CN109762924B - Molecular marker for salt tolerance in jute and application thereof - Google Patents
Molecular marker for salt tolerance in jute and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of plant breeding, in particular to a molecular marker for salt tolerance in jute and application thereof. The SNP site exists in the genome of the jute and is linked with QTL influencing the salt tolerance character of the jute, so that the SNP site can be used for the related research of the salt tolerance character of the jute. The SNP locus provided by the invention can be applied to the cultivation of jute salt-tolerant varieties and can also be used for the research on the aspects of population genetic structures, genetic variation levels and the like.
Description
Technical Field
The invention relates to the technical field of plant breeding, in particular to a molecular marker for salt tolerance in jute and application thereof.
Background
Soil salinization has evolved into a global problem, is one of the important factors leading to land desertification and arable land degradation, and has also become a major crisis faced by mankind. The problem of soil salinization seriously affects the growth and development of plants, and has become one of the major abiotic stresses in nature. At present, the area of the global saline-alkali soil reaches 9.5 hundred million hm & lt 2 & gt. Soil salinization has become an important factor that restricts grain production and affects grain safety. However, the contradiction between the rapid population growth and the shortage of food is increasingly aggravated, and the method for improving and utilizing the saline-alkali soil by screening and cultivating new varieties of salt-tolerant crops is considered to be a solution with economic and ecological benefits.
Jute is a Tiliaceae (Tiliaceae), jute (Corchorus) plant, annual herbaceous bast fibre crop, and there are two main types of jute mainly cultivated in the world at present, namely Corchorus rotundifolia (Corchorus, capsularis, white jute in india, bangladesh, nipaler and the like in south asia) and Corchorus longissima (Corchorus, olitorius, south asia is tossa jute).
Jute is one of the most important bast fiber crops in the world, has the characteristics of good fiber hygroscopicity, quick water dispersion, antibacterial and bacteriostatic properties, degradability, environmental friendliness and the like, is commercially attractive as golden fiber, is an important raw material in the bast fiber spinning industry, and is second only to cotton in yield and planting area in the world. Jute is also an important salt-tolerant economic crop and can be planted in large areas of coastal beaches and saline-alkali lands.
At present, the research on the salt tolerance of jute is slow, people still know the salt tolerance of jute in the initial stage, reports about the molecular mechanism of the salt tolerance of jute are less, and the previous reports on the salt tolerance of jute are relatively less. The biological basis of jute molecules is weaker overall. Analyzing the gene expression change of jute under the condition of salt stress, searching jute salt stress response genes, and carrying out function prediction and metabolic pathway analysis on the jute salt stress response genes to deepen the research on jute salt tolerance mechanism, which is favorable for cultivating excellent salt-tolerant varieties of jute and has important significance on the research on the salt tolerance of other plants.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention relates to a novel SNP site related to the salt tolerance of jute, and researches on the aspects of detection, application and the like of the SNP site.
The SNP locus exists in a jute genome and is linked with QTL influencing the salt tolerance character of jute, so that the SNP locus can be used for the related research of the salt tolerance character of jute.
The SNP locus can be used for cultivating and identifying the jute plant with salt resistance.
Compared with the prior art, the invention has the following beneficial effects:
the mk6160 SNP, the mk5633 SNP, the mk6484 SNP, the mk6723 SNP, the mk7047 SNP, the mk5945, the mk6330, the mk6391 and the mk6398 provided by the invention can be applied to the cultivation of jute salt-tolerant varieties and can also be used for the research of population genetic structures, genetic variation levels and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of a GBS technology route employed in an embodiment of the present invention;
FIG. 2 is a schematic flow chart of the content of genetic map information analysis in the example of the present invention;
FIG. 3 is a schematic diagram of the GBS library construction scheme in an embodiment of the present invention;
FIG. 4 shows 150F in one embodiment of the present invention 2：3 The STIG data for lines and the four day treatment under two salt stress conditions (FIG. 4A, 140mM salt concentration; FIG. 4B;
FIG. 5 is QTL mapping results of salt tolerance of jute under 140mM (n = 5) salt stress conditions;
FIG. 6 is QTL mapping results for salt tolerance of jute under 160mM (n = 8) salt stress conditions;
FIG. 7 is a partial linkage group tag distribution on LG 4;
FIG. 8 is a partial linkage group tag distribution on LG 4;
FIG. 9 is a partial linkage group marker distribution on LG 4;
FIG. 10 is a partial linkage group tag distribution on LG 4;
FIG. 11 is a partial linkage group tag distribution on LG 4;
FIG. 12 is a partial linkage group marker distribution on LG 4;
FIG. 13 is a partial linkage group tag distribution on LG 4;
FIG. 14 is a partial linkage group marker distribution on LG 4;
FIG. 15 is a partial linkage group marker distribution on LG 4.
Detailed Description
The present invention may be understood more readily by reference to the following description of certain embodiments of the invention and the detailed description of the examples included therein.
Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such embodiments are necessarily varied. It is also to be understood that the terminology used in the description is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Noun definitions
Before setting forth the details of the invention, it should be understood that several terms used in the specification are used.
Agronomically superior: as used herein refers to a genotype that has the best manifestation of many discernible traits such as seed yield, protrusions, germination vigour, nutritional vigour, disease resistance, seed set, stand ability (standability), and degranulation, which allows producers to harvest products of commercial importance.
And (3) hybridization: mating of the two parental plants.
F1 hybrid/F1 generation: the first generation progeny of a cross of two non-isogenic plants.
F2 hybrid/F2 generation: progeny produced by selfing of the F1 generation.
SNP: when 2 homologous sequences are compared, it refers to a single nucleotide polymorphism or a single nucleotide mutation.
Quantitative Trait Loci (QTL): quantitative Trait Loci (QTLs) refer to genetic loci that control, to some extent, a trait, usually distributed continuously, as a numerical value.
Linkage: a phenomenon in which alleles on the same chromosome are more prone to segregate together than would be expected by chance if transmission of the alleles were independent.
Exemplary embodiments of the invention
The present invention relates to a method for identifying and/or selecting jute plants exhibiting newly conferred or enhanced salt resistance comprising:
a. detecting the existence of an mk6160 SNP locus, an mk5633 SNP, an mk6484 SNP locus, an mk6723 SNP locus and an mk7047 SNP locus in the DNA of the jute plant; and
b. selecting said jute plant having said allele associated with newly conferred or enhanced salt tolerance;
wherein the Scaffold number of the mk6160 SNP site marker is AWUE01014572.1, the position on the Scaffold is 75788, the base of the reference sequence is C, and the variant base is T;
the number of the Scaffold where the mk5633 SNP site marker is located is AWUE01017764.1, the position on the Scaffold is 367, the base of the reference sequence is G, and the variant base is A;
the Scaffold number of the mk6484 SNP locus marker is AWUE01014576.1, the position on the Scaffold is 11416, the base of the reference sequence is G, and the variant base is A;
the number of the Scaffold where the mk6723 SNP site marker is located is AWUE01014578.1, the position on the Scaffold is 28342, the base of the reference sequence is T, and the variant base is G;
the Scaffold number of the mk7047 SNP locus marker is AWUE01014583.1, the position on the Scaffold is 47766, the base of the reference sequence is G, and the base of the mutation is T.
The invention relates to application of an mk6160 SNP site, an mk5633 SNP site, an mk6484 SNP site, an mk6723 SNP site and an mk7047 SNP site in identifying QTL (quantitative trait loci) for endowing salt tolerance in jute plants.
QTLs can be identified by using molecular markers. QTLs can be identified by location on a genetic map, or by indicating a location on a linkage group or chromosome. Therefore, the genetic traits conferred by the QTL can be identified and characterized by molecular markers.
In some embodiments, the method of detecting the presence of the mk6160 SNP site, the mk5633 SNP, the mk6484 SNP site, the mk6723 SNP site, the mk7047 SNP site is PCR amplification or sequencing.
In some embodiments, the nucleotide sequences of the upstream and downstream primers used for PCR amplification of the mk6160 SNP site, the mk6484 SNP site, and the mk5633 SNP site are set forth in SEQ ID NOs: 1 and 2, SEQ ID NO:3 and 4, SEQ ID NO:5 and 6.
According to an aspect of the present invention, the present invention also relates to a method for breeding jute, comprising the steps of:
1) Determining whether the jute plant has at least one of mk6160 SNP locus, mk5633 SNP, mk6484 SNP locus, mk6723 SNP locus and mk7047 SNP locus by using the method described above;
2) Selecting a first jute plant comprising at least one of said mk6160 SNP site, mk5633 SNP site, mk6484 SNP site, mk6723 SNP site, mk7047 SNP site and crossing it with a second jute plant to produce a progeny plant comprising at least one of said mk6160 SNP site, mk5633 SNP site, mk6484 SNP site, mk6723 SNP site, mk7047 SNP site.
According to an aspect of the invention, the method further comprises:
3) Repeating steps 1) -2) 2-10 times using the progeny plant described in step 2) as a starting material to produce further progeny plants.
According to an aspect of the present invention, the second jute plant is of an agronomically elite variety.
According to an aspect of the present invention, the method further comprises selecting a jute plant comprising said mk6160 SNP site, mk5633 SNP, mk6484 SNP site, mk6723 SNP site, mk7047 SNP site and agronomically superior characteristics.
According to another aspect of the present invention, the present invention also relates to the use of the jute plant produced by the method as described above for producing a jute propagation material having salt tolerance suitable for producing jute plants having salt tolerance and comprising the mk6160 SNP site, mk5633 SNP, mk6484 SNP site, mk6723 SNP site, mk7047 SNP site as mentioned above or seeds thereof;
wherein the propagation material is suitable for sexual propagation, vegetative propagation or tissue culture of regenerable cells.
In some embodiments, the propagation material suitable for sexual propagation is selected from the group consisting of microspores, pollen, ovaries, ovules, embryo sacs and egg cells;
said propagation material suitable for vegetative propagation is selected from cuttings, roots, stems, cells, protoplasts;
the propagation material suitable for tissue culture of regenerable cells is selected from the group consisting of leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, anthers, flowers, seeds and stems.
The present invention also relates to a method of seed production comprising growing jute plants comprising salt tolerance properties, allowing the plants to produce seeds, and harvesting those seeds. The production of seeds is suitably performed by crossing or selfing.
According to another aspect of the present invention, the present invention also relates to the use of the mk6160 SNP site, mk5633 SNP, mk6484 SNP site, mk6723 SNP site, mk7047 SNP site as mentioned above in the study of genetic diversity in jute populations.
The invention also relates to a method for detecting the salt tolerance of jute, which comprises the steps of detecting the genetic information of at least one of mk5945, mk6330, mk6391 and mk6398 in a jute sample, and judging whether the jute sample has the salt tolerance or not according to the genetic information of at least one of mk5945, mk6330, mk6391 and mk 6398;
the mk5945 is positioned at 164674 on AWUE01014570.1, the reference base is T, and the variant base is A;
the mk6330 is located at 13864 th position on AWUE01014574.1, the reference base is C, and the variant base is T;
the mk6391 is located at 2325 th position on AWUE01014575.1, the reference base is A, and the variant base is G;
the mk6398 is located at position 21609 on AWUE01014575.1, the reference base is A, and the variant base is G;
preferably, genetic information of at least one of mk5945, mk6330, mk6391, mk6398 is obtained by probe, PCR amplification or sequencing;
preferably, the primer pair used for PCR amplification is shown in SEQ ID NO:7~8, SEQ ID NO:9 to10, SEQ ID NO:11 to 12, SEQ ID NO:13 to 14.
The invention also relates to a jute breeding method, which comprises the following steps: (ii) (a) performing the method as described above; (b) Selecting a jute sample with salt tolerance according to the step (a), and taking the jute sample as a parent to breed offspring.
Preferably, the progeny is obtained by breeding by backcrossing, selfing and/or crossing with other jute having superior agronomic traits.
Use of a jute plant produced by a process as described above to produce a jute propagation material having salt tolerance, wherein the propagation material is suitable for sexual propagation, vegetative propagation or tissue culture of regenerable cells.
Preferably, said propagation material suitable for sexual propagation is selected from the group consisting of microspores, pollen, ovaries, ovules, embryo sacs and egg cells;
said propagation material suitable for vegetative propagation is selected from cuttings, roots, stems, cells, protoplasts;
said propagation material suitable for tissue culture of regenerable cells is selected from the group consisting of leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, anthers, flowers, seeds and stems.
The application of mk5945, mk6330, mk6391 and mk6398 mentioned above in the construction of jute genetic map or research of jute population genetic diversity.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Examples
1. Project background
The GBS (Genotyping-by-sequencing) technology refers to Genotyping through sequencing, constructs SNP molecular markers by selecting appropriate restriction enzymes and combining high-throughput colony sequencing, and can be used in the fields of molecular marker development, ultrahigh-density genetic map construction, colony genetic analysis, colony GWAS analysis and the like.
The route of GBS technology is shown in fig. 1 and mainly includes three parts:
1) Experimental part: performing electronic enzyme digestion evaluation, selecting proper endonuclease according to species and scientific research purposes, building a library after a sample is detected to be qualified, and sequencing on a HiSeq PE150 sequencing platform;
2) And (3) standard information analysis: after basic quality control, comparing the original sequencing data with a reference genome, and performing mutation detection and screening;
3) High-level information analysis: the method comprises the analysis of polymorphism among parents, the genotyping of offspring, the screening of markers, the construction of genetic maps, the QTL positioning by combining phenotypic characters and the like.
The genetic map information analysis content comprises: data quality control (removal of adaptors and low quality data), GBS outcome data statistics, comparison to reference genomes, population SNP detection, genetic marker development, genetic marker screening, genetic map construction, QTL mapping, other customized analyses, etc. (figure 2).
2. Experimental strategy
2.1 Design of experiments
The two parent plants used in this study were the wild c. Olitorius l. Germplasm J009 and c. Olitorias l. Variety gazey long fruit (GFG), which had a lower salt tolerance than J009. 150F 2 populations used to construct high resolution genetic maps and incorporating F 2：3 And carrying out salt tolerance QTL positioning on the salt tolerance character of the offspring. Young leaf tissues from 150F 2 individuals and both parents were collected for genomic DNA extraction. Total genomic DNA of each individual was extracted using DNeasy Plant Mini Kit (Tiangen Biochemical technology, beijing) Co., ltd.) and prepared as GBS library for sequencing. DNA degradation and contamination were monitored on a 1% agarose gel. The DNA purity was checked using a NanoPhotometer spectrophotometer (IMPLEN, CA, USA), and the DNA concentration was measured using a Qubit DNA assay kit and a Qubit 2.0 fluorometer (Life Technologies, CA, USA).
The wild plant germplasm resource usually carries a large amount of resistance genes and is a natural gene bank for breeding against adverse circumstances, so the wild plant germplasm resource is very suitable for screening the salt-tolerant genes of jute.
2.2 library construction and sequencing
A schematic representation of the GBS library construction scheme is shown in FIG. 3. Firstly, the genome is digested by restriction enzyme, each sample is amplified after a linker with barcode is added, then the samples are mixed, and the required fragment is selected for library construction. Double-ended (Paired-End) 150 sequencing was performed using the Illumina HiSeqTM sequencing platform:
1) Enzyme digestion: carrying out enzyme digestion on 0.1-1 microgram genome DNA by using restriction enzyme to obtain a proper marker density;
2) Adding P1 and P2 linkers: adding P1 and P2 adapters (complementary to the nicks of the digested DNA) at two ends of the digested fragment;
3) Fragment selection: PCR amplification of tag sequence with P1 and P2 joint at two ends, DNA fragment posing, electrophoresis recovery of DNA in required interval;
5) High-throughput sequencing: cluster preparation and machine sequencing.
3. Information analysis method and result
3.1 Sequencing data statistics and quality assessment
Qualified DNA libraries were tested for Illumina HiseqTM sequencing, yielding Raw data (i.e., raw data or Raw reads) and results stored in FASTQ file format (filename:. Fq). The original sequencing data contains joint information, low-quality bases and undetected bases (expressed by N), in order to ensure the quality of information analysis, the information can cause great interference on subsequent information analysis, the interference information needs to be removed before analysis, and finally obtained data is effective data which is called Clean data or Clean reads. The raw data filtering method is as follows:
(1) The reads pair containing the linker sequence needs to be filtered out;
(2) When the content of N contained in the single-ended sequencing read exceeds 10% of the length proportion of the read, the pair of paired reads needs to be removed;
(3) The pair of paired reads needs to be removed when the number of low-mass (quality Q. Ltoreq.5) bases contained in the single ended sequencing read exceeds 50% of the length proportion of the read.
And obtaining high-quality Clean data through the strict filtering of the sequencing data. And (4) counting sequencing data of all samples of parents and offspring, wherein the sequencing data comprise sequencing reads quantity, data yield, sequencing error rate, Q20, Q30, GC content and the like. The statistical results of the sequencing data quality of the parents and the offspring are shown in the table 1.
TABLE 1 sequencing data production and quality statistics
Note: sample: a sample name; raw base: the number of bases of the original data; clean base: the high-quality base number of the original data after being filtered; effective rate: effective utilization rate of data, namely the percentage of clean base and Raw base; q20, Q30: the percentage of bases with a Phred number greater than 20, 30 to the total bases; GC content (%): GC content.
In addition, when Clean data was compared with the nucleotide database of NCBI, no DNA contamination from other sources was found.
In the project, 2 jute parent samples are sequenced totally, the total sequencing data amount is 16.78Gb, and each sample is 83.88Gb on average; the amount of clean data with high quality is 16.68Gb, and the average data amount per sample is 83.41Gb. The sequencing quality is high (Q20 is more than or equal to 90 percent, Q30 is more than or equal to 85 percent), and the GC distribution is normal. 150 jute progeny samples, the total sequencing data volume is 113.48Gb, and each sample is 756.57Mb on average; the amount of clean data of high quality is 113.47Gb, averaging 756.51Mb per sample. The sequencing quality is high (Q20 is more than or equal to 90 percent, Q30 is more than or equal to 85 percent), and the GC distribution is normal.
In conclusion, the library construction and sequencing are successful.
3.2 Comparative analysis
The sequencing data was aligned to the reference genome. The sample alignment rate can reflect the similarity of the sample sequencing data and a reference genome, and the coverage depth and the coverage degree can directly reflect the uniformity of the sequencing data and the homology with a reference sequence.
The specific analysis steps are as follows:
(1) Using BWA alignment software (parameter: mem-t 4-k 32-M-R), aligning PE reads of parent and offspring Clean data with a reference genome;
(2) Carrying out format conversion on the comparison result by using SAMtools, and converting the comparison result into SAM/BAM files;
(3) Using a Perl script to count the comparison rate and the coverage;
(4) The results were ranked using SAMtools comparisons (parameter: sort) for subsequent analysis.
3.2.1 reference genome basic cases
The reference genome base case statistical details are as follows:
TABLE 2 reference genome Baseline statistics
3.3 Group SNP detection
SNP (single nucleotide polymorphism) mainly refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genome level, and includes single base conversion, peak conversion and the like. We adopt GATK and other software to detect group SNP.
The specific analysis steps are as follows:
(1) The BWA alignment results were filtered: selecting reads which are compared to the unique position on the genome, and carrying out subsequent analysis;
(2) SNP detection, adopting GATK (-type UnifiedGenotyper) to detect group SNP of the filtered bam file;
(3) SNP filtration: in order to reduce false positive SNP caused by sequencing errors, parents and filial generations require that the number of SNP base supports is not less than 4.
(4) Statistics of related information of SNP: number of heterozygous SNPs, number of homozygous SNPs, heterozygous SNP ratio.
3.3.1 Display of SNP detection results
Parental SNP detection results are shown in table 3.
TABLE 3 parental SNP detection statistics
Note: homozygous SNP: homozygous SNPs, such as AA;
heterozygosis SNP: heterozygous SNPs, such as AC;
Het rate： Heterozygosis SNP / Total；
total: number of all SNPs.
3.4 SNP marker development
3.4.1 inter-parent tag development
And (3) carrying out inter-parent polymorphism marker development based on the detection results of 2 parent genotypes. Filtering out sites with parent information deletion; and (2) screening sites of which parents and parents are all homozygous and have polymorphism (for example, at a certain SNP site, the genotype of the parent 1 is GG, the genotype of the parent 2 is AA, the genotypes of the parents are all homozygous, and the genotypes of the parents are different). The project obtains 3238 polymorphic sites, 3238 zxft, wherein the available marker types of the F2 population are 'aa x bb' type, and the polymorphic markers are 217356. All the types and amounts of markers developed are shown in Table 4. The inter-parental polymorphic marker display format is shown in Table 5.
TABLE 4 Mark development types
Note: the MarkerType refers to parental genotypes, such as ab x cc, and the genotypes taking ab and cc as male parents and female parents;
p1 genotype: parental 1 genotype;
p2 genetic type: parental 2 genotype;
marker number: the number of each type of mark;
percentage, each type of mark accounts for the total number of the effective marks; total: the total number of valid flags.
TABLE 5 partial polymorphic marker display between parents of F2 population
Note: and (2) Chr: number of chromosome (or Scaffold) on which the marker is located;
position: the position of the marker on the chromosome (or) the Scaffold;
ref: a reference genomic base type;
p1: the parental 1 genotype;
p2: parental 2 genotype.
3.4.2 progeny genotyping
After the inter-parent marker development is completed, the genotypes of the 150 offspring at the 2 parent polymorphic marker sites are extracted. The results of genotyping the progeny at the individual marker loci are shown in Table 6.
TABLE 6 progeny genotyping
Note: and (2) Chr: the serial number of the label is Scaffold;
position: marking the position of the Scaffold;
ref: a reference genomic base type;
p1: parental 1 genotype;
p2: parental 2 genotype;
J382-1-J382-102: the genotype of part of the progeny individuals at the marker locus;
"- -": indicating a deletion.
3.4.3 Genetic marker screening
Screening the classified filial generation markers, wherein the specific screening steps are as follows:
and (5) abnormal base detection.
In the progeny typing results, basetypes that do not appear in a few parents may appear. For example, at a SNP site, the parental genotypes are "AA" and "TT", respectively, and if a base (G or C) other than "A or T" appears in the progeny, that base is considered to be an abnormal base. The occurrence of abnormal bases may be influenced by the quality of reference genome assembly, the quality of parental sequencing data, the genotyping accuracy and other factors, and may also be variations occurring in progeny populations. For an abnormal base that appears in the progeny but is not present in the parent, we consider it as missing, indicated by the symbol "- -". The detection shows that no abnormal base is found, which indicates that the genotyping accuracy is better.
And (4) filtering the integrity.
The genotype is screened for markers that cover at least 75% or more of all progeny (this criterion is appropriately adjusted based on the actual marker data amount). That is, at least 75 individuals out of 100 progeny have a defined genotype for a single polymorphic marker site. By filtering the markers with poor genotype integrity coverage, 9019 markers were obtained.
Partial separation flag (Segregation departure) filtering.
Partial separation markers generally exist and can influence map construction results and QTL positioning, and by using a majority of literature to partial separation processing methods, the 9019 candidate markers are subjected to partial separation filtration by adopting chi-square test, and a threshold value set for partial separationpIs 0.001. A total of 8150 remaining valid markers will enter the linkage analysis by segregation analysis.
3.5 Genetic map construction
3.5.1 linkage group construction
Constructing the high-quality genetic marker obtained after screening by adopting a Joinmap 4.0 software genetic map: 1) Dividing the linkage groups, and setting the LOD value to be 2-30; 2) Sequencing each linkage group by adopting a maximum likelihood method; 3) The genetic distance between markers was calculated using the Kosambi function.
3.5.2 Genetic map results
The number of SNP markers per linkage group and the total genetic distance per linkage group are shown in Table 7.
TABLE 7 statistics of genetic linkage group information
Note: group: a linkage group number;
SNP markers: the number of SNP markers;
map length is genetic distance length;
average distance (cM): an average genetic distance;
gap (cM) maximum genetic distance between markers;
statistics were made on the distribution of gap on each chromosome and the results are shown in Table 8.
TABLE 8 statistics of the distribution of gap sizes on chromosomes for genetic maps
Note: and (2) Chr: a chromosome name;
<5cM: the number of genetic distances between markers is less than 5 cM;
5to10 cM: the number of gap with genetic distance between markers ranging from 5cM to10 cM;
10to20 cM: the number of gaps with genetic distance between markers ranging from 10cM to 20cM;
>20cM: the number of gap with genetic distance between markers being more than 20 cM;
ratio: the number of gap with genetic distance between markers less than 5cM is the percentage of the total markers;
3.5.3 marker profile
Drawing a linkage graph by using a perl SVG module; wherein, the distribution of the partially linked group markers on LG4 is shown in FIG. 7.
3.6 atlas quality assessment
Evaluating the linkage relation of the markers by using the linkage group heat map; if the marks are correct, the linkage between the marks becomes weaker as the distance between the marks increases.
The species of the item is jute, the F2 generation group, and the number of individuals is 150. The LOD value threshold value of each phenotype is determined by using PT (Permutation test) in MapQTL, QTL positioning is carried out by using CIM algorithm in WinQTL software, and the QTL section corresponding to each phenotype is determined according to the threshold value obtained by the previous replacement test.
four-SNP and QTL linkage analysis method
The salt tolerance test is carried out under the environment of two salt concentrations, 140mM and 160mM, both of which are 12 h-12 h day/night light cycles, and the culture is carried out in an illumination incubator, wherein the culture temperature is 28 +/-0.5 ℃/25 +/-0.5 ℃, and the humidity is 75%. Surface sterilized seeds of the population used for QTL mapping (150F) 2：3 l ines) to a clean and sterile filter paper with two uniform sterile filter papersOn a petri dish of bacteria. Each line was seeded on two petri dishes, each dish having 30 seeds. One dish was used for salt stress treatment and the other dish was used as control. For salt stress induction, 3ml NaCl solution and 3ml water were applied to the salt stress and control dishes, respectively, on the first day, and then 2ml NaCl solution and 2ml water were added to each corresponding plate for six days. A random complete design with three biotic repeats was used in each salt stress environment. Throughout the six day experiment, conditions for seed germination were observed and recorded daily. When the shoots were longer than 3mm, the seeds were defined as germinated seeds. The Salt Tolerance Index (STIG) of seed germination was evaluated according to the following equation:
salt tolerance index = (number of germinated seeds under salt stress ÷ total number of seeds)/(number of germinated seeds under control conditions ÷ total number of seeds).
Selecting an LOD threshold value of the experiment, wherein the LOD value corresponding to each linkage group is selected at 95% confidence (namely, the LOD of the second row corresponding to the sixth row at 0.95 is selected as the LOD threshold value of the linkage group, and when the sixth row does not have 0.95, the LOD threshold value is selected to be more than 0.95 and is closest to 0.95);
selection of LOD threshold for each phenotype: at the genome-wide level, the LOD at the 95% confidence Interval (i.e., the value in the Interval) was chosen as the threshold, and the results are shown in the following table:
TABLE 9 LOD thresholds
We recorded F numbers from 150 2：3 lines' STIG and two parents under two salt stress conditions for 6 days. The STIG data on day four showed normal distribution (fig. 4) and was used as phenotypic data for QTL. According to 1000 permutation tests, the LOD score threshold was determined to be about 3.5 under two salt stress conditions. We identified three significant QTLs on LG4 under two salt stress conditions and detected one major QTL at the same time in both environments, named qJST-1. QTL qJST-1 is located between the markers mk5633 and mk6723 on LG4 under 140mM salt stress conditions 11.4-23.7cMAnd 16.9-21.6cM between the markers mk6160 and mk6484 on LG4 under 160mM salt stress conditions. The LOD peak of qJST-1 was localized at 19.31cM on LG4 under 140mM and 160mM salt stress conditions, accounting for 11.81% and 19.61% of total phenotypic variation, respectively. QTL qJST-2 was located between markers mk7047 and mk5638 (at the 9-11.4cM position of LG 4) under 140mM salt stress conditions, and the LOD peak on LG4 was located at 10.01cM, accounting for 3.74% phenotypic variation. QTL qJST-3 was detected under 160mM salt stress conditions, which is located between markers mk6393 and mk6391 (10.4-16.9 cM) on LG4 that partially overlap with qJST-2. The LOD peak of qJST-3 was located at 13.41cM on LG4, accounting for 8.84% of phenotypic variation. All additive effects of QTL were negative, indicating that the J009 allele conferred an increased salt tolerance value.
TABLE 10 QTL associated with the salt tolerance trait of jute under two salt concentration stress conditions
Note:
additive _ effect: additive effect
Left Marker: marker name corresponding to QTL left boundary
Right Marker: marker name corresponding to QTL right boundary
PVE: percent phenotypic variation
TABLE 11 molecular markers closely linked to qJST-1, qJST-2 and qJST-3
Note: the character number is train; lg is the number of the linkage group; third column Position (cM): the genetic position of the LOD peak in the qtl section; LOD: the LOD value peak in section qtl; additive _ effect: additive effects; dominant _ effect: a dominant effect; r2: QTL accounts for the proportion of phenotypic variation; LOD2_ L (cM): QTL left boundary at 99% confidence interval; LOD2_ R (cM): QTL right boundary at 99% confidence interval; marker2_ L: a marker name corresponding to the left boundary of the QTL; marker2_ R: and marking names corresponding to the right boundary of the QTL.
TABLE 12 detailed information of molecular markers closely linked to qJST-1, qJST-2 and qJST-3
In this study, we developed the first high-density and most complete genetic map of jute using the GBS method. The genetic map contained 4839 markers on 7 LG, consistent with the number of chromosomes in jute (n = 7), spanning 1375.41 cM, with an average distance per locus of 0.28 cM, lower than previously reported values. Therefore, the genetic map will greatly enhance QTL/gene mapping and marker-assisted breeding in jute.
Salt tolerance is a quantitative genetic trait controlled by multiple genes in many crops. Accurate phenotyping is an important component of QTL localization; thus, we measured the relative seed germination percentage within six days under two salt stress environments for the salt stress and the control lines, three biological replicates. All F's in the first two days of the experiment under salt stress 2：3 The germination rate of the strain is very low. On the last two days, the salt tolerant phenotype was polarized to a secondary distribution and then most consistent with the positive distribution on day four. Therefore, we selected salt-tolerant phenotypic data for QTL mapping on day four.
Salt stress is a major environmental stress factor that limits plant development and limits crop yield. The salt tolerance of crops can be improved through accurate breeding, and the salt tolerance related genes or molecular markers need to be identified. However, most previous studies on salt tolerance of jute focused on only morphological, physiological and proteomic components. To our knowledge, only two studies related to salt tolerance of jute have been performed by transcriptome sequencing, revealing a large number of differentially expressed salt tolerance genes. However, the large number of differentially expressed genes makes it difficult to identify the best candidate gene associated with salt tolerance for practical applications.
Therefore, we performed QTL mapping to locate candidate genes within a limited interval of the genome. We succeeded in finding 16 QTLs associated with salt tolerance.
All additive effects were negative for the three significant QTLs, indicating that increased trait values were conferred by the J009 allele. Overall, these results highlight the presence of excellent resistance genes in wild jute material. The wild germplasm resources are utilized to facilitate the acceleration of the cultivation of jute disease-resistant varieties.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> institute of hemp, national academy of agricultural sciences
<120> molecular marker of salt tolerance character in jute and application thereof
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Claims (5)
1. A method of identifying and/or selecting a jute plant of the long fruit variety exhibiting newly conferred or enhanced salt tolerance comprising:
a. detecting an mk6160 SNP locus in the DNA of the jute plant with the long fruit seed; and
b. selecting said mulukhiya plant having an allele associated with newly conferred or enhanced salt tolerance;
in the allele, the bases of the two homologous chromosomes at the mk6160 SNP site are both C:
the number of the Scaffold where the mk6160 SNP site is located is AWUE01014572.1, the position on the Scaffold is 75788, and the base is C;
the method for detecting the genotype of the mk6160 SNP locus is PCR amplification or sequencing;
the nucleotide sequences of the upstream and downstream primers used for PCR amplification of the mk6160 SNP locus are respectively shown as SEQ ID NO:1 and 2;
the Corchorus olitorius Linne is J009.
2. A method for breeding jute of long fruit variety, which comprises the following steps:
1) Selecting a jute plant of the species sildenum having said allele using the method of claim 1;
2) Defining the mulukhiya plant obtained in step 1) as a first mulukhiya plant and crossing it with a second mulukhiya plant to produce a progeny plant comprising said allele;
the second jute plant of long fruit seed belongs to an agronomically excellent variety;
3) Repeating steps 1) -2) 2-10 times using the progeny plant described in step 2) as a starting material to produce further progeny plants;
the method further comprises selecting a jute plant of long fruit variety comprising the CC genotype at the mk6160 SNP locus and agronomically superior characteristics;
the Corchorus olitorius Linne is J009.
3. Use of a Mulukhiya plant produced by the method of claim 2 for producing Mulukhiya propagation material having salt tolerance, which propagation material is suitable for producing Mulukhiya plants or seeds thereof having salt tolerance and comprising the mk6160 SNP site CC genotype of claim 1;
wherein the propagation material is suitable for tissue culture of sexual propagation, vegetative propagation or regenerable cells;
the Corchorus olitorius Linne is J009.
4. The use according to claim 3, said propagation material suitable for sexual reproduction being selected from the group consisting of microspores, pollen, ovaries, ovules, embryo sacs and egg cells;
said propagation material suitable for vegetative propagation is selected from cuttings, roots, stems, cells, protoplasts;
the propagation material suitable for tissue culture of regenerable cells is selected from the group consisting of leaves, embryos, cotyledons, hypocotyls, meristematic cells, root tips, flowers and seeds.
5. The application of the mk6160 SNP locus in the method of claim 1 in researching salt tolerance resistance in jute of the long fruit seed;
the Corchorus olitorius Linne is J009.
Applications Claiming Priority (18)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810859543.7A CN108588271A (en) | 2018-08-01 | 2018-08-01 | A kind of SNP marker mk6398 and its application |
| CN201810859521.0A CN108570520A (en) | 2018-08-01 | 2018-08-01 | A kind of SNP marker mk6330 and its application |
| CN2018108595210 | 2018-08-01 | ||
| CN2018108594913 | 2018-08-01 | ||
| CN2018108595244 | 2018-08-01 | ||
| CN201810859542.2A CN108913803A (en) | 2018-08-01 | 2018-08-01 | The molecular labeling of salt-tolerance character and its application in jute |
| CN201810859475.4A CN108676912A (en) | 2018-08-01 | 2018-08-01 | The molecular labeling of salt-tolerance character and its application in jute |
| CN201810859524.4A CN108913802A (en) | 2018-08-01 | 2018-08-01 | A kind of SNP marker mk5945 and its application |
| CN201810859498.5A CN108570518A (en) | 2018-08-01 | 2018-08-01 | The molecular labeling of salt-tolerance character and its application in jute |
| CN201810859522.5A CN108913801A (en) | 2018-08-01 | 2018-08-01 | A kind of SNP marker mk6391 and its application |
| CN2018108594754 | 2018-08-01 | ||
| CN2018108595437 | 2018-08-01 | ||
| CN201810859499.XA CN108570519A (en) | 2018-08-01 | 2018-08-01 | The molecular labeling of salt-tolerance character and its application in jute |
| CN201810859499X | 2018-08-01 | ||
| CN2018108595225 | 2018-08-01 | ||
| CN201810859491.3A CN108611436A (en) | 2018-08-01 | 2018-08-01 | The molecular labeling of salt-tolerance character and its application in jute |
| CN2018108595422 | 2018-08-01 | ||
| CN2018108594985 | 2018-08-01 |
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| WO2017106274A1 (en) * | 2015-12-16 | 2017-06-22 | Syngenta Participations Ag | Genetic regions & genes associated with increased yield in plants |
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| WO2017106274A1 (en) * | 2015-12-16 | 2017-06-22 | Syngenta Participations Ag | Genetic regions & genes associated with increased yield in plants |
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