CN107604094B - Cauliflower SSR primer developed based on transcriptome sequencing - Google Patents
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Abstract
The invention provides a cauliflower SSR primer developed based on transcriptome sequencing, which comprises 17 pairs of primers, wherein the primer sequence is shown as SEQ ID NO. 1-34. According to the invention, a large amount of SSR primers are developed by utilizing a large amount of transcript data information obtained by sequencing of a cauliflower transcriptome, and the effectiveness of the developed SSR markers is verified by utilizing representative germplasm resources collected by the subject group, so that a foundation is laid for the subsequent aspects of cauliflower genetic diversity research, genome comparison research, genetic map construction, important trait QTL positioning, core resource utilization and storage, molecular assisted breeding and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a cauliflower SSR primer developed based on transcriptome sequencing.
Background
Cauliflower (A. cauliflower)Brassica oleraceaL.var. botrytis L.2n =2x = 18) is a cabbage vegetable of brassica of brassicaceae with a curd flower as an edible organ, and is native to the coast of the mediterranean (liu cabriosa, 2011). The cauliflower is planted in Fujian, Zhejiang, Hubei and other areas in China, and is one of the very important vegetables in China. Cauliflower has abundant nutrition and anticancer and cancer-preventing effects, and as a health-care vegetable used as both medicine and food, the demand is increasing and the planting area is expanding (Zhushiyang et al, 2012). It is reported that in China, cauliflower is at the top of the world in terms of cultivation area and yield (Lemna livinata et al, 2014).
The research on the breeding of the cauliflower in China starts late. Variety resources are the basis of breeding research, and identification and analysis of genetic relationship among cauliflower varieties are essential key steps for variety breeding. The DNA marking technology is an important auxiliary method for researching the genetic diversity of vegetables on a molecular level in the field of biology. In order to study the genetic diversity of cauliflower on a molecular level, in recent years, a number of researchers have carried out research works using AFLP (ligensqi, 2003), RAPD (forest a kind of jade, 2007; field source, 2007), InDel (liu cabona, 2011), ISSR (madeiyu et al, 2010), SRAP (lou yu et al, 2015; yao qin et al, 2016), and the like. Research reports on SSR markers on cauliflowers are few, and identification of the genetic relationship of the cauliflowers is carried out only by the aid of EST-SSR markers developed by cabbages according to the general principle of kindred species primers, such as winter plums, Liu Yuxia and the like (2011; Liu Yuxia and 2011).
The SSR (simple sequence repeat) molecular marker technology is a DNA molecular marker with the advantages of simple operation, good repeatability, high co-dominance, good stability, site specificity, etc., and is widely used in eukaryotes and prokaryotes (Tuler et al, 2015). According to its origin, it can be classified into genomic SSR (g-SSR) and expressed sequence tag SSR (EST-SSR). Traditional SSR markers are developed by designing SSR primers according to conserved regions on two sides of a gene sequence through genome or EST (expressed sequence tag) downloading, so that the traditional SSR markers have the advantages of large workload, long time and high cost (Zhang et, 2012), and limited cauliflower EST sequences in an NCBI database, and the quantity of the developed SSR primers is small. With the rapid development of molecular biology technology, the high-throughput transcriptome sequencing technology can obtain a large amount of transcript information of a certain species under the condition of no reference genome, and a large amount of SSR primers are developed by utilizing bioinformatics software, so that the method has the advantages of rapidness, high efficiency, reasonable cost, reliable data and the like. Currently, the development of SSR markers by transcriptome sequencing platforms has been applied to vegetables such as chinese cabbage (Ding et al, 2015), radish (Zhai et al, 2014), chili (liufeng et al, 2012), eggplant (weimingming et al, 2016), pumpkin (Wu et al, 2014), flowering cabbage (li rong hua et al, 2016), onion (li man tang et al, 2015), spinach (latent weiwei et al, 2016). According to the research, a large amount of transcript data information obtained by sequencing of a cauliflower transcriptome is utilized to develop a large amount of SSR primers, and representative germplasm resources collected by the subject matter group are utilized to carry out validity verification on the developed SSR markers, so that a foundation is laid for the subsequent aspects of cauliflower genetic diversity research, genome comparison research, genetic map construction, important trait QTL positioning, core resource utilization and storage, molecular assisted breeding and the like.
Disclosure of Invention
The invention aims to provide a cauliflower SSR primer developed based on transcriptome sequencing, which lays a foundation for the subsequent researches on genetic diversity of cauliflower, comparative genome research, genetic map construction, QTL (quantitative trait locus) positioning of important characters, utilization and storage of core resources, molecular assisted breeding and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
the primers comprise 17 pairs of primers, and the sequences of the primers are shown in SEQ ID NO. 1-34. The following table specifically shows:
SSR primer information of 17 pairs of cauliflowers
The invention has the advantages that: the invention carries out transcriptome sequencing research on cauliflower to obtain 66450 unigene sequences, which contain 10715 SSR sites, the occurrence frequency is 12.09%, and the average distribution distance is 5.9 kb. The predominant type in SSR sites is dinucleotide repeat motifs, accounting for 51.16% of total SSR; followed by a trinucleotide repeat motif with an occurrence frequency of 47.37%. The repeated sequence has 49 kinds of motifs, and the repeated motifs with high frequency are AG/CT, GA/TC and GAA/TTC. 1164 SSR length of the repetitive sequence is more than or equal to 20 bp, 6119 pairs of SSR primer combinations with potential high polymorphism are designed. Randomly screening 40 pairs of SSR primers for PCR amplification, effectively amplifying 31 pairs of primers, and showing polymorphism in 24 cauliflower varieties by 17 pairs of primers. UPGMA analysis showed that 24 cauliflower lots were classified into 3 categories with 17 pairs of polymorphic primers. The developed SSR markers of the cauliflower are rich in types, high in occurrence frequency and high in availability, and provide candidate markers for identification of genetic relationship of cauliflower varieties, drawing of genetic maps and the like.
Drawings
FIG. 1 shows the validation of the amplification of primers. Wherein M is Maker, and lanes 1-40 are PCR amplification products of primers 1-40.
FIG. 2 polymorphism of primer 1 in 24 broccoli material. M is marker, and the serial numbers of other varieties are shown in Table 1.
FIG. 3 UPGMA clustering plot of the broccoli material tested.
Detailed Description
1 materials and methods
1.1 sources of transcriptome data
Cauliflower transcriptome data was derived from the subject high throughput transcriptome deep sequencing of cauliflower. During sequencing, the cauliflower pedicel is placed in liquid nitrogen for quick freezing and storage, and then the cauliflower pedicel is quickly stored to the RNA-Seq transcriptome of Beijing Baimaike company for sequencing at low temperature by dry ice, the total data volume is 15.58 Gb, and 66450 unigene is contained as analysis background data.
1.2 plant Material and DNA extraction thereof
The material used to screen and validate the designed primers was 24 broccoli varieties collected and stored in this subject group (table 1). Broccoli DNA was extracted using a modified CTAB method (forest 29682etc., 2015). Total RNA of each material was extracted with Trizol reagent (Invitrogen, USA) (Guo et al, 2009).
TABLE 1 SSR polymorphic analysis Material of Cauliflower
1.3 transcriptome SSR locus identification and primer design
And (3) carrying out SSR locus search on the cauliflower transcriptome data by using MISA software, wherein the search criteria are as follows: the length of the repeated motif is 2-6 bp, and the minimum repeated times of the dinucleotide, the trinucleotide, the tetranucleotide, the pentanucleotide and the hexanucleotide are respectively 6 times, 5 times, 4 times and 4 times. Primer design is carried out on unigene with more than 1kb containing SSR sites by using batch design software Primer 3.0. The principle of primer design is as follows: (1) the length of the primer is 18-25 bp; (2) the size of the PCR product is 100-300 bp; (3) the annealing temperature (Tm) is between 55 and 65 ℃, and the Tm difference of the upstream primer and the downstream primer is less than or equal to 2 ℃; (4) the GC content is between 40% and 60%; (5) the occurrence of secondary structures of the primers such as hairpin structure, dimer, mismatch, primer dimer (Li uncing et al, 2014) was avoided as much as possible. Each SSR site generates 3 pairs of primers. 40 pairs of primer sequences were randomly selected from the designed primers and synthesized by Shanghai platinum Biotechnology Ltd.
1.4 PCR amplification
In a total volume of 20. mu.L PCR reaction, 100 ng. mu.L was contained-11.5. mu.L, 2.5. mu.L of 10 XPCR buffer, 10. mu. mol. L-1Forward primer 1. mu.L, 10. mu. mol. L-1Reverse primer 1. mu.L, 10 mmol. multidot.L-1，dNTP 4 μL，TaqDNA polymerase (5U. mu.L)-1）0.3 μL，ddH2O9.7. mu.L. PCR amplification was performed on an ABI9700 PCR instrument (Applied Biosystems, USA) using the following protocol: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30 s, annealing at 54 ℃ for 30 s, annealing at 72 ℃ for 1 min, and 35 cycles; finally, the extension is carried out for 7 min at 72 ℃ and the product is stored at 4 ℃. And observing and photographing after electrophoresis to detect the PCR amplification product.
1.5 data statistics
And (3) adopting a manual band reading method, marking a repeatable and clear band on the electrophoretogram as '1', marking a non-band or weak band which is not easy to distinguish at the same position as '0', and establishing an original data matrix by utilizing an EXCEL table. And (3) calculating the number of allelic loci, the polymorphism information amount and the genetic distance of each pair of primer amplification loci by using a software DPS (data processing system), and performing cluster mapping according to UPGMA (unified Power plant data processor).
2 results and analysis
2.1 number and distribution of SSR sites in cauliflower transcriptome
The MISA software is used for carrying out SSR search on 66450 unigene sequences (the total sequence length is 47671150 bp) of a cauliflower transcriptome, 10715 SSR sites are obtained in total, and the result shows that 8036 SSR sites meeting the conditions exist, the occurrence frequency (the ratio of the unigene number containing SSR to the total unigene number) is 12.09%, and the occurrence frequency (the ratio of the detected SSR number to the total unigene number) is 16.12%. Wherein 5950 unigene sequences containing 1 SSR locus, 550 unigene sequences containing compound SSR locus, and 1 SSR locus can be found by averaging 5.9 kb. The variety of the SSR of the cauliflower transcriptome is rich, and the repeated primitive appears in the highest frequency of 5 times of repetition seen from the repeated times, and 1149 repeated primitives account for 31.32% of the total SSR. The next 6 and 7 replicates, in turn 1117 and 602, representing 30.44% and 16.41% of the total SSR population, respectively (table 2). From the viewpoint of frequency of occurrence, the two-nucleotide and three-nucleotide repeat motifs are mainly focused, and the predominant repeat motif type is the two-nucleotide repeat motif, which accounts for 51.16% of the total SSR, and the three-nucleotide repeat motif, which accounts for 47.37% of the total SSR.
TABLE 2 type, quantity and distribution frequency of cauliflower EST-SSR
2.2 transcriptome SSR motif repeat types and frequency characteristics
From the type of broccoli transcriptome SSR nucleotide motifs, the eligible 8036 SSR sites contain 49 repeat motifs with 3, 20, 12, 8 and 6 dinucleotide to hexanucleotide repeats, respectively. From the distribution frequency (Table 3), the dinucleotide repeat type AG/CT appeared the most, accounting for 39.47% of the total SSR, and 77.14% of the total dinucleotide repeat motifs. Among the trinucleotides, AAG/CTT was the most abundant in repeat type, accounting for 7.17% of the total SSR, accounting for 22.56% of the total trinucleotide repeat motifs. In addition, the tetranucleotides have ACAA/TTGT and AAAC/GTTT repeating motifs as main components, accounting for 20 percent of the total number; five nucleotides contain five repeating motifs, CAAAG, TCCGT, CACGC, CGAAT, TGGAC, TGATT, ttttttg, CTCTT, accounting for 0.10% of the total SSR. The repeated motifs in the hexanucleotide are CACACACC, AGAGAC, GTGCCG, CAGGTG, TCATGC and TGGAG respectively, and account for 0.07 percent of the total number of the SSR.
TABLE 3 frequency of occurrence of different microsatellite repeat motifs (motifs) in cauliflower transcription
2.3 evaluation of availability of the cauliflower transcriptome SSR
One of the important criteria for judging the availability of SSR molecular markers is their polymorphism (Lissang et al, 2010). The length of the SSR site repeat sequence is an important factor influencing the polymorphism height, when the length of the SSR site repeat sequence is more than or equal to 20 bp, the polymorphism is higher, the polymorphism between 12 and 20 bp is moderate, and the polymorphism smaller than 12 bp is extremely low (Singh H et al, 2010). Therefore, SSR sites below 12 bp were deleted in this study. From the distribution of the lengths of the repeated sequences of all SSR loci (table 4), the SSR loci with the lengths of 12-15 bp are the most, and the number of the SSR loci reaches 3310 and accounts for 41.19 percent of the total SSR loci; secondly, 2329 SSR loci with the length less than 12 bp account for 28.98 percent of the total SSR loci; 1233 SSR loci with 16-19 bp account for 15.34% of the total SSR loci; the length of the SSR is equal to or more than 20 bp, and the number of the SSR loci is 1164, wherein the number of the SSR loci is 592 and 572, which respectively account for 7.37 percent and 7.12 percent of the total number of the SSR loci, and the SSR loci of the two parts have abundant polymorphism.
TABLE 4 Length distribution of the repeat sequences of the SSR-loci of the cauliflower transcriptome
2.4 design and screening of SSR primers for cauliflower transcriptome
The primer design is carried out on 8036 cauliflower unigene sequences containing SSR sites, the length of the primer sequence is 18-25 bp, the length of an amplification product is 100-280 bp, the GC content is 40-60%, the annealing temperature is 55-65 ℃, the difference between the annealing temperature values of an upstream primer and a downstream primer is not more than 5 ℃, the occurrence of a hairpin structure, a dimer, a mismatch and a primer dimer is avoided, the mismatch of 3 bases is allowed at the 5 'end of the primer, the mismatch of 1 base is allowed at the 3' end of the primer and the like. Primers which do not meet the conditions are removed, and 6119 pairs of high-quality labeled primers are successfully designed. Randomly selecting 40 pairs of nucleotide primers with different repeat units of 2-6 bp above from the nucleotide primers to synthesize the nucleotide primers. The SSR locus comprises dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide repeated motif, and DNA of a cauliflower strain 'Qingnong 80 days' is used for amplification to verify the effectiveness of the primer. The results showed that 31 pairs of primers achieved efficient amplification (bright white band in FIG. 1), accounting for 77.5% of the 40 SSR primers. Of the 31 effective amplification primers, 28 (90.32%) of the PCR amplification products matched the expected size, 2 (6.45%) of the amplification products exceeded the expected length, and 1 (3.23%) of the amplification products was less than the expected length.
2.5 polymorphism analysis
And selecting 24 parts of cauliflower materials, and performing amplification and polymorphism evaluation on EST-SSR primers conforming to expectations by using 28 parts of cauliflower materials. Among them, 17 pairs of primers had polymorphism differences (Table 4) and accounted for 60.71% of the effective amplification primers. The number of polymorphic fragments generated by each pair of primers is between 1 and 6. 17 pairs of primers yielded 35 bands with 29 polymorphic fragments, each pair of primers yielded an average of 1.5 polymorphic fragments, and FIG. 2 shows the amplification of primer 8.
Using 17 pairs of polymorphic SSR primers to perform cluster analysis on 24 broccoli material, at genetic distance 0.625, the test material was clustered into 3 major groups (fig. 3), 18 materials from group 1, at genetic distance 0.5, were divided into 3 subclasses, subclass 1, containing 11 varieties, varieties 1, 7, 2, 10, 11, 3, 6, 9, 4, 5, and 8, varieties 1, 3, 6, 4, and 5, 4 varieties derived from taiwan, varieties 2 and 9 derived from xiamen, varieties 7 and 9 derived from japan, and varieties 8 and 11 derived from france; subclass 2, which contains 5 varieties, varieties 12, 15, 13, 19 and 20, wherein varieties 12 and 13 are derived from france, varieties 19 and 20 are derived from xiamen, and varieties are introduced in taiwan; subclass 3 contains 2 varieties, with varieties 22 and 24 grouped into one, both varieties being from xiamen. Group 2 materials, varieties 14 and 17, were 2 parts of group 2 material, both from taiwan. Group 3 total 4 materials, 16, 18, 21 and 23 respectively, where 21, 23 and 18 are from mansion and variety 16 is from france. From the clustering result, most of the varieties from the same region are gathered together, which is in accordance with the distribution of the varieties on the geographical position, and a small part of the varieties from the countries such as Taiwan, Japan, France and the like are gathered together, which is related to the mutual introduction among the countries.
TABLE 417 pairs of SSR primer information for cauliflower
Discussion of 3
The study applied the information of cauliflower transcriptome biological data to develop 66450 unigene sequences containing 10715 SSR sites with an SSR site frequency of 12.09%, which was higher than 4.69% of masson pine (meina et al, 2017), 5.57% of onion (plumfull hall et al, 2015)), 7.83% of pepper (liu peak et al, 2012), 9.52% of squash (royal ocean et al, 2016), but slightly lower than 16.82% of chinese cabbage (Ding et al, 2015), 12.35% of wintersweet (plum et al, 2013), 15.62% of cherry (zongyu et al, 2016), 16.49% of highland barley (xujinqing et al, 2017). On average, 1 SSR locus can be found by 5.9kb in the research, which is higher than that of onion (plumping hall and the like, 2015)), pepper (Liufeng and the like, 2012) and masson pine (Meiliana and the like, 2017), and thus, the SSR markers of the cauliflower are very rich. The frequency and density of SSR occurrences are caused to vary, which may be related to differences in species genomes, methods of transcriptome sequencing, size of data volume, number and length of unigene sequences, criteria for SSR searches, and the like (Varshney RK et al, 2005; Huanghai Swallow et al, 2013). Previous studies found that most of the developed SSR markers are most abundant in di-and trinucleotides (Liang X et al, 2009; Zhuhaisheng et al, 2016). The research shows that the dominant types of the repeated elements in the different types of SSR repeated elements of cauliflowers are mainly dinucleotide and trinucleotide repeated elements which respectively account for 51.16% and 47.37% of the total SSR, and the results are consistent with the results of previous researches on brassica vegetable crops such as radish (ZHai et al, 2014), Chinese cabbage (Ding et al, 2015), and flowering Chinese cabbage (Lironghua, etc., 2016). Of the dinucleotides, the repeat types AG/CT and GA/TC appeared most frequently, consistent with the findings from Lonicera caerulea (Zhang Qingtian et al, 2016), Rosa roxburghii (\37154;. Saume et al, 2015), and Populus diversifolia (Du et al, 2013). Trinucleotides have the repeat type AAG/CTT as the predominant repeat motif, which is consistent with the notion that AAG/CTT is the predominant repeat type of trinucleotide in dicotyledonous plants (Morgante M, 2002).
High polymorphism is an important investigation index of SSR marker technology. Research shows that the length of a repeated sequence in an SSR molecular marker is related to the polymorphism of the marker, and the longer the repeated sequence, the higher the polymorphism. The research shows that 1164 SSR site repeat motif lengths in the cauliflower SSR sites are more than or equal to 20 bp. The primers can be designed through the SSR sites to obtain a large number of SSR primers with high polymorphism. The method comprises the steps of designing primers for 8036 SSR locus unigene sequences by using software to obtain 6119 pairs of SSR primers, randomly synthesizing 40 pairs of SSR primers, verifying the effectiveness of the SSR primers by using DNA of a cauliflower strain Qingnong 80 days', amplifying 31 pairs of primers to obtain strips meeting requirements, wherein 9 pairs of primers of the non-amplified strips are possibly related to the annealing temperature of the primers or the quality of the primers. The polymorphism ratio of 17 pairs of primers screened out by 40 pairs of primers is 60.71%, which is lower than 73.33% of towel gourd (cinnabar, etc., 2016), but higher than 55.9% of quinoa (Zhang Pai, etc., 2016), 38.71% of taxus chinensis (Liyanlin, etc., 2014) and 33% of mung bean (Chen et al, 2015). This also correlates with the randomness of primer synthesis and the amount of test material (Zhang Qingtian et al, 2016). The results of 17 pairs of polymorphic primers, the length of the repetitive sequence of which is 21-25 bp and is more than or equal to 20 bp, verify the previous research results (Singh H et al, 2010). 24 cauliflower varieties are subjected to cluster analysis by using 17 pairs of differential primers, at the genetic distance of 0.625, 24 cauliflower varieties are divided into 3 categories, Taiwan varieties are basically grouped into one category, Xiamen varieties are close in distance, the categories can be basically classified according to geographical positions, a small number of Japanese and French varieties or Japanese and Xiamen varieties are grouped into a cluster in a small category, and the cluster is possibly related to mutual introduction among domains to cause gene drift. But also reflects that the genetic variability of the cauliflower germplasm resources is relatively narrow, and the cauliflower germplasm resources are urgently required to be enriched and expanded. The research can accurately map genetic diversity and genetic relationship among 24 cauliflower materials, but deep analysis of genetic codes and genetic relationship of cauliflower in a deeper layer needs more molecular markers and larger sample groups to be detected.
The research results show that the SSR can provide candidate markers for research works such as digging of functional genes of the cauliflower, molecular marker assisted breeding, genetic diversity analysis, comparative genome research, genetic map drawing and the like due to rich types and high occurrence frequency of the cauliflower transcriptome.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> institute of agricultural sciences college of Fujian province
<120> SSR primers for cauliflower developed based on transcriptome sequencing
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Claims (2)
1. The SSR primers of cauliflower based on transcriptome sequencing development are characterized in that: the primers comprise 17 pairs of primers, and the sequences of the primers are shown in SEQ ID NO. 1-34.
2. Use of a broccoli SSR primer developed based on transcriptome sequencing as claimed in claim 1 for identification of genetic relationship of broccoli variety, mapping genetic map.
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