CN103602743B - Method for detecting resistance of cotton plants to greensickness by molecular marker-assisted selection - Google Patents
Method for detecting resistance of cotton plants to greensickness by molecular marker-assisted selection Download PDFInfo
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Abstract
The invention provides a method for detecting the resistance of cotton plants to greensickness by molecular marker-assisted selection. The method comprises the following steps: (1), soaking seeds of cotton plants to be detected in a soaking solution; (2), planting the soaked seeds in a disease garden; (3), selecting a cotton plant capable of surviving in the disease garden as a preliminary screened cotton plant; and (4), respectively carrying out PCR (Polymerase Chain Reaction) amplification on genome DNA (Desoxvribose Nucleic Acid) of the cotton plant by a first primer pair and a second primer pair to obtain a first amplification product and a second amplification product, wherein the first primer pair is shown as SEQ ID NO:1 and SEQ ID NO:2, the second primer pair is shown as SEQ ID NO:3 and SEQ ID NO:4, and if the first amplification product has nucleic acid shown as SEQ ID NO:5 and the second amplification product has nucleic acid shown as SEQ ID NO:6, the cotton plant to be detected is a greensickness-resistant plant. According to the method, the resistance to greensickness is predicted, the cotton plants are screened to discard plants which do not show symptoms but are susceptible in a disease garden screening process, the waste of manpower and material resources is reduced, and the breeding efficiency is increased.
Description
Technical field
The present invention relates to agricultural biological technical field, particularly, relate to a kind of method of cotton plants to resistance to verticillium wilt that detect.
Background technology
Cotton verticillium wilt is global great Plant diseases, causes the production loss of 10-15% to Cotton Production throughout the year, and the serious time surpasses the whole nation and plants 60% of cotton area, to Cotton Production, causes heavy blow.Its pathogenic bacteria loses leaf and soil propagation with seed, residual branch, and sclerotium can be survived for many years in soil, and the means such as chemicals, tillage and cultivation are invalid to it, are called " cancer " of cotton by plant pathology circle, become pendent global problem for a long time.Cultivate disease-resistant variety and be acknowledged as the most economical effective means of cotton verticillium wilt that solve; study carefully the controlled by multiple genes hereditary pattern that resistance that its reason always failing is cotton verticillium wilt is subject to a plurality of major gene co-controllings; comparatively complicated (the Qi Weiyan of genetic mechanism; Zhang Yongjun; Zhang Tianzhen, Chen Jieyin, Dai little Feng. based on Artificial disease nursery screening and the auxiliary research of verticillium wilt resistance of cotton by same breeding method and application of molecule marker. Molecular Plant Breeding; 2012,10 (5): 607-612; Jiang Feng, Zhao Jun, Zhou Lei, Guo Wangzhen, Zhang Tianzhen. the molecule marker location of upland cotton resisting verticillium gene. Chinese science, 2009,39 (9): 849-861; Ge Haiyan, Wang Yechun, Guo Wangzhen, Zhang Tianzhen. heredity and the molecule marking research of upland cotton resisting verticillium proterties. Cotton Science, 2008,20 (1): 19-22; Jiang F; Zhao J; Zhou L; Guo WZ; Zhang TZ.Molecular mapping of Verticillium wilt resistance QTL on chromosomes D7and D9in upland cotton.Science China SerC-Life Science; 2009,52 (9): 872-884; Yang Chang, Guo Wangzhen, Zhang Tianzhen. the QTL location of the economical characters such as upland cotton resisting verticillium, fibrous quality and output. Molecular Plant Breeding, 2007,5 (6): 797-805), lack the molecule marker effectively can be used for stable screening and accurately to follow the tracks of its resistant gene, identify very difficultly, cause verticillium wilt resistance of cotton by same Advances in Breeding slow.
As far back as the forties in 20th century, China just starts cotton breeding, carries out cross-breeding after the fifties, and cotton field disease was light at that time, substantially according to output performance Cultivars.After the seventies, the generation of cotton wilt increases the weight of gradually, started anti-blight breeding, but breeding technique adopts the susceptible experimental plot of nature to carry out Disease Resistance Identification substantially, because pathogenic bacteria is not enough, field morbidity is inhomogeneous, between the time, difference is large, and breeding parent and filial generation material qualification result are inaccurate, and material is elected or superseded blindness is larger.After the nineties, verticillium occurs gradually seriously, and in the urgent need to cultivating resisting verticillium kind, but Disease Resistance Identification and seed selection still adopt and the similar method of anti-blight, breeding efficiency is low, progeny material is selected inaccurate, and improved variety disease resistance is poor, and disease-resistant variety output is lower.
Verticillium wilt resistance of cotton by same molecular mark technology is applied in fiber species improvement, sterile line improvement etc., but on resisting verticillium breed breeding owing to lacking reliable and stable, closely linked molecule marker, not yet application with produce actual.Lei Mengdeshi cotton is the wild cotton seed to verticillium high resistance, also be the genomic common ancestor of D of cultivation cotton seed upland cotton and sea island cotton, by to the analysis of the cotton genome disease-resistant gene of Lei Mengdeshi and the system development of SSR mark thereof, can obtain the resisting verticillium gene cluster of genetic stability in cultivation cotton seed and chain SSR mark thereof, and then to materials different in cotton breeding process, strain, kind is carried out early generation screening, eliminate not disease-resistant plant, save production cost, improve breeding and efficiency of selection, set up thus the verticillium wilt resistance of cotton by same breeding technique of efficiently and accurately.
Summary of the invention
The object of this invention is to provide a kind of method of cotton plants to verticillium wilt pathogen resistance that detect.
To achieve these goals, the invention provides a kind of method of cotton plants to verticillium wilt pathogen resistance that detect, the method comprises:
(1) seed of cotton plants to be measured is soaked with seed-soaking liquid; Described seed-soaking liquid is that the tissue of dying from the cotton plants of verticillium is pulverized, and is soaked in water, and filters the bacteria suspension obtaining;
(2) seed after soaking is planted in sick garden; Described sick garden is the tissue pulverizing of dying from the cotton plants of verticillium to be mixed to soil build afterwards;
(3) cotton plants that selection can be survived in sick garden is as primary dcreening operation cotton plants;
(4) use respectively the genomic dna of the first primer pair and the second primer pair primary dcreening operation cotton plants to carry out pcr amplification, obtain the first amplified production and the second amplified production; The first primer pair is as shown in SEQ ID NO:1 and SEQ ID NO:2, and the second primer pair is as shown in SEQ ID NO:3 and SEQ ID NO:4;
If have in the first amplified production in the nucleic acid shown in SEQ ID NO:5 and the second amplified production and have the nucleic acid shown in SEQ ID NO:6, indicating cotton plants to be measured is resisting verticillium plant.
By technique scheme, the present invention can realize cotton plants is detected easily and quickly to verticillium wilt pathogen resistance.
Other features and advantages of the present invention partly in detail are described the embodiment subsequently.
Accompanying drawing explanation
Accompanying drawing is to be used to provide a further understanding of the present invention, and forms a part for specification sheets, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.In the accompanying drawings:
Fig. 1 is the first amplified production electrophorogram of the cotton plants of cotton No. 1 of variety name sea island cotton 7124 and army.
Fig. 2 is the second amplified production electrophorogram of the cotton plants of cotton No. 1 of variety name sea island cotton 7124 and army.
Fig. 3 is the first amplified production electrophorogram of 12 primary dcreening operation cotton plants.
Fig. 4 is the second amplified production electrophorogram of 12 primary dcreening operation cotton plants.
Embodiment
Below the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only, for description and interpretation the present invention, is not limited to the present invention.
The invention provides a kind of method of cotton plants to verticillium wilt pathogen resistance that detect, the method comprises:
(1) seed of cotton plants to be measured is soaked with seed-soaking liquid; Described seed-soaking liquid is that the tissue of dying from the cotton plants of verticillium is pulverized, and is soaked in water, and filters the bacteria suspension obtaining;
(2) seed after soaking is planted in sick garden; Described sick garden is the tissue pulverizing of dying from the cotton plants of verticillium to be mixed to soil build afterwards;
(3) cotton plants that selection can be survived in sick garden is as primary dcreening operation cotton plants;
(4) use respectively the genomic dna of the first primer pair and the second primer pair primary dcreening operation cotton plants to carry out pcr amplification, obtain the first amplified production and the second amplified production; The first primer pair is as shown in SEQ ID NO:1 and SEQ ID NO:2, and the second primer pair is as shown in SEQ ID NO:3 and SEQ ID NO:4;
If have in the first amplified production in the nucleic acid shown in SEQ ID NO:5 and the second amplified production and have the nucleic acid shown in SEQ ID NO:6, indicating cotton plants to be measured is resisting verticillium plant.
Wherein, if do not had in the first amplified production in the nucleic acid shown in SEQ ID NO:5 or the second amplified production, do not there is the nucleic acid shown in SEQ ID NO:6, indicate cotton plants to be measured for sense verticillium plant.
Wherein, the condition of described pcr amplification can comprise: denaturation temperature is 93.5-94.5 ℃; Annealing temperature is 54.5-55.5 ℃.
Wherein, the condition of described pcr amplification can also comprise: PCR cycle number is 28-32 wheel.
Wherein, while preparing seed-soaking liquid, with respect to the tissue of the cotton plants of dying from verticillium of every weight part, the consumption of water can be 5 weight parts.
Wherein, with seed-soaking liquid, soak kind of a period of the day from 11 p.m. to 1 a.m, with respect to the seed of every weight part, the consumption of seed-soaking liquid can be 2 weight parts.
Wherein, with the time of seed-soaking liquid immersion seed, it can be 6 hours.
Wherein, while building disease garden, with respect to the soil of every square metre, the incorporation of tissue of dying from the cotton plants of verticillium can be 3kg.
Wherein, the genomic dna of cotton plants to be measured can be extracted and obtain by conventional DNA extraction method from cotton plants to be measured.
The present invention can predict and screen the resistance of verticillium wilt pathogen cotton plants by detection molecules mark, though eliminate, do not fall ill but still be susceptible plant in the screening process of sick garden, reduces the waste of manpower and materials, improves breeding efficiency.
Below will describe the present invention by embodiment.
Embodiment 1
1) collect 31 kinds of cotton seed materials, according to document (Zhang Xinghua, Resistance Strain of Cotton is withered, verticillium progress and Resistance Identification method thereof, Agriculture in Jiangxi journal, 2008,20 (3): sick garden, the artificial seedbed method 43-49), identify their disease resistance, result as listed in Table 1, wherein, 3 parts of disease-resistant germplasms, are expressed as " R " on resisting verticillium material one hurdle of table 1; 28 parts of susceptible germplasms, are expressed as " S " on sense verticillium material one hurdle of table 1.
(2) according to the method in < < molecular cloning experiment guide > >, the genomic dna of separated every part of germplasm.
The genomic dna of every part of germplasm of take is template, adopt forward primer (5 '-AAACAATAATAAACGAGTTGAATTA-3 ') and reverse primer first primer pair of (5 '-TTGTTTCATAATTTTAAAGTATGTA-3 ') as shown in SEQ ID NO:2 as shown in SEQ ID NO:1 to carry out pcr amplification, denaturation temperature is 94 ℃; Annealing temperature is 55 ℃: PCR cycle number is 30 to take turns; Obtain respectively the first amplified production of every part of material.
The genomic dna of every part of germplasm of take is template, adopt forward primer (5 '-AAGGGTAAGTAATAAATCATAGAAC-3 ') and reverse primer second primer pair of (5 '-GCATTTATCCATTTCTTTACTTTTC-3 ') as shown in SEQ ID NO:4 as shown in SEQ ID NO:3 to carry out pcr amplification, denaturation temperature is 94 ℃; Annealing temperature is 55 ℃: PCR cycle number is 30 to take turns; Obtain respectively the second amplified production of every part of material.
(3) according to the method in < < molecular cloning experiment guide > >, the first amplified production in step (2) is carried out to electrophoresis detection on polyacrylamide gel; Fig. 1 is the first amplified production electrophorogram of the cotton plants of cotton No. 1 of variety name sea island cotton 7124 and army; Consistent with sea island cotton 7124 banding patterns is expressed as "+" in Markers for Detection result one hurdle of table 1; Consistent with army cotton No. 1 banding pattern is expressed as "-" in Markers for Detection result one hurdle of table 1.In the electrophorogram of sea island cotton 7124 (the left swimming lane of Fig. 1), the size of the 3rd band is 185bp from top to bottom, sequence through order-checking is SEQ ID NO:5(AAACAATAATAAACGAGTTGAATTAAAATGCTTTTGTAATTAAATGGGCAT AAAAAGATAGACATTTTGCGAATCAACAAGACCATAAAATATATGAAAATTTTAAT ATATATATATATGTATGTGTATGTACATAAAATATGAAAAAAATATGTAGGTATAC ATACTTTAAAATTATGAAACAA), be the differential band with respect to cotton No. 1 of army.
According to the method in < < molecular cloning experiment guide > >, the second amplified production in step (2) is carried out to electrophoresis detection on polyacrylamide gel; Fig. 2 is the second amplified production electrophorogram of the cotton plants of cotton No. 1 of variety name sea island cotton 7124 and army; Consistent with sea island cotton 7124 banding patterns is expressed as "+" in Markers for Detection result one hurdle of table 1; Consistent with army cotton No. 1 banding pattern is expressed as "-" in Markers for Detection result one hurdle of table 1.In the electrophorogram of sea island cotton 7124 (the left swimming lane of Fig. 2), the size of the 1st band is 249bp from top to bottom, sequence through order-checking is SEQ ID NO:6(5 '-AAGGGTAAGTAATAAATCATAGAACACAATAATAATGACGTAATTATAATAATAAT CACCAAATAATAATAACAATGACCATATTAATTACTATTCTAATACTAAAAATATA AAAATGAATTAAACACTAATAAAAATGATAATAATAAAATAATAATAATAGTGATA ATAATAATGAAATGTATAAATAAAGGAATAAATAACAATATAAATTCTAAACACAA GAAAAGTAAAGAAATGGATAAATGC-3 '), it is the differential band with respect to cotton No. 1 of army.
Table 1
The result of table 1 proves: if had in the first amplified production in the nucleic acid shown in SEQ ID NO:5 and the second amplified production, have the nucleic acid shown in SEQ ID NO:6, indicating cotton plants to be measured is resisting verticillium plant; If do not have in the first amplified production in the nucleic acid shown in SEQ ID NO:5 or the second amplified production and do not there is the nucleic acid shown in SEQ ID NO:6, indicate cotton plants to be measured for sense verticillium plant.
Embodiment 2
In the present embodiment, cotton plants to be measured is planted the F2 of cotton No. 2 (GK44) choosing system and new 59-4 for seed in being.In plant cotton No. 2 (GK44) choosing system and new 59-4 F2 for seed, to the resistance of verticillium, be ignorant in advance.
Will die from the tissue of cotton plants of verticillium pulverize, the water soaking 48 hours of weight such as use, filter and obtain the seed-soaking liquid that contains verticillium pathogenic bacterium.
By in plant cotton No. 2 (GK44) choosing system and new 59-4 F2 for seed, be soaked in the above-mentioned seed-soaking liquid that contains verticillium pathogenic bacterium 24 hours, the seed after being soaked, wherein, with respect to the seed of every weight part, the consumption of seed-soaking liquid is 5 weight parts.
The tissue of dying from the cotton plants of verticillium is pulverized, mixed in soil, with respect to the soil of every square metre, the incorporation of tissue of dying from the cotton plants of verticillium is 3kg, builds disease garden.Seed after soaking is planted in sick garden, grow altogether the cotton seedling of 197 strains, but only have 12 strain survivals final results seed, all the other all die from verticillium, obtain the cotton plants of 12 resisting verticilliums, are recorded as respectively primary dcreening operation cotton plants 1-12.
Extract the DNA of 12 primary dcreening operation cotton plants, adopt forward primer (5 '-AAACAATAATAAACGAGTTGAATTA-3 ') and reverse primer first primer pair of (5 '-TTGTTTCATAATTTTAAAGTATGTA-3 ') as shown in SEQ ID NO:2 as shown in SEQ ID NO:1 to carry out pcr amplification, obtain respectively the first amplified production of every part of material, and adopt forward primer (5 '-AAGGGTAAGTAATAAATCATAGAAC-3 ') and reverse primer second primer pair of (5 '-GCATTTATCCATTTCTTTACTTTTC-3 ') as shown in SEQ ID NO:4 as shown in SEQ ID NO:3 to carry out pcr amplification, obtain respectively the second amplified production of every part of material, the first amplified production and the second amplified production are carried out to electrophoresis detection on polyacrylamide gel, Fig. 3 is the first amplified production electrophorogram of 12 primary dcreening operation cotton plants.The electrophoresis result of amplified production shows, plant 8,9 to be measured is consistent with sea island cotton 7124 with 12 molecule marker band, having sequence is SEQ ID NO:5(5 '-AAACAATAATAAACGAGTTGAATTAAAATGCTTTTGTAATTAAATGGGCATAAAAA GATAGACATTTTGCGAATCAACAAGACCATAAAATATATGAAAATTTTAATATATA TATATATGTATGTGTATGTACATAAAATATGAAAAAAATATGTAGGTATACATACT TTAAAATTATGAAACAA-3 ') differential band, for "+", plant 1-7 to be measured, 10 and 12 molecule marker are "-".Fig. 4 is the second amplified production electrophorogram of 12 primary dcreening operation cotton plants.The electrophoresis result of amplified production shows, plant 8 to be measured, 9 is consistent with sea island cotton 7124 with 12 molecule marker band, having sequence is SEQ ID NO:6(5 '-AAGGGTAAGTAATAAATCATAGAACACAATAATAATGACGTAATTATAATAATAAT CACCAAATAATAATAACAATGACCATATTAATTACTATTCTAATACTAAAAATATA AAAATGAATTAAACACTAATAAAAATGATAATAATAAAATAATAATAATAGTGATA ATAATAATGAAATGTATAAATAAAGGAATAAATAACAATATAAATTCTAAACACAA GAAAAGTAAAGAAATGGATAAATGC-3 ') differential band, for "+", plant 1-7 to be measured, 10 and 12 molecule marker is "-".
According to the above results, indicating plant 8,9 to be measured and 12 is the disease-resistant plant of verticillium, and plant 1-7,10 and 12 is verticillium disease plant.
Comparative example 1
According to document (Zhang Xinghua, Resistance Strain of Cotton is withered, verticillium progress and Resistance Identification method thereof, Agriculture in Jiangxi journal, 2008,20 (3): sick garden, the artificial seedbed method 43-49), primary dcreening operation cotton plants 1-12 to embodiment 2 carries out Disease Resistance Identification again, plant 8,9 and 12 verticillium feelings index are all less than 20, are verticillium wilt-resistant cotton plant, and plant 1-7,10 and 12 verticillium feelings index are all greater than 40, for verticillium disease plant, come to the same thing with the indication of embodiment 2.
More visible by embodiment 2 and comparative example 1, method of the present invention can realize the detection accurately to resistance to verticillium wilt to cotton plants.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characterictic described in above-mentioned embodiment, in reconcilable situation, can combine by any suitable mode, for fear of unnecessary repetition, the present invention is to the explanation no longer separately of various possible array modes.
In addition, between various embodiment of the present invention, also can carry out arbitrary combination, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (6)
1. detect the method for cotton plants to resistance to verticillium wilt, it is characterized in that, the method comprises:
(1) seed of cotton plants to be measured is soaked with seed-soaking liquid; Described seed-soaking liquid is that the tissue of dying from the cotton plants of verticillium is pulverized, and is soaked in water, and filters the bacteria suspension obtaining;
(2) seed after soaking is planted in sick garden; Described sick garden is the tissue pulverizing of dying from the cotton plants of verticillium to be mixed to soil build afterwards;
(3) cotton plants that selection can be survived in sick garden is as primary dcreening operation cotton plants;
(4) use respectively the genomic dna of the first primer pair and the second primer pair primary dcreening operation cotton plants to carry out pcr amplification, obtain the first amplified production and the second amplified production; The first primer pair is as shown in SEQ ID NO:1 and SEQ ID NO:2, and the second primer pair is as shown in SEQ ID NO:3 and SEQ ID NO:4;
If contained in the first amplified production just like containing just like the nucleic acid shown in SEQ ID NO:6 in the nucleic acid shown in SEQ ID NO:5 and the second amplified production, indicating cotton plants to be measured is resisting verticillium plant;
Wherein, the condition of described pcr amplification comprises: denaturation temperature is 93.5-94.5 ℃; Annealing temperature is 54.5-55.5 ℃.
2. method according to claim 1, wherein, the condition of described pcr amplification also comprises: PCR cycle number is 28-32 wheel.
3. method according to claim 1, wherein, while preparing seed-soaking liquid, with respect to the tissue of the cotton plants of dying from verticillium of every weight part, the consumption of water is 5 weight parts.
4. according to the method described in claim 1 or 3, wherein, with seed-soaking liquid, soak kind of a period of the day from 11 p.m. to 1 a.m, with respect to the seed of every weight part, the consumption of seed-soaking liquid is 2 weight parts.
5. method according to claim 4, wherein, the time of soaking seed with seed-soaking liquid is 6 hours.
6. method according to claim 1, wherein, while building disease garden, with respect to the soil of every square metre, the incorporation of tissue of dying from the cotton plants of verticillium is 3kg.
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| CN108018373B (en) * | 2014-09-26 | 2021-06-15 | 中国农业科学院棉花研究所 | Molecular marker of QTL/major gene related to verticillium wilt resistance from sea island cotton Hai1 |
| CN104846109B (en) * | 2015-05-29 | 2017-04-19 | 江苏省农业科学院 | Molecular marker with cotton verticillium wilt functional gene and application of molecular marker |
| CN104878003A (en) * | 2015-06-15 | 2015-09-02 | 南京农业大学 | Cotton verticillium wilt resistance major QTL (Quantitative Trait Locus) and linked molecular marker |
| CN106755369B (en) * | 2016-12-09 | 2018-10-23 | 中国农业科学院农产品加工研究所 | Molecular labeling and primer pair and detection cotton plants are to the method and kit of verticillium wilt pathogen resistance |
| CN106520990B (en) * | 2016-12-09 | 2017-11-07 | 中国农业科学院农产品加工研究所 | Primer pair and molecular labeling and detection cotton plants are to the method and kit of verticillium wilt pathogen resistance |
| CN111349712B (en) * | 2020-03-12 | 2022-07-12 | 江苏省农业科学院 | Drought-resistant related SSR (simple sequence repeat) sequence from abnormal cotton and application thereof |
| CN110129475B (en) * | 2019-05-09 | 2022-07-12 | 江苏省农业科学院 | SSR nucleic acid sequence related to cotton high coat score and application thereof |
| CN110093442B (en) * | 2019-05-09 | 2022-07-12 | 江苏省农业科学院 | SSR molecular markers related to cotton short stalk and high coat |
| CN111763757B (en) * | 2020-06-24 | 2022-06-28 | 江苏省农业科学院 | Abnormal cotton chromosome fragment capable of causing cotton upland death, molecular marker and application |
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| 《"湘农大棉1号"的SSR数字指纹图谱构建及纯度鉴定》;葛瑞华 等;《中国农学通报》;20121231;第28卷(第6期);79-83 * |
| 《QTLs mapping for Verticillium wilt resistance at seedling and maturity stages in Gossypium barbadense》;Yang C.等;《Plant Science》;20081231;第174卷(第3期);第290-298页 * |
| 《基于人工病圃筛选和分子标记辅助的棉花抗黄萎病育种方法研究与应用》;祈伟彦 等;《分子植物育种》;20121231;第10卷(第5期);第607-612页 * |
| 《海岛棉品种抗黄萎病基因SSR标记的验证及克隆》;王省芬 等;《植物遗传资源学报》;20071231;第8卷(第2期);第149-152页 * |
| Yang C.等.《QTLs mapping for Verticillium wilt resistance at seedling and maturity stages in Gossypium barbadense》.《Plant Science》.2008,第174卷(第3期),第290-298页. |
| 王省芬 等.《海岛棉品种抗黄萎病基因SSR标记的验证及克隆》.《植物遗传资源学报》.2007,第8卷(第2期),第149-152页. |
| 祈伟彦 等.《基于人工病圃筛选和分子标记辅助的棉花抗黄萎病育种方法研究与应用》.《分子植物育种》.2012,第10卷(第5期),第607-612页. |
| 葛瑞华 等.《"湘农大棉1号"的SSR数字指纹图谱构建及纯度鉴定》.《中国农学通报》.2012,第28卷(第6期),79-83. |
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