CN109554498B - Molecular marker for identifying early-late maturing characteristics of single-cropping water bamboo and application and acquisition method thereof - Google Patents
Molecular marker for identifying early-late maturing characteristics of single-cropping water bamboo and application and acquisition method thereof Download PDFInfo
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Abstract
A molecular marker for identifying early and late maturing characteristics of single-cropping water bamboo and an application and acquisition method thereof, belonging to the technical field of molecular biology. The molecular marker comprises molecular markers UeSE1 and UeSE2, wherein the nucleotide sequence of a forward primer UeSNPSE1-F of the molecular marker UeSE1 is shown as SEQ ID No.1, and the nucleotide sequence of a reverse primer UeSNPSE1-R is shown as SEQ ID No. 2; the nucleotide sequence of a forward primer UeSNPSE2-F of the molecular marker UeSE2 is shown as SEQ ID NO.3, and the nucleotide sequence of a reverse primer UeSNPSE2-R is shown as SEQ ID NO. 4. The early-maturing single-cropping water bamboo is screened by the SNP marker, so that a good water bamboo variety can be selected in advance, the workload is reduced, and the screening efficiency is improved.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a molecular marker for identifying the early-late maturing characteristic of single-cropping water bamboo, and an application and acquisition method thereof.
Background
Zizania latifolia (Zizania latifolia Turcz.) is a perennial root herbaceous plant of Gramineae, native to China and southeast Asia, and is the second largest aquatic vegetable in China. Zizania latifolia is cultivated in China early, before the West week, zizania latifolia seeds are eaten as grains, and fungus galls formed by infection of stems of zizania latifolia (Ustilago esculenta) are discovered to be not only edible, but also fleshy and tender, and excellent in taste, so that the zizania latifolia is eaten as a vegetable, and the zizania latifolia is widely planted as a vegetable in the late stage of Tang Dynasty. Besides being used as vegetables, the wild rice shoots can also be used as medicines and have the effects of promoting urination, quenching thirst and clearing away heat and toxic materials. The single-cropping water bamboo is a water bamboo variety harvested once a year, and the harvest season is generally harvested at the beginning of-10 months before and after 9 months. The single cropping water bamboo is a short-day crop, and the water bamboo is pregnant only after the sunshine turns short in autumn. The single-cropping water bamboo is generally tall and big in plant, excellent in color and taste and wide in planting range. In China, from Beijing to Guangzhou, Taiwan to Sichuan can be cultivated. In the period of the early-maturing single-cropping water bamboos from the sunstroke to the autumn, some single-cropping water bamboos are bred into double-cropping cultivated varieties (such as beautiful water bamboos, water treasure No.1, golden water bamboos No.2 and the like) for multiple years. The first harvesting period of the single-cropping water bamboos cultivated in double cropping is generally in the period from summer to summer, and the second harvesting period is 9-10 months, so that the single-cropping water bamboos have no difference with other single-cropping water bamboos. The early-maturing single-cropping water bamboo has the characteristic that the double-cropping water bamboo is harvested twice a year, and also has the characteristics that the double-cropping water bamboo is large in size and tender in color and luster and the like, so that the market price is higher than that of the double-cropping water bamboo. Taking the board-like zizania latifolia cooperative society of Tongxiang city as an example, the average purchase price of the beauty zizania latifolia harvested in the middle and late days of 6 months is 2-4 times that of the dragon zizania latifolia harvested at the beginning of 6 months. Therefore, the breeding of the early-maturing single-cropping water bamboo can be used for double-cropping cultivation, not only can the advantages of the single-cropping water bamboo in the quality of the water bamboo relative to the double-cropping water bamboo be kept, but also the field utilization rate can be improved, and the income of water bamboo farmers can be increased.
The principle of SNP labeling is: single nucleotide variations (deletions, insertions, frameshifts) often occur in organisms, and the result of such variations is single nucleotide polymorphisms. Although most SNPs are located in non-coding regions, they are very closely related to the phenotype of the organism. With the development of high-throughput sequencing technology, a large amount of SNPs are analyzed, and the research of evaluating genetic diversity at the genome level is also greatly promoted. Because the data volume of the SNP is very huge, the molecular marker developed based on the SNP has higher precision.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to design and provide a molecular marker for identifying the early-late maturing characteristic of single-cropping water bamboo and a technical scheme of an application and acquisition method thereof. According to the invention, through carrying out SNP scanning on the Ustilago esculenta of the internal wild rice of the wild rice, a plurality of molecular markers related to the early-late maturing characteristic of the single-cropping wild rice can are screened out, so that an auxiliary selection system of the molecular markers of the early-late maturing characteristic of the single-cropping wild rice can be established, the selection efficiency of the single-cropping early-maturing wild rice can variety is improved, and a foundation is laid for efficient breeding of the wild rice can.
The molecular marker for identifying the early-late maturing characteristic of the single cropping water bamboo is characterized by comprising molecular markers UeSE1 and UeSE2, wherein the nucleotide sequence of a forward primer UeSSPSE 1-F of the molecular marker UeSE1 is shown as SEQ ID No.1, and the nucleotide sequence of a reverse primer UeSSPSE 1-R is shown as SEQ ID No. 2; the nucleotide sequence of a forward primer UeSNPSE2-F of the molecular marker UeSE2 is shown as SEQ ID NO.3, and the nucleotide sequence of a reverse primer UeSNPSE2-R is shown as SEQ ID NO. 4.
The molecular marker is applied to breeding of the wild rice shoots.
The molecular marker is applied to the identification of the early and late maturing of the single-cropping water bamboo in water bamboo breeding.
The method for obtaining the molecular marker is characterized by comprising the following steps of:
a. identification of the single cropping water bamboo maturity: collecting single-cropping water bamboo varieties in different areas in batches and recording the single-cropping water bamboo varieties;
b. population analysis
1) Separating and culturing Ustilago esculenta in the wild rice stem;
2) extracting genome DNA of Ustilago esculenta by a CTAB method;
3) performing genome second-generation re-sequencing on the Ustilago esculenta genome DNA of 5 single-cropping water bamboos;
4) scanning to obtain all SNPs;
5) screening out different SNP sites of the early-maturing variety and the late-maturing variety of the Ustilago esculenta of the single-cropping wild rice shoots;
6) selecting 20 wild rice smut separated from zizania aquatica collected from different areas to verify the SNP sites obtained by screening;
c. identifying the screened SNP locus by using other varieties with the early-late maturing characteristic of the single-cropping water bamboo and obtaining a map; in total, 2 pairs of molecular markers UeSE1 and UeSE2 with the early-late maturing characteristic of the single cropping wild rice shoots are obtained.
The obtaining method is characterized in that the identification of the early-late maturing characteristic of the single cropping water bamboo in the step a is specifically as follows: collecting single-season Zizania latifolia in 8-9 months from Zhejiang province Tungxiang city, Zhejiang province Jinhua city, Zhejiang province Yuyao city, Zhejiang province Hangzhou city and Jiangsu province Suzhou city, and recording the collection period and weight of Zizania latifolia; when in collection, the zizania latifolia with complete shape, medium size and disease-free zizania latifolia leaves is selected.
The obtaining method is characterized in that the Ustilago esculenta in the step 1) of separating and culturing the zizania aquatica specifically comprises the following steps:
a. adding sterile water into stem of Zizania latifolia, and grinding;
b. spread on YEPS medium containing carbenicillin and kanamycin and culture for 4-6 d.
The obtaining method is characterized in that the differential SNP loci screened out in the step b 5) of the single-season early-maturing zizania aquatica smut bacteria and the single-season late-maturing zizania aquatica smut bacteria are specifically as follows:
a. marking all SNP data as A, B types, representing two characteristics of single-season precocity and single-season late maturity;
b. automatically identifying A, B all SNPs in two classes and classifying; the output format is Genotyping simple-A/B.txt; the data content is SNP _ site a/b, wherein a represents the number of the SNPs, and b represents the total number of one type;
c. copying the data to an Excel worksheet, and selecting SNPs with 1 in the A class and 0 in the B class by using a screening function.
The obtaining method is characterized in that 20 zizania aquatica separated zizania aquatica collected from different areas in the step b 6) are selected to verify the SNP sites obtained by screening, and the verification method specifically comprises the following steps:
a. grinding and separating 20 zizania latifolia collected from different areas;
b. extracting all genome DNA of the Ustilago esculenta;
c. designing an AS-PCR primer aiming at the obtained SNP data;
d. selecting the zizania latifolia smut DNA of a typical single-cropping zizania latifolia variety with early and late maturity to perform AS-PCR verification, and verifying to obtain 3 SNP sites with polymorphism; designing HRM detection amplification primers aiming at the 3 SNP sites;
e. amplifying 20 zizania aquatica smut DNA by using 3 pairs of HRM primers, and using PCR products for HRM detection;
f. the early-late maturing characteristic of the single-cropping water bamboo is marked by curves with different colors.
The invention has the following beneficial effects:
1) the method is based on genome re-sequencing data of 5 single-cropping water bamboos with different early and late maturing characteristics, SNP data are obtained through scanning, and single-cropping water bamboos maturity associated loci are obtained through screening by an effective means. Combining the identification results of 20 single-cropping zizania latifolia smut bacteria with different ripeness in different areas as objects, and screening out the molecular markers related to the early and late ripeness of the single-cropping zizania latifolia.
2) The research period is short, and the wild rice shoots varieties in various regions are used as materials, so that the interference of environmental factors on the maturity of the wild rice shoots can be eliminated.
3) The SNP marker is based on genome re-sequencing data, has good accuracy, rich sites and large information amount, and the screened sites have higher reliability.
4) The breeding period of the wild rice shoots is long, the breeding process is complicated, the early-maturing single-cropping wild rice shoots are screened by SNP markers, good wild rice shoot varieties can be selected in advance, the workload is reduced, and the screening efficiency is improved.
Drawings
FIG. 1 shows polymorphism results of 3 pairs of primers AS-PCR of 4 typical single-cropping zizania aquatica smut with early and late maturity, and sites with polymorphism are screened;
FIG. 2 is a LightScanner high resolution melting curve of example 3;
FIG. 3 is a distribution diagram of 3 SNP sites in 20 single-cropping zizania aquatica smut.
Detailed Description
The present invention will be further illustrated with reference to the following examples, which are intended to be exemplary only and are not intended to limit the scope of the invention. Examples other than the experimental materials were commercially available.
Example 1: test materials and Classification of maturity
The following test materials were respectively obtained from agriculture science research institute of Jinhua city, Zhejiang province, Jinshui city, Jinyun county, agricultural science research institute of Yuyao city, Nibo city, Zhejiang province, Suzhou city, Jiangsu province, Jiangxing city, Tungxiang city, Dong city, Zizania rice stem cooperation society, Shaoxing Zhongxing city, Zhejiang province, Huangjiang province, Taizhou city, Huangyan district vegetable research institute, and Hangzhou city, Zhejiang province, China Life science institute of metering university. The sample collection time is 2016, 9, 2017, 9 and 2018, 9.
TABLE 1 variety, maturity and source of test samples
Example 2: population analysis
1) Extracting genome DNA of the Ustilago esculenta by a CTAB method, measuring the DNA concentration by a nucleic acid micro-analyzer, and detecting the DNA quality by agarose gel electrophoresis. By ddH2O diluted to 100 ng/. mu.L and stored at-20 ℃.
2) And (3) sending the DNA of the 5 single-cropping water bamboo varieties with determined maturity stages to second-generation re-sequencing. The reference genome is a Ustilago esculenta MT type genome, and WGS INSDC: JTLW 00000000.1.
3) And (3) comparing sequencing data to a reference genome by using GATK, SAMTOOLS and BWA software to obtain an SNP data file. 21529 SNP sites are obtained in total.
4) All smut samples of zizania esculenta participating in SNP scanning are numbered as A, B types, which respectively represent that the single-cropping zizania esculenta is early-maturing and the single-cropping zizania esculenta is late-maturing. And (3) outputting A, B types of SNP data into SNP c a/b format by extracting Genotype data in the SNP scanning result, wherein a represents the number of the types of SNPs at the c-th SNP site, and b represents the total number of the types of samples. And pasting the output results to an Excel worksheet, and screening the SNP sites of b/b in the early maturity and 0/b in the late maturity by using an Excel screening function. (in this example, since a is 10, B is 18, and C is 20, a total of 14 SNP sites were selected, with SNP C1 being (a row 3/3, B row 0/2).
Example 3: SNP site identification
1) Selecting proper SNP sites from 14 screened SNP sites for verification: due to the problem of the sequencing quality of the reference genome, SNP sites in contig which is relatively behind the reference genome need to be excluded, 3 SNP sites are selected as verification sites in total through screening, and related primers are designed.
2) AS-PCR (allele specific PCR), which is a principle that the base mismatch at the 3' end of a primer can interrupt a PCR extension program to cause no amplification product, plays an important role in detecting SNP and allele genotypes. In this example, 3 pairs of AS-PCR primers were designed, all of which are shown in Table 2.
TABLE 2 allele-specific PCR primer sequence Listing
| Forward primer | Primer sequence (5 '-3') | Reverse primer | Primer sequence (5 '-3') |
| SNPSE 1-F | AGTCACATCACATCACACTGGTCCAT | SNPSE 1-R | TGGAGACTGCTGGTCAAATGAAATTAT |
| SNPSE 2-F | GCGACACTCACACCTCCACGTTT | SNPSE 2-R | GCCCCTGCTCCTTTCCTGAA |
| SNPSE 3-F | CATTTACCCAGTTTTGGCTATCACTCTG | SNPSE 3-R | TCAAGGAAGGGGAATGGTTGC |
3) In the example, 4 typical single-cropping zizania latifolia smut DNA is selected as a template to verify the polymorphism of the SNP sites on the table.
4) AS-PCR System: 1 μ L of Ustilago esculenta genomic DNA; 0.4. mu.M forward primer; 0.4. mu.M reverse primer; 5 μ L of 2 × Taq Master Plus Mix; 3.2 μ L ddH2O。
5) The AS-PCR program was set up AS: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 65 ℃ for 30 s; extension at 72 ℃ for 15 s; a total of 34 cycles; completely extending for 7min at 72 ℃; storing at 12 deg.C.
6) All PCR products were electrophoresed on 2% agarose gel with check bands.
7) Electrophoretic verification results (as shown in fig. 1): 1 represents a band; 0 represents a band; bands indicate that the site has polymorphism in single cropping cane maturity.
8) In this example, 3 sites having polymorphisms were further detected by using an HRM curve (high resolution melting curve).
9) The HRM is mainly based on the length of a DNA sequence, GC content and base complementarity difference, and a high-resolution melting curve is used for analyzing a sample, and the extremely high temperature uniformity and temperature resolution enable the resolution precision to be capable of distinguishing single base difference. The method has the advantages of low cost, large single detection amount and high accuracy, and the detected sample can be continuously used for other experiments and can be used for distinguishing homozygotes and heterozygotes of alleles.
10) In this example, 3 pairs of HRM detection fragment amplification primers were designed for the above 3 SNP sites. All primers are attached to Table 3.
TABLE 3 HRM detection fragment amplification primers
| Forward primer | Primer sequence (5 '-3') | Reverse primer | Primer sequence (5 '-3') |
| HRMSE 1-F | CCAACGGTCCAATCCAACTTCTG | HRMSE 1-R | GCACCTGTCAAGGGCTGTCA |
| HRMSE 2-F | ACTCCAGCGACACTCACACCTC | HRMSE 2-R | GTGGCACACCGCTAGTGATAGTC |
| HRMSE 3-F | AAGCCTATCTGGACACCACACTTG | HRMSE 3-R | GCCCACACTCATCTGACACTAATTG |
11) In this example, 20 kinds of DNA of the unicompartmental zizania aquatica (numbered 6-25 in Table 1) having different maturity characteristics were used as template to verify the 3 polymorphic SNP sites obtained in step 7).
12) HRM-PCR System: 1 μ L of Ustilago esculenta genomic DNA; 0.6. mu.M forward primer; 0.6. mu.M reverse primer; 7.5 μ L2 × Taq Master Plus Mix; 5.3 μ L ddH2O。
13) HRM-PCR program set to: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 65 ℃ for 30 s; extension at 72 ℃ for 15 s; a total of 40 cycles; completely extending for 7min at 72 ℃; storing at 12 deg.C.
14) mu.L of 5 × Eva Green fluorescent dye was added to wells of all PCR products, sealed and centrifuged at 1000rpm for 15s, then sealed with 20. mu.L of mineral oil.
15) Denaturation at 94 ℃ for 2min allowed the fluorescent dye to bind fully to the DNA duplex.
16) The product was measured using the LightScanner high resolution melting curve system and the results are shown in figure 2.
17) The results show that HRMSE-1 and HRMSE-2 have obvious classification on the ripeness characteristics of single-season cane shoots, so that HRMSE-1 and HRMSE-2 correspond to molecular markers UeSE1 and UeSE 2.
18) The distribution map of 2 SNP loci in 20 zizania aquatica smut is shown in FIG. 3.
Sequence listing
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Claims (8)
1. A molecular marker for identifying the early-late maturing characteristics of single-cropping water bamboos is characterized by comprising molecular markers UeSE1 and UeSE2, wherein the nucleotide sequence of a forward primer UeSSPSE 1-F of the molecular marker UeSE1 is shown as SEQ ID No.1, and the nucleotide sequence of a reverse primer UeSSPSE 1-R is shown as SEQ ID No. 2; the nucleotide sequence of a forward primer UeSNPSE2-F of the molecular marker UeSE2 is shown as SEQ ID NO.3, and the nucleotide sequence of a reverse primer UeSNPSE2-R is shown as SEQ ID NO. 4.
2. The application of the molecular marker of claim 1 in breeding of zizania latifolia.
3. The application of the molecular marker of claim 1 in identifying early and late maturing single cropping water bamboo in water bamboo breeding.
4. The method for obtaining a molecular marker according to claim 1, comprising the steps of:
a. identification of the single cropping water bamboo maturity: collecting single-cropping water bamboo varieties in different areas in batches and recording the single-cropping water bamboo varieties;
b. population analysis
1) Separating and culturing Ustilago esculenta in the wild rice stem;
2) extracting genome DNA of Ustilago esculenta by a CTAB method;
3) performing genome second-generation re-sequencing on the Ustilago esculenta genome DNA of 5 single-cropping water bamboos;
4) scanning to obtain all SNPs;
5) screening out different SNP sites of Ustilago esculenta of a single-cropping water bamboo early-maturing variety and Ustilago esculenta of a single-cropping water bamboo late-maturing variety;
6) selecting 20 wild rice smut separated from zizania aquatica collected from different areas to verify the SNP sites obtained by screening;
c. identifying the screened SNP locus by using other varieties with the early-late maturing characteristic of the single-cropping water bamboo and obtaining a map; in total, 2 pairs of molecular markers UeSE1 and UeSE2 with the early-late maturing characteristic of the single cropping wild rice shoots are obtained.
5. The obtaining method of claim 4, wherein the identification of the single cropping cane shoot maturity in step a is specifically: collecting single-season Zizania latifolia in 8-9 months from Zhejiang province Tungxiang city, Zhejiang province Jinhua city, Zhejiang province Yuyao city, Zhejiang province Hangzhou city and Jiangsu province Suzhou city, and recording the collection period and weight of Zizania latifolia; when in collection, the zizania latifolia with complete shape, medium size and disease-free zizania latifolia leaves is selected.
6. The obtaining method of claim 4, wherein the smut bacteria isolated and cultured from zizania aquatica in step b 1) is specifically:
a. adding sterile water into stem of Zizania latifolia, and grinding;
b. spread on YEPS medium containing carbenicillin and kanamycin and culture for 4-6 d.
7. The obtaining method of claim 4, wherein the differential SNP sites of the early-maturing zizania latifolia smut and the late-maturing zizania latifolia smut screened in step b 5) are specifically:
a. marking all SNP data as A, B types, representing two maturity characteristics of single-cropping water bamboo, namely early-maturing and late-maturing;
b. automatically identifying A, B all SNPs in two classes and classifying; the output format is Genotyping simple-A/B.txt; the data content is SNP _ site a/b, wherein a represents the number of the SNPs, and b represents the total number of one type;
c. copying the data to an Excel worksheet, and selecting SNPs with 1 in the A class and 0 in the B class by using a screening function.
8. The obtaining method of claim 4, wherein the step of selecting 20 Ustilago esculenta isolated from zizania aquatica collected from different regions in step b 6) to verify the SNP sites obtained by screening specifically comprises:
a. grinding and separating 20 zizania latifolia collected from different areas;
b. extracting all genome DNA of the Ustilago esculenta;
c. designing an AS-PCR primer aiming at the obtained SNP data;
d. selecting the Ustilago esculenta DNA of a typical single-cropping water bamboo variety to carry out AS-PCR verification, and obtaining 3 SNP sites with polymorphism through verification; designing HRM detection amplification primers aiming at the 3 SNP sites;
e. amplifying 20 zizania aquatica smut DNA by using 3 pairs of HRM primers, and using PCR products for HRM detection;
f. the early-late maturing characteristic of the single-cropping water bamboo is marked by curves with different colors.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1849871A (en) * | 2006-05-26 | 2006-10-25 | 扬州大学 | Method for promoting earliness of autumn water bamboo |
| CN105838616A (en) * | 2016-01-28 | 2016-08-10 | 中国计量学院 | Ustilago esculenta haploid strain UET1 and use thereof |
| CN105838782A (en) * | 2016-01-28 | 2016-08-10 | 中国计量学院 | Method for fast and stable separation and identification of Ustilago esculenta sexual compatible haploid strain |
| CN106085880A (en) * | 2016-07-11 | 2016-11-09 | 中国农业科学院麻类研究所 | The separation method of a kind of smut and used medium |
| CN109022290A (en) * | 2018-07-25 | 2018-12-18 | 中国计量大学 | A kind of wild rice smut haploid strains UeMTSP and its application |
-
2018
- 2018-12-21 CN CN201811571172.9A patent/CN109554498B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1849871A (en) * | 2006-05-26 | 2006-10-25 | 扬州大学 | Method for promoting earliness of autumn water bamboo |
| CN105838616A (en) * | 2016-01-28 | 2016-08-10 | 中国计量学院 | Ustilago esculenta haploid strain UET1 and use thereof |
| CN105838782A (en) * | 2016-01-28 | 2016-08-10 | 中国计量学院 | Method for fast and stable separation and identification of Ustilago esculenta sexual compatible haploid strain |
| CN106085880A (en) * | 2016-07-11 | 2016-11-09 | 中国农业科学院麻类研究所 | The separation method of a kind of smut and used medium |
| CN109022290A (en) * | 2018-07-25 | 2018-12-18 | 中国计量大学 | A kind of wild rice smut haploid strains UeMTSP and its application |
Non-Patent Citations (7)
| Title |
|---|
| Comparative whole-genome analysis reveals artificial selection effects on Ustilago esculenta genome;Ye Z, Pan Y, Zhang Y等;《DNA Res》;20170719;第24卷(第6期);第635-648页 * |
| Investigation on the differentiation of two Ustilago esculenta strains - implications of a relationship with the host phenotypes appearing in the fields;Zhang Y, Cao Q, Hu P等;《BMC Microbiol》;20171206;第17卷(第1期);第1-16页 * |
| RNA-seq analysis provides insight into reprogramming of culm development in Zizania latifolia induced by Ustilago esculenta.;Wang ZD, Yan N, Wang ZH等;《Plant Mol Biol》;20171026;第95卷(第6期);第533-547页 * |
| 不同茭白品种中菰黑粉菌的鉴定及ISSR多态性分析;康璐瑶，崔海峰，张雅芬等;《菌物学报》;20170922;第36卷(第9期);第1210-1221页 * |
| 茭白品种间基因组ISSR多态性分析;李帅，崔海峰，张雅芬等;《中国计量学院学报》;20160630;第27卷(第2期);第161-166页 * |
| 茭白黑粉菌(Ustilago esculenta)研究进展;张治平，耿园，戴海博等;《江苏农业科学》;20161231;第44卷(第12期);第45-48页 * |
| 茭白黑粉菌核相变化及ISSR分子标记筛选的研究;赵煦泓，尤文雨，崔海峰等;《中国计量学院学报》;20110630;第22卷(第2期);第176-180页 * |
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