humid area in southern China, and is preferably an area with 105 degrees 92 to 121 degrees 08 'of east longitude, 30 degrees 39 to 36 degrees 23' of north latitude, 4000 to 1300mm of annual precipitation and more than 2300 hours of annual sunshine hours.

Wherein, the northwest climate zone belongs to temperate continental climate, the northeast climate zone belongs to temperate monsoon climate, and the south climate zone belongs to temperate monsoon climate-subtropical monsoon climate.

In a second aspect of the present invention, there is provided a method for obtaining the molecular marker of the first aspect, the method comprising the steps of:

step 1, determining the phenotypic characters of the poplar group.

According to a preferred embodiment of the invention, the poplar population is a populus tomentosa population,

preferably, the populus tomentosa population comprises three sub-populations of different climatic regions, namely a northwest climatic region sub-population, a northeast climatic region sub-population and a southern climatic region sub-population.

According to an embodiment of the invention, the selected populus tomentosa population comprises 209 individuals, wherein the northwest climate zone has 69 individuals, the northeast climate zone has 72 individuals, and the south climate zone has 68 individuals.

In a further preferred embodiment, the phenotypic trait comprises stomatal width, stomatal aspect ratio and photosynthetic efficiency.

Among them, it is preferable to determine the photosynthetic efficiency of poplar population by using LI6400 portable photosynthesis system (LI-COR inc., Lincoln, NE, USA), and to determine the width and aspect ratio of air holes by using a method commonly used in the art, specifically: collecting 3 functional leaves of each individual of a poplar group for determination, cleaning cilia on the back of each poplar leaf by using an adhesive tape, uniformly coating nail polish on the leaf back, tearing down the poplar leaf after air drying, observing the poplar leaf under a microscope, selecting three non-overlapping areas for taking a picture for each sample, and extracting the picture phenotype by using ImageJ software to obtain the numerical values of the air hole width and the air hole length-width ratio of each individual.

And 2, extracting genome DNA of the poplar group.

Wherein, the DNA extraction of the leaf tissue of each individual in the population can be performed by the methods or DNA extraction kits commonly used in the prior art, such as: the DNeasy Plant Mini Kit (Qiagen China, Shanghai, China) Kit was used.

Preferably, the DNA quality detection is carried out by adopting an ultraviolet spectrophotometer and gel electrophoresis, and the qualified DNA is stored at the temperature of-20 ℃.

And 3, obtaining the molecular marker which is obviously related to the phenotypic character.

In the present invention, the molecular markers related to poplar stomata morphology (stomata width, stomata aspect ratio) and photosynthetic efficiency are obtained by using selective elimination and whole genome correlation analysis methods, preferably according to the following steps:

and 3-1, performing re-sequencing on DNA of each individual in the poplar group, and performing quality control on the obtained original data.

Preferably, the DNA re-sequencing is double-ended sequencing with a sequencing depth of 30 ×, the sequencing using Illumina GA2 re-sequencing platform.

Wherein, the quality control standard is as follows: (i) reads (reads) containing > 10% unidentified nucleotides are removed; (ii) removing reads with the base quality less than 5 and the quantity more than 50%; (iii) reads with more than 10nt compared to the adapter (adapter) are removed, and mismatch less than or equal to 10 percent is allowed; (iv) putative PCR repeats (two identical paired reads 1 and 2) generated by PCR amplification during library construction were deleted.

And 3-2, identifying the single nucleotide polymorphism sites and the genotypes of the single nucleotide polymorphism sites at the whole genome level by using the data after quality control.

Preferably, the data after quality control is aligned to the poplar reference genome v3.0, single nucleotide polymorphism sites are identified at the whole genome level and the genotype of the SNP sites is obtained.

And 3-3, screening the SNPs in the whole genome level to obtain a high-quality SNPs label set.

Preferably, VCFTOOLS software is used for screening the SNPs at the whole genome level, and the screening conditions are as follows: minimum Allele Frequency (MAF) >0.05, deletion genotype (MG) < 0.2.

And 3-4, obtaining the SNP marker which is obviously related to the stomatal morphology and the photosynthetic efficiency.

Preferably, a Mixed Linear Model (MLM) in Efficient Mixed-Model Association Edxpedicted (EMMAX) software is used to perform Association analysis on the whole genome high-quality SNPs collection and poplar phenotypic traits, so as to obtain SNP sites significantly associated with stomatal morphology (stomatal width, stomatal aspect ratio) and photosynthetic efficiency.

And 3-5, carrying out selective clearance analysis on the obtained significantly associated SNP markers.

The selective elimination refers to a phenomenon that in the natural selection or artificial selection process, the frequency of some dominant alleles in a population is increased, and polymorphism of a surrounding linked chromosome region is reduced due to a car-carrying effect.

Preferably, population differentiation coefficients (Fst) and nucleotide diversity indices (pi) of three climatic region sub-populations of poplar are calculated by sliding window algorithm using a sliding window of 2kb and a step of 1kb on each chromosome using VCFTOOLS software to detect selective regions, respectively. After removing the window with pi <1E-03, the window with the most significant pi and Fst values of the first 5 percent is taken as the most significant selective clearing area and combined with a single scanning area with the interval distance less than 5kb, and the gene overlapped with the scanning area is defined as the selective clearing gene.

More preferably, it is determined whether the SNP marker significantly associated with stomatal morphology and photosynthetic efficiency obtained in step 3-4 is located within the selectively cleared region obtained above,

wherein, if the SNP marker is located in a selective clearing region, the marker is a marker subjected to natural selection, has environmental adaptability and can be used for region breeding;

if the SNP marker is not located in the selectively clearing region, the marker is a marker which is not subjected to natural selection, and is not environmentally adaptive and cannot be used for region breeding.

The inventor finds that the molecular marker obtained through double verification of whole genome association analysis and selective elimination has definite function and obvious effect, can be directly applied to breeding practice of genetic improvement of poplar stomata morphology, has environmental adaptability, and is suitable for regional breeding and climatic adaptation breeding.

According to the embodiment of the invention, the SNP markers, which are remarkably related to the pore width, the pore aspect ratio and the photosynthetic efficiency of the populus tomentosa, are also positioned in a selective clearance area while being related to a target trait, and are subjected to remarkable natural selection. The selected gene is shown to possibly determine the variation of the population stomata morphology of different habitats, so that the difference of the allele frequency of the SNP in three sub-populations is caused, the photosynthetic capacity of the tree is improved to the maximum extent to respond to the change of the environment, and the molecular marker provided by the invention can be used for regional breeding.

In a third aspect of the present invention, there is provided a primer pair for amplifying the molecular marker related to poplar stomatal morphology and photosynthetic efficiency of the first aspect, wherein the primer pair comprises a primer P1 and a primer P2,

wherein, the primer P1 has the sequence shown in SEQ ID NO: 3, primer P2 has the nucleotide sequence shown as SEQ ID NO: 4.

In the present invention, in SEQ ID NO: 3 and SEQ ID NO: 4 by adding 1-20 bases to the 5 'end and the 3' end respectively and obtaining primer pairs with basically the same DNA fragments (the DNA sequences between the upstream primer and the downstream primer are the same), and the primer pairs are all included in the primer pairs.

Preferably, the nucleotide sequence of the primer P1 is shown as SEQ ID NO: 3, the nucleotide sequence of the primer P2 is shown as SEQ ID NO: 4, respectively.

The inventor researches and discovers that the pore width, the pore aspect ratio and the photosynthetic efficiency can be effectively determined by detecting whether the genomic DNA of the poplar individual has the molecular marker. Specifically, as described above, the poplar individual whose genotype at the SNP site is CC has a large stomatal width, stomatal length-width ratio, and high photosynthetic efficiency; the poplar individual with the genotype TT at the SNP locus has smaller pore width, pore length-width ratio and lower photosynthetic efficiency. Therefore, when the molecular marker of the poplar individual to be detected is detected to be the CC genotype, the width and the length-width ratio of the air hole can be determined to be larger, and the photosynthetic efficiency is higher; when the molecular marker of the poplar individual to be detected is TT genotype, the width, length and width of the air hole can be determined to be small, and the photosynthetic efficiency is low.

In the invention, the primer is adopted to carry out PCR amplification on the nucleotide fragment of the poplar to be detected, wherein the nucleotide fragment is the molecular marker related to stomatal morphology and photosynthetic efficiency, and further, the detection of the molecular marker can be effectively realized by methods such as direct sequencing and the like, so that whether the poplar to be detected has the molecular marker is determined.

Therefore, the primer pair provided by the invention can be effectively used for poplar molecular marker assisted breeding, so that the method can assist in early screening of excellent tree species with high stomatal width, stomatal length-width ratio and photosynthetic efficiency in a short time at low cost and high accuracy, and the screened tree species can improve photosynthetic capacity to the maximum extent under different habitats.

In a fourth aspect of the present invention, there is provided a molecular marker according to the first aspect, a molecular marker obtained by the method according to the second aspect, or a primer pair according to the third aspect, wherein the molecular marker is used in any one of the following aspects (1) to (4):

(1) screening or auxiliary screening of a new poplar variety with high air hole width, air hole length-width ratio and photosynthetic efficiency; (2) molecular marker assisted breeding of poplar; (3) preparing and screening or auxiliary screening products of new poplar varieties with high air hole width, air hole length-width ratio and photosynthetic efficiency; (4) preparing a product of poplar molecular marker assisted breeding.

In the fifth aspect of the invention, a poplar genetic improvement method is provided, the method comprises the steps of subculturing and breeding poplar individuals of CC genotype of the molecular marker in the first aspect, and eliminating all other genotype individuals of the molecular marker.

Preferably, the method for genetic improvement of poplar comprises the following steps:

step i, extracting genomic DNA of poplar;

step ii, using poplar genome DNA as a template to perform PCR amplification;

step iii, determining the genotype of the molecular marker of the poplar to be detected;

step iv, determining the stomatal morphology and the photosynthetic efficiency of the poplar to be detected according to the genotype detection result;

and v, subculturing and breeding the CC genotype individuals of the molecular markers.

The sixth aspect of the present invention provides a system for predicting stomatal morphology and photosynthetic efficiency of poplar, comprising an amplification subsystem, a genotype identification subsystem and a prediction subsystem connected in sequence, wherein,

the amplification subsystem is used for amplifying the genomic DNA of the poplar to be detected;

a genotype identification subsystem, which is used for determining the genotype of the molecular marker of the first aspect of the poplar to be detected;

and the prediction subsystem is used for predicting the stomatal morphology and the photosynthetic efficiency of the poplar according to the genotype of the molecular marker.

Wherein, preferably, the prediction subsystem is used for predicting the air pore width, the air pore length-width ratio and the photosynthetic efficiency of the Chinese white poplar.

In the present invention, the primer pair of the third aspect is preferably used for amplification, and the features and advantages described above for the molecular marker related to the stomatal morphology and photosynthetic efficiency of poplar and the primer pair for amplifying the molecular marker are also applicable to the system for predicting the stomatal morphology and photosynthetic efficiency of poplar, and are not repeated herein.

Examples

The present invention is further described below by way of specific examples, which are merely exemplary and do not limit the scope of the present invention in any way.

Example 1 acquisition of molecular markers associated with Populus tomentosa pore width, pore aspect ratio, and photosynthetic efficiency

Step 1, 209 individuals from three habitability areas of populus tomentosa constitute a test population, wherein the population comprises 69 individuals in northwest climate areas (NW), 72 individuals in northeast climate areas (NE) and 68 individuals in south climate areas (S), and the specific division standard of the climate areas is shown in fig. 1;

the photosynthetic efficiency (photosynthetic rate) of the above test population was determined using LI6400 portable photosynthesis system (LI-COR inc., Lincoln, NE, USA);

collecting 3 functional leaves of each individual of a poplar group for determination, cleaning cilia on the back of each poplar leaf by using an adhesive tape, uniformly coating nail polish on the leaf back, tearing down the poplar leaf after air drying, observing the poplar leaf under a microscope, selecting three non-overlapping areas for taking a picture for each sample, and extracting the picture phenotype by using ImageJ software to obtain the numerical values of the air hole width and the air hole length-width ratio of each individual.

Step 2, extracting the genome DNA of 209 individual leaf tissues of a test population by using a DNeasy Plant Mini Kit (Qiagen China, Shanghai, China) Kit;

and (3) detecting the DNA quality by adopting an ultraviolet spectrophotometer and gel electrophoresis, and storing the qualified DNA at the temperature of-20 ℃.

And 3, obtaining molecular markers related to the air pore width, the air pore length-width ratio and the photosynthetic efficiency of the poplar by adopting a selective elimination and whole genome correlation analysis method:

3-1, performing re-sequencing on DNA of each individual in the populus tomentosa test population, and performing quality control on the obtained original data:

the DNA re-sequencing is double-end sequencing, the sequencing depth is 30 x, and an Illumina GA2 re-sequencing platform is adopted for sequencing;

the quality control criteria are: (i) reads (reads) containing > 10% unidentified nucleotides are removed; (ii) removing reads with the base quality less than 5 and the quantity more than 50%; (iii) reads with more than 10nt compared to the adapter (adapter) are removed, and mismatch less than or equal to 10 percent is allowed; (iv) putative PCR repeats (two identical paired reads 1 and 2) generated by PCR amplification during library construction were deleted.

Step 3-2, identifying the single nucleotide polymorphism sites and the genotypes thereof at the whole genome level by using the data after quality control:

aligning cleardata (data after quality control) of each sample to a poplar reference genome v3.0(http:// popgynie. org /) using Burrows-Wheeleralignerv0.7.5a-r405 (default parameters); filtering out low quality reads (MQ < 20) using SAMtolsvv 1.1; genome wide Single Nucleotide Polymorphism Sites (SNPs) were identified using Genome Analysis Toolkit (GATK) v4.0, with the following parameters: SNP, QD <5.0| | | MQ <40.0| | | FS >60.0| | SOR >3.0| | MQRankSum < -12.5| | ReadPosRankSum < -8.0; QD <5.0| | FS >200.0| | SOR >10.0| | MQRankSum < -12.5| | ReadPosRankSum < -8.0; obtaining biallelic SNPs by using Vcftools _ 0.1.13;

finally, a total of 13,063,406 SNPs with Minimum Allele Frequency (MAF) >0.05 and deletion Genotype (MG) <0.2 were obtained.

Step 3-3, screening the SNPs in the whole genome level to obtain a high-quality SNPs label set:

preferably, the whole genome level SNPs are screened by VCFTOOLS software under the following conditions: minimum Allele Frequency (MAF)>0.05, deletion genotype (Missinggenotype, MG)<0.2; linkage disequilibrium (r)²)<0.2, heterozygosity frequency<95 percent; group structure analysis is carried out by Admixure v1.3.0 software by utilizing the SNP set; the K value ranges from 1 to 10, and the minimum cross validation error value is obtained when K equals 3.

And 3-4, obtaining SNP markers which are obviously related to stomatal morphology and photosynthetic efficiency:

performing Association analysis on a whole-genome high-quality SNPs set and Populus tomentosa phenotypic characters by using a Mixed Linear Model (MLM) in Efficient Mixed-Model Association EXpeded (EMMAX) software to obtain SNP sites which are obviously associated with stomatal width, stomatal aspect ratio and photosynthetic efficiency;

through the screening, the SNP locus at 3140 th base downstream of ZCWCC3 gene is found to be obviously related to the pore width, pore length-width ratio and photosynthetic efficiency of populus tomentosa, and specifically, the SNP locus is located in a position shown in SEQ ID NO: 1 at position 7897 of the nucleotide sequence set forth in seq id no.

And 3-5, carrying out selective clearance analysis on the obtained significantly associated SNP markers:

the population differentiation coefficient (Fst) and nucleotide diversity index (pi) of three climatic region sub-populations of the poplar are respectively calculated by using VCFTOOLS software through a sliding window algorithm. After removing the window with pi <1E-03, the window with the most significant pi and Fst values of the first 5 percent is taken as the most significant selective clearing area and combined with a single scanning area with the interval distance less than 5kb, and the gene overlapped with the scanning area is defined as the selective clearing gene.

By the method of combining whole genome association analysis and selective elimination, the SNP site located at 3140 th base downstream of the zcwccc 3 gene was determined to be located in the selective elimination region while being associated with the target traits (stomatal width, stomatal aspect ratio, and photosynthetic efficiency), and thus, the method was significantly naturally selected.

Wherein the genotypes, the pore widths, the pore aspect ratios and the photosynthetic efficiency values of the SNP sites of the populus tomentosa in the three subgroups are shown in Table 1 and FIGS. 2 to 4:

TABLE 1

As can be seen from Table 1, the genomic DNA of Populus tomentosa individuals from the southern climate zone (S) subpopulation encoded the gene at the 3140 th base of ZCWCCC 3, which showed the largest stomata width and stomata aspect ratio and the highest photosynthetic efficiency, the stomata width (4.01 + -0.73) and stomata aspect ratio (3.68 + -0.63) of the candidate individuals in this subpopulation were 10.92% and 11.48% higher than the overall level (3.62 + -0.71; 3.43 + -0.57), and the photosynthetic rate (18.36 + -4.64) was 7.38% higher than the overall level (17.10 + -3.54); the genotype of the 3140 th base downstream of the gene ZCWCC3 encoded by the genomic DNA of an individual Chinese white poplar from a northwest climate zone (NW) sub-population is TT, the stomatal width and the stomatal aspect ratio are the smallest, the photosynthetic efficiency is the lowest, the stomatal width (2.89 +/-0.71) and the stomatal aspect ratio (2.66 +/-0.54) of the candidate individuals in the population are 20.06 percent and 19.38 percent lower than the overall level (3.62 +/-0.71; 3.43 +/-0.57), and the photosynthetic rate (15.77 +/-2.9) is 7.79 percent lower than the overall level (17.10 +/-3.54); the genotype of 3140 th base downstream of ZCWCC3 gene encoded by genomic DNA of individual populus tomentosa from the northeast climate zone (NE) subpopulation is CT, and the stomatal width, stomatal aspect ratio and photosynthetic rate (3.95 + -0.69; 3.95 + -0.55; 17.17 + -3.09) are moderate compared with the overall level (3.62 + -0.71; 3.43 + -0.57; 17.10 + -3.54).

Furthermore, the 3140 th base upstream of ZCWCC3 gene in the above three subgroups of Chinese white poplar was genotyped, and the distribution results of allele and genotype frequencies are shown in Table 2.

Table 2:

as can be seen from table 2, three genotypes were detected at the above SNP sites, TT, CT and CC, respectively, where CC is the dominant genotype, mainly concentrated in the southern climate zone sub-population, and the frequency of CC genotype of this sub-population is significantly higher than those of the northwest climate zone sub-population (0.07) and the northeast climate zone sub-population (0.14).

Example 2 validation of molecular markers

Randomly selecting 3 groups of Chinese white poplar individuals carrying different genotype combinations from 2-year-old Chinese white poplar germplasm resource groups (300 plants, 300 nurseries in Shandong Guangxi county), wherein 10 individuals in northwest climate region, northeast climate region and south climate region are respectively marked as vNW, vNE and vS, carrying out tissue culture by collecting leaves, obtaining corresponding tissue culture seedlings after 2 months, and measuring the stomatal width, stomatal aspect ratio and photosynthetic efficiency of the tissue culture seedlings, wherein the results are shown in Table 3.

TABLE 3

As can be seen from table 3, in the vNW subgroup, 10 populus tomentosa individuals randomly selected mainly included TT genotype (CC: CT: TT ═ 0:1:4), the stomatal width and stomatal aspect ratio of the corresponding tissue culture seedling samples were minimal, and the photosynthetic efficiency (photosynthetic rate) was minimal, which is reflected in that the stomatal aspect ratio and the stomatal width of the candidate random individual were 22.07% and 26.56% lower than the overall level, and the net photosynthetic rate of the candidate random individual was 6.46% lower than the overall level; in the vS subgroup, the CC genotype is mainly included (CC: CT: TT ═ 0:0:10), the stomatal width and stomatal aspect ratio of the corresponding tissue culture seedling sample are the largest, the photosynthetic efficiency (photosynthetic rate) is the highest, the stomatal aspect ratio and the stomatal width of the candidate random individual are 14.96% and 23.12% higher than the whole level, and the net photosynthetic rate of the candidate random individual is 4.88% higher than the whole level; in the vNE subgroup, mainly including the CT genotype (CC: CT: TT ═ 1:6:3), the stomatal width, stomatal aspect ratio and photosynthetic efficiency of the corresponding tissue culture seedling samples were moderate.

The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention.

SEQUENCE LISTING

<110> Beijing university of forestry

<120> molecular marker related to poplar stomatal morphology and photosynthetic efficiency and application thereof

<130> 2021

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 8256

<212> DNA

<213> nucleotide sequence (Populus tominosa)

<400> 1

aactaacaaa tacttttttt ttttctttca aaatggcaat gcccaaaaac ccaataatca 60

tggaatttaa ctgaatcaat aaaacgatgt cgttgctact ttctcctcgg aaataacact 120

tcccttaacc cctacaagaa ctgctaaacc acagaagctc acaaaaaatc acaggaactt 180

ccactgcaaa agaagcttca atggcatcct tgttagtctc catcagtttc cctatgcctc 240

tgtccgcccc taaattctca ttcaaggtaa aaaaatcatt catgcccatc ttcttgtaag 300

cgacaattgt tatatttaac ttatccttct tgaatttctt tgtcatttta tttcattgag 360

tgatccaaga attgaacttt cttaattgaa aggttcttta tttgagcacc gtgaattctt 420

gttgctattc ttatgaagct gagtaaataa tttggtgggt tttgtttttt aaaagagtgg 480

tttttttcat aaaggaatga actttatgga gtaaagtagt gctagagatg gtatagtgag 540

gttgggatgt ttataaattt gtggggtaag agttgtttta tagtttgatt cagttggtgt 600

tttggatgtg atttaggaaa attatgggtt gtcatcttgt aggattagga gtttggtcgt 660

ccgtttgttt cctttaaagg gttctgtgta tgatatggat atgttgtctg aaagtttaga 720

gagactgttt ttttcggcaa aaaaattgat ggcttttttg aaggatacta tgaatgcgta 780

ttgtgtgaca agagatgagt tattgtagat taatattccc atcaaattct tggaatataa 840

tgttgctaac cattgtatac aagtattagt tagttgttaa atgagaaacc agcattgaaa 900

gtgaacaatg tgatcatatt tcctttgagc tacattatga tagggagaga tcttgtagaa 960

tagtccatat acagacaact acttgatggt tcattgctgc acgaaagtgt agagaagata 1020

aatgttttca ctttagaagt caattaaaag atttgagatt gaacaacttt tggcatggaa 1080

ttgtgtaaaa ttgtagtttt gttatagaag ctgactgggt ggattaatca cacagtctgt 1140

gtttttaaat tgtttccacc tattggcccg catgaaaata aattttcaat tctcttgttg 1200

aaaactaata acgagttcct cttacagttc tggggtaaat gactatttta cttctgttct 1260

ttaattatga taagatcata tcgaatgtaa gaaactagac atgaaatttg aagttcacgt 1320

ttgtcttaaa gtttggtttg aaccaatagt caacatcata ggttaattca tagaaatgct 1380

gactttttgt atatatctgg ttgcatgttc aagactgtta ctgcttgcaa aaatgagcct 1440

atgtcaatgt agagttgatg tttcatggtg atttagattc ttcctgccaa ccactagtaa 1500

tggtttatgc gtaaaatcca tcacttacca catcatgagt tggtaacctg tccccaacta 1560

ttattatttt gtattatcac gttgtgctcc ccaagttctt gacttgagtt aataagtttg 1620

tgttttgatt tgggactgtt ttgtgactgg tttctcagga attacaacta aggaaatcag 1680

ctgttactag actttcaggc caagcaacat ctgggacagg tatcttaatt tcacgccaac 1740

taccaaaaaa gagttgaagc aagatgattt atcatattca gttgatagtt tttaccattt 1800

ttgcctgtga tgggaacata agcttgtgaa aataagtaac tgttatagca taacatgttg 1860

agttgaatta gaagtgcacc atggattgtg ggtttgtaag agttaaatta tgtaatgaaa 1920

acttctattt aaagtcaaaa aagaaaagta aaggaaaagg gagaagttca ctggtaatgc 1980

taacagataa tgcataagaa acgtgaagac ctgccttcta cttgctctct tatatgactt 2040

caaattttgt ttacagcaac gaaccttttg gtcccttgca acgcaactgg agagatgctt 2100

tctgtgaatc agagctgcgg tggatgtcta gcaactccca ccaaccattt ttaccgactt 2160

gtttctagtt gtatgcattt aagctcttgt actcacaaaa taataactgg ttattgggtg 2220

gggccagata tagatgatgg ctggggattt gtagaaggtt tcgttaatca aattacttga 2280

ttttatttta tcatgaatga tctgactggc tcatttcatt tttcttgtac tgtagacttt 2340

gatgttgaag tgaatatcag aaatatcgat cgatcagaga atgatgaatt tctttttata 2400

attggaccaa agtaaaataa taatgaaatc tttagttttc cttgataaac attttctatc 2460

atatatatga gctcctctga aatgttagga tgatttcttg gaaggagcca tggacttcta 2520

gagaaagaac aaagcttgct tacacaccta ccatgatatt atgttttatg ctcttggtag 2580

agtcgatggt ggagggtttt aacgaaattc attttgaagt gattatagct atttgtttca 2640

gtcagagcta cgttataaca gcatcatttt cttagcactt atattgcaga agtgttggca 2700

tgtcatgctg tgcatggaat aatttatgtt tatgcatacc cattgtcatt gctgaatggc 2760

attggtgcat tttggcttgc atcaaattag ttcctggtac cgttgaggat taaggtgagg 2820

actttttttc aaacttgaaa gaccaatgca gctcttgaat tttggtgtct cagggctcgt 2880

aacaatttgc tgcgcttgct tatggcaagc tgctgcgttg caaatggaca acccatgatt 2940

ctattaccat ctatcctgta aaattggtgc acaaaacgtg gtccaatgct tgttctgttt 3000

agtttaattc ggatcctatg ttgtcgagtg ctctctagcg agcaatgcaa gcaaccagat 3060

tatgaaaccc aatgccgaga ctcatggtca aagcccgtgg aagcaacttt agttgaaatc 3120

attctttcaa gaatgccgta attagttgcc taatattgca ttgacatagt tgatcctaga 3180

gtctcgaggt tatcagataa ttcattctat caagaaaacc cttaatggtg ttatttgcat 3240

cggtatagtt ggtacttaga acgttttcaa ttagtttcag cttttgtctc tcactttgtc 3300

tcatatcatg tatgatttct ctcattttgt tgggaaacaa ttttaagtgg gtctttgcat 3360

tcctctcccc cttttctatt aggcacaact aagattccat cttttgtcaa aacagatgat 3420

agttgagaag tggtcccaaa gagctcagtg aagctcttgg tggtatgttg aagcttgaaa 3480

cctaactcaa tttcaattct aacattaatt tatcagggtt ctatcatcat ctttttacgg 3540

ggagattacc aatccatttg ctgatatagt taaaaataag catgcgtcag attttgattg 3600

gattttctcg acaaaacatg ctatgtaaag ccgcaatagt agtggtcccg attctagggt 3660

tatgcaaact ctgaatctag tactcaagta atctttttta gccctaatta gttgactaat 3720

taattatgca tctggtaact tcacttgctt tcgagaggga agaatcagaa caacgaaggt 3780

tcagctacat ccaggtgtcc atttagatat gttgtgtagc tctagcattt tcaggtatgt 3840

atttgctggt tggacttgct taaatggtca cggagagccc cggggggcat gcaagagtgc 3900

tcaaagaacc aggtggtgta cgtttgacgc ctattgctgg ggaacccttg aaatgcacgt 3960

actctccacg taaccaatta ctgatgaatg aatttgcctc ataggttggc atgcttcatt 4020

agcatgattt aacatcaggg tttacataaa tatgtgaaca aataatgaac attactggtt 4080

ttagcaagct gctgttgctg tcttttggtc tggttcagag accgttcttg agtttttgga 4140

tggcttcgtc aattatcaag tctttcctct ctaagaacct tcagttcctt tgttcaattc 4200

aatttgagtt atcaaaataa caaagttatt gtcaatgtgt tatagtgatt gatcagaaaa 4260

ttcatcacaa gttctgagca ggagcagtac tgttcgtcat ccttataaga gcgtgagcgt 4320

gcgtgtgcaa gagagagaga gataagagaa acgtctgaac tgtaaagacg aaaagaagaa 4380

acaaaatcat tttgattaat ggtaaagggt tgagaagaaa atttagattc caatatattc 4440

acactgaaac taaagttttt ccagcaatct gatgcaccca acttcctttc tgaaatctat 4500

gaacaggaaa ctagagggtt cttactcaaa agcaacaaca aaaaggttct attatagctt 4560

gagcattgct tacagtacaa atttcaaaga tctttcttgc gaagctcgta tttctcaaag 4620

ttatgaacaa gaagctaact tgtcatatgt gtactctaat cacaggcctc aagctagaaa 4680

ttatgagaaa atgtcagaca ccaaaaactt tgctacttaa gtgaagtatg cgattcgttg 4740

catagctaaa attatacaga tttcccccct tcactgtaaa ggagaagtta agagataaga 4800

cgagaagaaa ccctaagctc aggagaaata tatatcaaat attacctcag attatacaga 4860

tttgtcaaag cttttttaag tcaaattaaa acttcaaata tcaaaccttg tcaagagatc 4920

atcaagcagg taaatctcca aaatttgtct tttttgtaca tcacggtctt catgttcttc 4980

ttcttctcga tcttcttctt cattacttca tcatcaagag tggacattat ttctcaggaa 5040

ggatttgtgg aagagctgct gttgccctta tcaagaaccc tagattctac tccgttatta 5100

ggagcttcaa ttggctgctt actttcatgg ttggattctt caccttggtc agtgccagaa 5160

gctatatgtt ggttagtgga tctctctctt tcagcctctc tatatttatg caagtacctt 5220

accatagcct ctgcatggtc gtcaaaccct agagaaatga gagcccaaca gatgtcatct 5280

ccgttcactg tcttgcggtt ctccttctga cacttatcag atgcctcgcc agttacaaaa 5340

cttataaatt ctgtcgcgca ttcttgcatg gtttgttttg cttctttgga gatcttggca 5400

gttggtggca ggattttctt catcatccga cccacattgg caacaggcaa caatctatct 5460

tgctcatcat ccatttcttc ttaataagaa attgctcttt gctttgcaaa ctctcttgtt 5520

aatagttgcc taaatatatg tgtgtgtatg catgtgtatg tgtatgtgtc tccacacggt 5580

atgcacatgg atgcatatcg atacatataa caagttgttg aattatatag aaaagaatca 5640

tgttgcagat gatttgacaa aaaaaaaaga ttttaaaaag ttgatttagt tgtctggtaa 5700

cgctatagtt actttaattt attcaggatt ttttatactt gattttgatt gtgtgtttag 5760

ttttgttagt aagttttttt atattctact gattgttaag tcatcaattg attaatatat 5820

ctcttgttat taataaataa acaaattgaa ttaatgattg tcactaatac aaaagacaag 5880

attaaataaa aaagattgtt agagtaacta ataccaaaga ctatgatttc ttaaatttat 5940

tttaaaatta aatgacgact cacgattctc gatattaaat atataaatat tcagtgtcac 6000

gtaatatatt gagatataat attaatgtta tatttaatac agttaaaatg accattcata 6060

attattgaga ttttttattg aaatttgaaa tcaatgcttt gttattataa agttataaaa 6120

atcaaaatag gttgcttcac caatggaatt ccaagaaaat ataatggatg tgttattaat 6180

tcaaattgca taacctaatt ctaatttgat tgaaaaggaa gaaaagaaat tagaacacaa 6240

taaaaagaaa caaaaaaaca ccgttgagct cttcatgagt ttgatattgt gatagatttt 6300

ataattatta ttttaaaaaa atataaatta tatctttttc gtaatcaaaa tttaaagaaa 6360

gaatttttaa tagagctcat gtaaaactat aatgcatatt aattaggtaa acatcttcaa 6420

aattataatt aaaacaaaat acaattttaa tcgcatctct ttacactaca agaaaaaaga 6480

tgaggattag tttggtcaac ctaaatatta ataatcctag taacttagct agttccccag 6540

ctgcaaagct gattaggtat tggtctccca tacagcacca ccttttttta aaatatccag 6600

ataatcggca gccctttctg cttgcagtgt attaggactg ttcatataat actattttct 6660

acaaattgaa atgagtggac tccgtttgtg cacgtttgca tgttttggga ttaacctaag 6720

caatacctaa aaagaactga ttttaagcac tagcttgcaa gtagcctctg atttacctca 6780

agcttaatta ggctagattt taggtttctc tctatacggc atcagagcat ttgcatcatg 6840

agtataatga aacagtttgg ttggacatac tgctagtggg aactcaaaaa ctttaactat 6900

tgattatagc aagaggcaag atcagttaat ttaattactt gtagtgaact gaaattaggt 6960

tttcaaaggg attctttagt gatcaaaact gttttttttt tttttttttg aagtttgatg 7020

atcaatgcca aatcttgaaa tgggctattt tttttttttt gtttagattt atgtattaga 7080

aactaatata aattattttc tgagatctca cctaaccgtt tatgttttta ggttgaattg 7140

attttttaat atggtatccg agacttgatg atcaagcggt catgaatttg aatcttatta 7200

tctttattta tttgataaaa tattaatcac aagcttttag gttgagataa ttttttaata 7260

ttataccttt aaatagttca atgatgcata ttctcttctt ttagtggcta ttttgtgcca 7320

tgcttagttg aattttgtta gtaaataatt ttttaggcat aataatctat gaatcgaatc 7380

aagctcattt ctttttaggc atatcctcgt ttcttatatt aggtataatc gtacgataga 7440

tgataaacta ttcaacagag gttgcactta atattactaa cttttgaacg tgctggtcta 7500

atttataatt aggagcaata ttggtggggc cagtaatcta gatttttggt atgctatatc 7560

tttgaataaa acctagtacg aggttattta caatttaaac aaattaatat ttaagtagct 7620

cgtggttatt atctcgataa taaaaagaaa ttcattttaa aattatttaa aaaatagatt 7680

tatcttatgc atttatcctt tcctctttaa accaaacatg ttttcgtctt ttctatatcc 7740

gtccctccag tattgagacg ctaaccaaaa ttttaaaact tagtttcttt cacattcttg 7800

taatctgatt tgattatcat ccattagcac taattaatgc cttatatata gcaaagtatt 7860

aacattgggg tcagccattg atatatagtc cgaattcaac taactcatga ttgttaaaat 7920

atgttagctt tacactttaa tcattatttt ctgcgaactt taaacaatct tcatgtatag 7980

agcttcgaaa aaacctcttg ttgtgtagaa aacatctcat gcatactttc tataggtata 8040

taagcttaaa atgtcttcca tattctatct acttacattg ctgagatttg ctttatgcat 8100

cattatttca ggttttaatc aaaagggttg gctgaaaaaa agacattttt catgaatata 8160

atggatgacg actagcttaa aaacggacaa aatgagatca acttaattta tgtgtgaaag 8220

ataacatggg aataaatgcc ttaattgtta aagaaa 8256

<210> 2

<211> 116

<212> PRT

<213> amino acid sequence (Populus tominosa)

<400> 2

Met Ala Ser Leu Leu Val Ser Ile Ser Phe Pro Met Pro Leu Ser Ala

1 5 10 15

Pro Lys Phe Ser Phe Lys Glu Leu Gln Leu Arg Lys Ser Ala Val Thr

20 25 30

Arg Leu Ser Gly Gln Ala Thr Ser Gly Thr Ala Thr Asn Leu Leu Val

35 40 45

Pro Cys Asn Ala Thr Gly Glu Ile Leu Ser Val Asn Gln Ser Cys Gly

50 55 60

Gly Cys Leu Ala Thr Pro Thr Asn His Phe Tyr Arg Leu Val Ser Ser

65 70 75 80

Cys Met His Val Ser Ser Cys Thr His Lys Ile Ile Thr Gly Tyr Trp

85 90 95

Val Gly Pro Asp Ile Asp Asp Gly Trp Gly Phe Val Glu Gly Phe Val

100 105 110

Asn Gln Ile Thr

115

<210> 3

<211> 38

<212> DNA

<213> primer P1 (Artificial sequence)

<400> 3

agaacacggg ggactatggc atccttgtta gtctccat 38

<210> 4

<211> 40

<212> DNA

<213> primer P2 (Artificial sequence)

<400> 4

acccccgggg atcctagtaa tttgattaac gaaaccttct 40

Claims (10)

1. A molecular marker related to the stomata morphology and the photosynthetic efficiency of a poplar is characterized in that the molecular marker is an SNP marker, is positioned at 3140 th base downstream of a ZCWCCC 3 gene of a poplar genome, and has C/T polymorphism.

2. The molecular marker related to poplar stomatal morphology and photosynthetic efficiency according to claim 1, characterized in that the molecular marker is located in the nucleotide sequence as shown in SEQ ID NO: 1 at position 7897 of the nucleotide sequence set forth in seq id no.

3. The molecular marker related to poplar stomatal morphology, photosynthetic efficiency according to claim 1 characterized in that the stomatal morphology comprises stomatal width and stomatal aspect ratio.

4. The molecular marker related to stomatal morphology and photosynthetic efficiency of poplar according to claim 1, wherein the genotype of the molecular marker is CC, TT and CT, wherein,

the molecular marker is a poplar individual with CC genotype and has larger pore width, pore length-width ratio and higher photosynthetic efficiency;

the poplar individual with TT as the genotype of the molecular marker has smaller pore width, pore length-width ratio and lower photosynthetic efficiency.

5. A method for obtaining the molecular marker of any one of claims 1 to 4, wherein the method comprises the following steps:

step 1, determining the phenotypic character of a poplar group;

step 2, extracting genome DNA of the poplar group;

and 3, obtaining the molecular marker which is obviously related to the phenotypic character.

6. The method according to claim 5, characterized in that step 3 comprises the following sub-steps:

3-1, performing re-sequencing on DNA of each individual in the poplar group, and performing quality control on the obtained original data;

3-2, identifying the single nucleotide polymorphism sites and the genotypes thereof at the whole genome level by using the data after quality control;

3-3, screening the SNPs in the whole genome level to obtain a high-quality SNPs label set;

3-4, obtaining SNP markers which are obviously related to stomatal morphology and photosynthetic efficiency;

and 3-5, carrying out selective clearance analysis on the obtained significantly associated SNP markers.

7. Primer pair for amplifying molecular markers related to stomatal morphology and photosynthetic efficiency of poplar according to one of claims 1 to 4, characterized in that the primer pair comprises primer P1 and primer P2,

wherein, the primer P1 has the sequence shown in SEQ ID NO: 3, primer P2 has the nucleotide sequence shown as SEQ ID NO: 4.

8. Use of the molecular marker of any one of claims 1 to 4, the molecular marker obtained by the method of claim 5 or 6, or the primer pair of claim 7 in any one of the following aspects (1) to (4):

(1) screening or auxiliary screening of a new poplar variety with high air hole width, air hole length-width ratio and photosynthetic efficiency; (2) molecular marker assisted breeding of poplar; (3) preparing and screening or auxiliary screening products of new poplar varieties with high air hole width, air hole length-width ratio and photosynthetic efficiency; (4) preparing a product of poplar molecular marker assisted breeding.

9. A method for genetic improvement of poplar, comprising the steps of subculturing an individual poplar with CC genotype of the molecular marker according to any one of claims 1 to 4, and eliminating all other genotype individuals of the molecular marker;

preferably, the method for genetic improvement of poplar comprises the following steps:

step i, extracting genomic DNA of poplar;

step ii, using poplar genome DNA as a template to perform PCR amplification;

step iii, determining the genotype of the molecular marker of the poplar to be detected;

step iv, determining the stomatal morphology and the photosynthetic efficiency of the poplar to be detected according to the genotype detection result;

and v, subculturing and breeding the CC genotype individuals of the molecular markers.

10. A system for predicting stomatal morphology and photosynthetic efficiency of poplar is characterized by comprising an amplification subsystem, a genotype identification subsystem and a prediction subsystem which are connected in sequence, wherein,

the amplification subsystem is used for amplifying the genomic DNA of the poplar to be detected;

a genotype identification subsystem for determining the genotype of the molecular marker of one of claims 1 to 4 of the poplar to be tested;

and the prediction subsystem is used for predicting the stomatal morphology and the photosynthetic efficiency of the poplar according to the genotype of the molecular marker.

Images