WO2021259025A1 - 能引起陆地棉致死的异常棉染色体片段及其分子标记 - Google Patents

能引起陆地棉致死的异常棉染色体片段及其分子标记 Download PDF

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WO2021259025A1
WO2021259025A1 PCT/CN2021/097861 CN2021097861W WO2021259025A1 WO 2021259025 A1 WO2021259025 A1 WO 2021259025A1 CN 2021097861 W CN2021097861 W CN 2021097861W WO 2021259025 A1 WO2021259025 A1 WO 2021259025A1
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seq
primer sequence
cotton
abnormal
reverse primer
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徐鹏
沈新莲
郭琪
徐珍珍
孟珊
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江苏省农业科学院
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Priority to US17/783,664 priority Critical patent/US20230037213A1/en
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the invention belongs to the technical field of molecular breeding, and specifically relates to an abnormal cotton chromosome fragment and its molecular marker that can cause death in upland cotton.
  • Stepbbins 1950.
  • the first category is called geographic isolation.
  • the same organism is divided into different populations due to geographical obstacles, so that genetic exchanges cannot occur between populations.
  • the second category is reproductive isolation before fertilization, which occurs before fertilization.
  • This isolation barrier is due to different flowering times between species, or pollen tubes cannot germinate after pollination or the pollen tubes cannot completely pass through the style to reach the ovary. , Or because of incompatibility between male and female gametes and other reasons, hybrid zygote cannot be formed.
  • the third category is reproductive isolation after fertilization, including hybrid lethality or hybrid weakness and hybrid breakdown.
  • hybrid lethality or hybrid disadvantage occurs in the F 1 generation, showing plant death, sterility, shortness, and reduced viability; while some interspecific hybrids can produce normal hybrid F 1 , but the F 2 generation plants often show disadvantages Or fatal, this phenomenon is called hybrid decline.
  • lethal genes are easily ignored and eliminated by breeders in production.
  • their special functions and mechanism of action still endow them with significance in scientific research and production.
  • reproductive isolation can break the reproductive barriers for traditional crop breeding and promote the exchange of genomes between species, which will help promote the application of excellent traits of wild species in modern breeding.
  • hybrid lethality and hybrid decline have been reported in different plants, and they have simple genetic methods.
  • Bateson (1909), Dobzhansky (1937) and Muller (1942) proposed the Bateson-Dobzhansky-Muller (BDM) model to explain the genetic basis of hybrid lethality and hybrid decline.
  • BDM Bateson-Dobzhansky-Muller
  • the classic BDM model includes the epistasis interaction of two points. Each branch derived from a common ancestor can form a different new site during the evolution process. These sites are harmless in their respective genomes, but in hybridization In species, these evolved genes interact.
  • the F 1 hybrid When the new mutant gene is dominant, the F 1 hybrid will be lethal; when the new mutant gene is recessive, the F 2 generation will show hybrid decline.
  • Dominant BDM interaction patterns can also occur between individual sites.
  • the uncoordinated evolutionary process of unit point means that a gene locus of an ancestor evolves in different directions in different branches, thus forming different genes in different populations.
  • ancestor genes and genes formed after evolution It is coordinated. There is no fundamental difference between a single point and two-point interaction mode. These genes evolve independently during the evolution process, and these sites have no negative effects in their respective genomes.
  • One of the objectives of the present invention is to provide an abnormal cotton chromosome fragment A11-9, which can cause death in upland cotton.
  • Another object of the present invention is to provide a molecular marker and primer sequence of the abnormal cotton chromosome segment A11-9, which molecular marker is closely linked to the lethal trait of the upland cotton-abnormal cotton introgression line.
  • the invention also protects the application of molecular markers and primers of the above-mentioned abnormal cotton chromosome fragment A11-9.
  • the present invention adopts the following technical solutions:
  • the abnormal cotton chromosome fragment A11-9 that can cause death in upland cotton.
  • the abnormal cotton chromosome fragment A11-9 is derived from abnormal cotton and is located on chromosome 11 of the cotton genome. It has 6 pairs of SSR markers: NAU5192, A11_175, JAAS3191, A11_243, JAAS3310, A11_193 markers; using abnormal cotton DNA as a template, the 6 pairs of SSR markers are used to simultaneously amplify the abnormal cotton DNA, and the chromosomal fragments containing the 6 SSR marker target bands are the abnormal cotton chromosomes.
  • Fragment A11-9; the 6 SSR marker primer sequences and their amplified fragment lengths are as follows:
  • NAU5192 The forward primer sequence is SEQ ID NO. 1, the reverse primer sequence is SEQ ID NO. 2, and the length of the amplified target fragment is 280 bp;
  • A11_175 The forward primer sequence is SEQ ID NO. 3, the reverse primer sequence is SEQ ID NO. 4, and the length of the amplified target fragment is 210 bp;
  • JAAS3191 The forward primer sequence is SEQ ID NO.5, the reverse primer sequence is SEQ ID NO.6, and the length of the amplified target fragment is 270bp;
  • A11_243 The forward primer sequence is SEQ ID NO. 7, the reverse primer sequence is SEQ ID NO. 8, and the length of the amplified target fragment is 280 bp;
  • JAAS3310 The forward primer sequence is SEQ ID NO. 9, the reverse primer sequence is SEQ ID NO. 10, and the length of the amplified target fragment is 250 bp;
  • A11_193 The forward primer sequence is SEQ ID NO.11, the reverse primer sequence is SEQ ID NO.12, and the length of the amplified target fragment is 250 bp;
  • the present invention also discloses the molecular markers of the abnormal cotton chromosome fragment A11-9.
  • the molecular markers are NAU5192, A11_175, JAAS3191, A11_243, JAAS3310, A11_193, and the primer sequences of each molecular marker and the lengths of the amplified bands are as follows :
  • NAU5192 The forward primer sequence is SEQ ID NO. 1, and the reverse primer sequence is SEQ ID NO. 2.
  • the length of the amplified target fragment in the abnormal cotton genome is 280 bp;
  • A11_175 The forward primer sequence is SEQ ID NO.3, the reverse primer sequence is SEQ ID NO.4, and the length of the amplified target fragment in the abnormal cotton genome is 210 bp;
  • JAAS3191 The forward primer sequence is SEQ ID NO.5, and the reverse primer sequence is SEQ ID NO.6.
  • the length of the amplified target fragment in the abnormal cotton genome is 270bp;
  • A11_243 The forward primer sequence is SEQ ID NO.7, the reverse primer sequence is SEQ ID NO.8, and the length of the amplified target fragment in the abnormal cotton genome is 280bp;
  • JAAS3310 The forward primer sequence is SEQ ID NO.9, the reverse primer sequence is SEQ ID NO.10, and the length of the amplified target fragment in the abnormal cotton genome is 250bp;
  • A11_193 The forward primer sequence is SEQ ID NO.11, the reverse primer sequence is SEQ ID NO.12, and the length of the amplified target fragment in the abnormal cotton genome is 250 bp;
  • the SSR marker primer on the abnormal cotton chromosome segment A11-9 is also disclosed, and the sequence of the marker primer is:
  • the SSR marker NAU5192 forward primer sequence is SEQ ID NO.1, and the reverse primer sequence is SEQ ID NO.2;
  • the SSR marker A11_175 forward primer sequence is SEQ ID NO. 3, and the reverse primer sequence is SEQ ID NO. 4;
  • SSR marker JAAS3191 forward primer sequence is SEQ ID NO.5, and reverse primer sequence is SEQ ID NO.6;
  • the SSR marker A11_243 forward primer sequence is SEQ ID NO.7, and the reverse primer sequence is SEQ ID NO.8;
  • SSR marker JAAS3310 forward primer sequence is SEQ ID NO.9
  • reverse primer sequence is SEQ ID NO.10;
  • the SSR marker A11_193 forward primer sequence is SEQ ID NO.11
  • the reverse primer sequence is SEQ ID NO.12.
  • the present invention also discloses the application of the above-mentioned molecular markers or molecular marker primers, including the application in the location of abnormal cotton lethal genes and the application in identifying the lethal traits of cotton plants.
  • the above-mentioned applications can be carried out in accordance with conventional methods.
  • kits containing the above-mentioned SSR-labeled primers can be used to identify whether the cotton material has lethal traits.
  • the kits can be selected to contain the above-mentioned primers.
  • the reagents used to detect the presence of the above-mentioned molecular markers The application in cotton lethal gene mapping, the molecular marker of the present invention can be used to locate the cotton lethal gene, and the above-mentioned applications can be carried out in accordance with conventional methods.
  • the method for constructing the upland cotton-abnormal cotton introgression system adopted in the present invention includes the following steps:
  • hexaploid F1 hybrids were obtained by doubling colchicine. Using morphology, cytology, molecular markers and other techniques to identify hexaploid hybrids that proved our double success (Zhang et al., 2014). Using hexaploid F 1 as the female parent and Su 8289 as the reincarnation parent, continue backcrossing for four consecutive times. In each generation combined with marker-assisted selection, a batch of abnormal cotton-derived introgression line populations were obtained, including a single fragment on chromosome A11 The introgression system CSSL11-9 is lethal.
  • lethal traits are divided into two types: lethal and normal.
  • the specific lethal traits are as follows: when the plant grows to about 7-8 fruit branches, the top leaves turn red and gradually spread throughout the plant, then the whole leaves wither and fall off, and the top of the plant is necrotic, and finally the plant is bare-rod-shaped; there are a few plants After the apex is necrotic, two new side ends emerge from the side, which can bloom normally and become bolls, but there are fewer bolls. Individual plants with red leaves on both sides and lethal symptoms are classified as lethal, while individual plants with normal green leaves on the entire plant are classified as normal.
  • the present invention obtains a single-fragment introgression line derived from abnormal cotton lethal traits, and creates important materials for promoting the fine positioning of target genes and subsequent map-based cloning.
  • the use of the molecular marker of the present invention can lay a technical foundation for the study of the molecular mechanism of lethal formation of introgression lines, and the use of the molecular marker can quickly identify whether a cotton plant is a lethal trait.
  • the molecular marker of the present invention has the characteristics of convenient detection, stable amplification product, and high specificity, and can be applied to cotton material identification simply, quickly and with high throughput.
  • Figure 1 shows the target bands amplified by the molecular markers on the 6 pairs of abnormal cotton chromosome fragments A11-9 of the present invention in the genome of Su 8289, CSSL11-9 and abnormal cotton;
  • lanes 1, 2, and 3 represent Su-8289, CSSL11-9 and abnormal cotton, respectively, and the arrows indicate the target bands specifically amplified by abnormal cotton.
  • Figure 2 shows the field phenotypes of normal plants and lethal plants of the present invention
  • panels a and c are plants with a normal phenotype
  • panels b and d are plants with a lethal phenotype.
  • the upland cotton Su 8289 (reincarnation parent) and abnormal cotton (donor parent) used in this experiment were introduced by the Jiangsu Academy of Agricultural Sciences and were recorded in "Caijiao Zhai, Peng Xu, Xia Zhang, etc. Development of Gossypium anomalum derived microsatellite markers and their use for gene-wide identification of recombination between the G. anomalum and G. hirsutum genes.
  • Theoretical and Applied Genetics, 2015, 128(8): 1531-1540 the public can obtain from the applicant, and can only be used to repeat the experiment of the present invention Use it for no other purpose, and it can also be obtained through purchase.
  • the method of extracting genomic DNA is:
  • the Taq enzyme and dNTPs used in the PCR reaction were purchased from Dalian Bao Bioengineering Co., Ltd.
  • the reagents used in the PAGE gel include acrylamide, methylene acrylamide, Tris-alkali, boric acid, silver nitrate, sodium hydroxide, TEMED and other reagents from Rongshengda Procurement by Experimental Instruments Co., Ltd.
  • the main instruments include Applied Biosystems PCR machine, Eppendor high-speed refrigerated centrifuge, water bath, shaker and electrophoresis tank and electrophoresis machine produced by Beijing Liuyi Instrument Factory.
  • the PCR reaction system is shown in Table 3.
  • the PCR reaction is carried out on the Applied Biosystems PCR machine, and the reaction procedure is:
  • the amplified products are electrophoresed by non-denaturing polyacrylamide gel: gel concentration is 9%, electrophoresis buffer is 0.5 ⁇ TBE, 180V constant pressure electrophoresis for 1.5 to 2 hours.
  • Loading buffer 0.25g bromophenol blue + 0.25g xylene cyanide + 40g sucrose, distilled water to make the volume to 100ml.
  • Dyeing solution 1g silver nitrate + 500ml distilled water.
  • Color developing solution 7.5g sodium hydroxide + 750 ⁇ L formaldehyde + 500ml distilled water.
  • Fixation Put the removed gel into the fixative solution (10% ethanol + 0.5% glacial acetic acid) for 12 minutes,
  • Color development add 1.5% sodium hydroxide + 0.4% formaldehyde, shake, until the strips on the film are clearly displayed, it can be finished, it is suitable to pour out the color development liquid, rinse 4 times with tap water, and place the film on the light box to take pictures .
  • hexaploid F1 hybrids were obtained by doubling colchicine. Using morphology, cytology, molecular markers and other techniques to identify hexaploid hybrids that proved that we have doubled success (Zhang et al., 2014). Using the hexaploid F1 as the female parent and Su 8289 as the reincarnation parent, continue backcrossing four times in succession. In each generation combined with marker-assisted selection, a batch of introgressive line populations derived from abnormal cotton was obtained. Among them, the single fragment on the A11 chromosome gradually Infiltration system CSSL11-9 is lethal.
  • the molecular weights of specific bands amplified by 6 molecular markers (NAU5192, A11_175, JAAS3191, A11_243, JAAS3310, A11_193) in the abnormal cotton genome are: 280bp, 210bp, 270bp, 280bp, 250bp, 250bp ( Figure 1). And it has been preliminarily located, and it is located on chromosome 11 of the cotton genome. Select individual plants that also contain the fragment (see Table 4).
  • lethal traits are divided into two types: lethal and normal.
  • the specific lethal traits are as follows: first, when the plant grows to about 7-8 fruiting branches, the top leaves turn red (Figure 2b) and gradually spread throughout the plant, then the whole leaves wither and fall off, and the top of the plant is necrotic at the same time, and finally the plant becomes a bare rod. (Figure 2d); a small part of the plant tops have two new lateral ends after necrosis, which can bloom normally, form bolls and spit out, but there are fewer bolls. Individual plants with red leaves on the front and back and with lethal symptoms are classified as lethal, while individual plants with normal green leaves in the entire plant are classified as normal ( Figure 2a, Figure 2c).

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Abstract

提供一种能引起陆地棉致死的异常棉染色体片段及分子标记，所述染色体片段A11-9来自于异常棉，位于棉花染色体组的第11号染色体，通过6对SSR标记：NAU5192、A11_175、JAAS3191、A11_243、JAAS3310、A11_193标记，以异常棉的DNA为模板，使用6对SSR标记同时对异常棉的DNA扩增，同时含有6对SSR标记的目的条带的染色体片段即为异常棉的染色体片段A11-9；还提供了源于异常棉致死性状的单片段渐渗系，为促进目标基因的精细定位及其以后的图位克隆创造了重要材料。

Description

能引起陆地棉致死的异常棉染色体片段及其分子标记 技术领域
本发明属于分子育种技术领域，具体涉及一种能引起陆地棉致死的异常棉染色体片段及其分子标记。
背景技术
早在20世纪初人们就意识到利用野生资源可以补偿现代育种失去的优异等位基因，但是种间生殖隔离限制了野生资源在育种中的利用。生殖隔离主要分为3种类型(Stebbins，1950)。第一类叫做地理隔离，同一种生物由于地理上的障碍而分成不同的种群，使种群间不能发生基因交流的现象。第二类是受精前的生殖隔离，它发生在受精以前，这种隔离障碍是由于物种之间不同的开花时间，或植物授粉后由于花粉管不能萌发或者花粉管不能完全穿过花柱达到子房，或者因为雌雄配子间不相容等原因而导致杂种合子不能形成。第三类是受精后生殖隔离，包括杂种致死(hybrid lethality)或称为杂种劣势(hybrid weakness)和杂种衰退(hybrid breakdown)两种类型。其中杂种致死或杂种劣势发生在F 1代，表现出植株死亡、不育、矮小以及生活力下降等；而有些种间杂交能产生正常的杂种F 1，然而其F 2代的植株往往表现劣势或者致死，这种现象称为杂种衰退。致死基因作为有害的遗传物质，在生产上很容易被育种家忽视而淘汰，然而其特殊的功能和作用机制仍然赋予其在科研和生产中的意义。阐明生殖隔离背后的机制可以为传统作物育种打破生殖障碍，促进种间基因组的交换，将有利于促进野生种优异性状在现代育种中的应用。
目前，有关杂种致死和杂种衰退在不同植物中均有报道，它们具有简单的遗传方式。杂种致死表型发生在F 1代，大多由一对或者两对显性基因控制，而杂种衰退则在F 2代表现，大多由一对或者两对隐性基因控制。Bateson(1909)、Dobzhansky(1937)和Muller(1942)提出Bateson-Dobzhansky-Muller(BDM)模型解释了杂种致死及杂种衰退产生的遗传基础。经典的BDM模型包括两位点的上位性互作，从共同祖先分化出来的每一个分支可以在进化过程中形成不同的新位点，这些位点在各自基因组中是无害的，然而在杂交种中，这些进化而来的基因产生互作，当新突变基因为显性时，从而导致F 1杂种致死；新突变基因为隐性时，则在F 2代表现杂种衰退。显性BDM 互作模式也可以发生在单个位点之间。单位点不协调的进化过程，表示祖先的一个基因位点在不同的分支中向不同的方向进化，从而在不同的种群中形成了不同的基因，在进化过程中祖先基因与进化后形成的基因是协调的。单位点与两位点互作模式没有根本区别，在进化过程中这些基因独立进化，而且这些位点在各自的基因组内是没有负作用的。大多数杂种致死和杂种衰退的报道(Bomblies et al.，2007；Yamamoto et al.，2007；Chen et al.，2013；Tikhenko et al.，2017)都是由两位点模型作用的结果。而单位点模式的报道则较少(Smith et al.，2011)。
植物杂种致死基因的克隆是解析杂种致死机理的分子基础，也是在根本上理解植物杂种致死现象的发生作用机理的关键，并有助于了解杂种致死基因的进化在生殖隔离与物种形成中的重要作用。现有研究已对杂种致死和杂种衰退相关基因在不同物种中进行了基因定位，部分基因已经被克隆。关于其致死分子机制，普遍认为杂种致死或衰退主要涉及到植物的免疫系统。目前棉花杂种致死研究相对滞后，不少学者相继发现了杂种致死现象，部分致死基因被染色体定位，未见其分子机制的研究报道。
发明内容
本发明的目的之一是提供一种异常棉染色体片段A11-9，所述片段能引起陆地棉致死。
本发明的另一目的是提供一种异常棉染色体片段A11-9的分子标记及引物序列，该分子标记与陆地棉-异常棉渐渗系致死性状紧密连锁。
本发明还保护上述异常棉染色体片段A11-9的分子标记及引物的应用。
为解决上述技术问题，本发明采用如下技术方案：
一种能引起陆地棉致死的异常棉染色体片段A11-9，所述异常棉染色体片段A11-9来自于异常棉，位于棉花染色体组的第11号染色体，通过6对SSR标记：NAU5192，A11_175，JAAS3191，A11_243，JAAS3310，A11_193标记；以异常棉的DNA为模板，使用所述6对SSR标记同时对异常棉的DNA扩增，同时含有6个SSR标记目的条带的染色体片段即为异常棉染色体片段A11-9；所述6个SSR标记引物序列及其扩增片段长度如下：
NAU5192：正向引物序列为SEQ ID NO.1，反向引物序列为SEQ ID NO.2，扩增目的片段长度为280bp；
A11_175：正向引物序列为SEQ ID NO.3，反向引物序列为SEQ ID NO.4，扩增目的片段长度为210bp；
JAAS3191：正向引物序列为SEQ ID NO.5，反向引物序列为SEQ ID NO.6，扩增目的片段长度为270bp；
A11_243：正向引物序列为SEQ ID NO.7，反向引物序列为SEQ ID NO.8，扩增目的片段长度为280bp；
JAAS3310：正向引物序列为SEQ ID NO.9，反向引物序列为SEQ ID NO.10，扩增目的片段长度为250bp；
A11_193：正向引物序列为SEQ ID NO.11，反向引物序列为SEQ ID NO.12，扩增目的片段长度为250bp；
本发明还公开了异常棉染色体片段A11-9的分子标记，所述分子标记分别为NAU5192，A11_175，JAAS3191，A11_243，JAAS3310，A11_193，各分子标记的引物序列及其扩增目的条带的长度如下：
NAU5192：正向引物序列为SEQ ID NO.1，反向引物序列为SEQ ID NO.2，在异常棉基因组中扩增目的片段长度为280bp；
A11_175：正向引物序列为SEQ ID NO.3，反向引物序列为SEQ ID NO.4，，在异常棉基因组中扩增目的片段长度为210bp；
JAAS3191：正向引物序列为SEQ ID NO.5，反向引物序列为SEQ ID NO.6，在异常棉基因组中扩增目的片段长度为270bp；
A11_243：正向引物序列为SEQ ID NO.7，反向引物序列为SEQ ID NO.8，在异常棉基因组中扩增目的片段长度为280bp；
JAAS3310：正向引物序列为SEQ ID NO.9，反向引物序列为SEQ ID NO.10，在异常棉基因组中扩增目的片段长度为250bp；
A11_193：正向引物序列为SEQ ID NO.11，反向引物序列为SEQ ID NO.12，在异常棉基因组中扩增目的片段长度为250bp；
另一方面，还公开了异常棉染色体片段A11-9上的SSR标记引物，所述标记引物的序列为：
SSR标记NAU5192正向引物序列为SEQ ID NO.1，反向引物序列为SEQ ID NO.2；
SSR标记A11_175正向引物序列为SEQ ID NO.3，反向引物序列为SEQ ID NO.4；
SSR标记JAAS3191正向引物序列为SEQ ID NO.5，反向引物序列为SEQ ID NO.6；
SSR标记A11_243正向引物序列为SEQ ID NO.7，反向引物序列为SEQ ID NO.8；
SSR标记JAAS3310正向引物序列为SEQ ID NO.9，反向引物序列为SEQ ID NO.10；
SSR标记A11_193正向引物序列为SEQ ID NO.11，反向引物序列为SEQ ID NO.12。
另外，本发明还公开了上述分子标记或分子标记引物的应用，包括在异常棉致死基因定位中的应用以及在鉴定棉花植株致死性状中的应用。上述这些应用均可以按照常规的方法进行。
另外，包含上述SSR标记引物的试剂盒，可以用来鉴定棉花材料是否具有致死性状，具体应用时，可以选择含有上述引物做成试剂盒，再者，用于检测上述分子标记是否存在的试剂在棉花致死基因定位中的应用，利用本发明的分子标记，可以对棉花致死基因进行定位，上述这些应用均可以按照常规的方法进行。
本发明采用的陆地棉-异常棉渐渗系的构建方法，包括如下步骤：
(1)以陆地棉86-1为母本，异常棉为父本，通过秋水仙素加倍获得了六倍体F1杂种。利用形态学、细胞学、分子标记等技术鉴定证明我们获得加倍成功的六倍体杂交种(Zhang et al.，2014)。以六倍体F 1为母本、苏8289为轮回亲本继续连续回交四次，每一世代结合标记辅助选择，获得了一批异常棉来源的渐渗系群体，其中A11染色体上的单片段渐渗系CSSL11-9存在致死现象。
(2)种植单片段渐渗系CSSL11-9产生的BC4F2植株，取幼嫩叶片，用CTAB法提取DNA作为模板，用所述的6对SSR标记为引物，进行PCR扩增，所述6对SSR标记在异常棉基因组中扩增的特异条带分子量分别是：280bp，210bp，270bp，280bp，250bp，250bp，选择同时包含6个分子标记特异条带的染色体片段即为异常棉染色体片段A11-9，仅包含纯合的异常棉分子标记特异条带的植株表现为致死性状。
(3)致死性状调查在大田分单株进行，从移栽前的幼苗期开始至成熟期，每隔两星期调查一次。表型性状划分为致死型和正常型两种。其致死性状具体表现为：植株大约长至7-8果枝时，顶端叶片变为红色，逐渐蔓延整个植株，随后整株叶片枯萎脱落，同时植株顶端坏死，最后植株呈光杆状；有少部分植株顶端在坏死后从侧面新发出两个侧端，能正常开花、成铃吐絮，但成铃较少。凡叶片正反面表现红色，并出现致死症状的单株归为致死型，而整个植株具有正常绿色叶片的单株归为正常型。
本发明具有如下优点：
本发明获得了源于异常棉致死性状的单片段渐渗系，为促进目标基因的精细定位及其以后的图位克隆创造了重要材料。
利用本发明的分子标记，可为渐渗系致死形成的分子机制研究奠定技术基础，并且，利用该分子标记可以快速的鉴定棉花植株是否为致死性状。
本发明的分子标记具有检测方便、扩增产物稳定、特异性高的特点，可以简便、快速、高通量地应用于棉花材料鉴定。
附图说明
图1是本发明6对异常棉染色体片段A11-9上分子标记在苏8289、CSSL11-9和异常棉基因组中扩增的目的条带；
图中，泳道1、2、3分别代表苏8289、CSSL11-9和异常棉，箭头所示为异常棉特异扩增的目的条带。
图2是本发明正常植株和致死植株的田间表型；
图中，图a和图c为正常表型植株，图b和图d为致死表型植株。
具体实施方式
下面将通过具体实施例对本发明进行详细的描述。提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
如在通篇说明书及权利要求当中所提及的“包含”或“包括”为一开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本发明的较佳实施方式，然所述描述乃以说明书的一般原则为目的，并非用以限定本发明的范围。本发明的保护范围当视所附权利要求所界定者为准。
1.试验材料
本实验所用的陆地棉苏8289(轮回亲本)以及异常棉(供体亲本)由江苏省农业科学院引进，均记载于“Caijiao Zhai，Peng Xu，Xia Zhang等.Development of Gossypium anomalum derived microsatellite markers and theiruse for genome-wide identification of recombination between the G.anomalumand G.hirsutum genomes.Theoretical and Applied Genetics，2015，128(8)：1531-1540”中，公众可从申请人处获得，仅可用于重复本发明实验使用，不得他用，也可以通过购买的途径获得。
2.试验方法
2.1提取基因组DNA的方法
种植CSSL11-9、苏8289和异常棉种子长成植株，提取基因组DNA。提取基因组DNA的方法为：
(1)将5g冻叶(或鲜叶)放入预冷的研钵中，加入液氮研磨。分2次加入10ml新鲜配制的提取缓冲液(表1)，转入50ml的离心管中，涡旋混匀，冰浴保存10min。4000rpm离心20min(4℃)，弃上清；
(2)于沉淀中加入15ml 65℃预热的裂解缓冲液(表2)，并用铜丝搅松，涡旋混匀，65℃水浴30min；
(3)加入15ml氯仿∶异戊醇(24∶1，体积比)混合液，翻转50次以上，4000rpm离心20min(15℃)，将上清转入50ml的离心管中，加0.6体积的预冷的异丙醇，缓慢翻转30次，混匀，静置10min，4000rpm，离心10min(室温)，弃上清，于沉淀中加入2ml 70％的乙醇洗涤，并转入10ml的离心管中，10000rpm离心5min。倒掉上清液，通风干燥20min；
(4)加3ml TE缓冲液(PH＝8.0，1.0M Tris-HCl溶液2.5ml和0.5M EDTA溶液0.5ml混匀，蒸馏水定容至250ml灭菌)溶解，65℃培养10～30min，加等体积的氯仿∶异戊醇(24∶1，体积比)，缓慢翻转50次，混匀，室温静置5min，10000rpm离心10min；
(5)上清液转入10ml离心管中，加0.1体积的3M醋酸钠(pH 5.2)，加等体积的异丙醇后翻转30次，放置30min，10000rpm离心5min。弃上清液，加2ml 70％乙醇洗DNA小团，10000rpm离心5min。倒掉上清液，通风干燥20min；
(6)加3ml TE缓冲液溶解，65℃培养10～30min；
(7)加5μl RNAase A(10mg/ml)，37℃温育30～60min。加等体积的氯仿∶异戊醇(24∶1，体积比)，缓慢翻转50次，混匀，室温静置5min，10000rpm离心10min；
(8)上清液转入10ml离心管中，加0.1体积的3M醋酸钠(pH 5.2)，加等体积的异丙醇后翻转30次；
(9)将絮状沉淀挑入加有800μl 70％乙醇的1.5ml的离心管中，10000rpm离心5min。弃上清液，自然通风干燥DNA小团；
(10)加200μl TE缓冲液[10mM Tris/HCl(pH 8.0)，1mM EDTA(pH 8.0)]溶解DNA(4℃，1～2天)，-20℃保存。
(11)以5μl大连宝生物50bp DNA Ladder作为对照，将DNA依次稀释5倍，10倍和20倍，通过1％琼脂糖凝胶电泳，确定DNA的浓度、纯度以及完整性。
(12)根据提取DNA的浓度，用TE将DNA稀释至20ng/μl工作液，混匀备用。
表1 DNA提取缓冲液配方
Figure PCTCN2021097861-appb-000001
表2裂解缓冲液配方
Figure PCTCN2021097861-appb-000002
2.2 PCR方法
(1)所用试剂及主要仪器
PCR反应所用Taq酶和dNTPs均购自大连宝生物工程有限公司，PAGE胶所用试剂包括丙烯酰胺，甲叉丙烯酰胺，Tris-碱，硼酸，硝酸银，氢氧化钠，TEMED等试剂从荣胜达实验仪器有限公司采购。主要仪器包括Applied Biosystems PCR仪，Eppendor高速冷冻离心机，水浴锅，摇床和北京六一仪器厂生产的电泳槽和电泳仪。
(2)PCR反应体系及扩增程序
PCR反应体系见表3。
表3 PCR反应体系
Figure PCTCN2021097861-appb-000003
Figure PCTCN2021097861-appb-000004
PCR反应在Applied Biosystems PCR仪上进行，反应程序为：
Figure PCTCN2021097861-appb-000005
电泳溶液配制：
扩增产物采用非变性聚丙烯酰胺凝胶电泳：凝胶浓度9％，电泳缓冲液为0.5×TBE，180V恒压电泳1.5～2小时。
9％PAGE胶：43.5g丙烯酰胺，1.5g甲叉丙烯酰胺，100ml 5×TBE，加蒸馏水定容至500ml，4℃保存。
10％过硫酸铵：10g过硫酸铵溶解在100ml蒸馏水中，4℃保存。
上样缓冲液：0.25g溴酚蓝+0.25g二甲苯氰+40g蔗糖，蒸馏水定容至100ml。
5×TBE：54g Tris-碱，27.5g硼酸，0.5M EDTA(PH＝8.0)20ml，蒸馏水定容至1L，室温保存。
染色液：1g硝酸银+500ml蒸馏水。
显色液：7.5g氢氧化钠+750μL甲醛+500ml蒸馏水。
凝胶制备及电泳过程：
(1)用清水将玻璃板、胶条和梳子洗净，晾干备用。
(2)将玻璃板、胶条和梳子按要求安装好，并用1％的琼脂糖凝胶封底，待凝胶凝固，将其固定在电泳槽上。
(3)在锥形瓶中倒入已经配制好的9％的PAGE胶，加入10％AP和TEMED，迅速灌胶，灌满后插入梳子，15-20分钟后，拔出梳子，准备电泳。
(4)电泳前在PCR扩增产物中加入2μL上样缓冲液，拔掉梳子，将电泳缓冲液(1×TBE)加入正负极电泳槽中，缓冲液高度要超过短玻璃板，每个点样孔上样2μL，180V恒压电泳1.5～2小时。到蓝色指示标记距胶下面2cm即可停止，小心拆开玻璃板去除凝胶，并对凝胶进行标记。
染色及显色过程：
(1)固定：将拆下来的凝胶放入固定液中(10％乙醇+0.5％冰乙酸)12min，
(2)染色：固定结束后，将固定液到出去，将染色液倒入(0.2％硝酸银水溶液)，12min后，蒸馏 水漂洗3次。
(3)显色：1.5％氢氧化钠+0.4％甲醛加入，摇晃，到胶片上条带显示清楚即可结束，适倒掉显色液，用自来水冲洗4次，将胶片放置在灯箱上拍照。
2.3标记的选择与鉴定
(1)以陆地棉86-1为母本，异常棉为父本，通过秋水仙素加倍获得了六倍体F1杂种。利用形态学、细胞学、分子标记等技术鉴定证明我们获得加倍成功的六倍体杂交种(Zhang et al.，2014)。以六倍体F1为母本、苏8289为轮回亲本继续连续回交四次，每一世代结合标记辅助选择，获得了一批异常棉来源的渐渗系群体，其中A11染色体上的单片段渐渗系CSSL11-9存在致死现象。
(2)种植单片段渐渗系CSSL11-9产生的BC4F2植株、轮回亲本苏8289以及异常棉，分别取幼嫩叶片，用CTAB法提取DNA作为模板，用本课题组开发的均匀覆盖异常棉染色体组的230对SSR引物进行检测，对单片段渐渗系CSSL11-9进行全基因组前景与背景鉴定。其中仅如表4中的6对SSR分子标记引物的扩增产物在单片段渐渗系CSSL11-9和轮回亲本苏8289之间有差异。然后取幼嫩叶片，用CTAB法提取DNA作为模板，用所述的6对SSR标记为引物，进行PCR扩增，选择同时包含6个分子标记特异条带的染色体片段即为异常棉染色体片段A11-9，仅包含纯合的异常棉分子标记特异条带的植株表现为致死性状。
6个分子标记(NAU5192，A11_175，JAAS3191，A11_243，JAAS3310，A11_193)在异常棉基因组中扩增的特异条带分子量分别是：280bp，210bp，270bp，280bp，250bp，250bp(图1)。并且对其进行了初步的定位，其在棉花染色体组第11号染色体上。选择同时包含该片段的单株(见表4)。通过标记鉴定，我们确定BC 4F 2单株中可扩 增出6个分子标记的目的片段，仅同时包含纯合的异常棉分子标记特异条带的植株表现为致死性状，即致死植株包含了异常棉染色体片段A11-9。
表4 A11-9染色体片段上的SSR分子标记及其序列
Figure PCTCN2021097861-appb-000006
然后再以苏8289为母本，CSSL11-9为父本构建了一个含有2337个单株的F 2群体，在大田分单株进行性状调查，从移栽前的幼苗期开始至成熟期，每隔两星期调查一次。表型性状划分为致死型和正常型两种。凡叶片正反面表现红色，并出现致死症状的单株归为致死型，而整个植株具有正常绿色叶片的单株归为正常型。结果显示2337个F 2单株中，表现为致死表型的单株545株，正常表型单株1792株，χ c 2＝3.516＜χ c 2 0.05，1＝3.84，表明正常表型和致死型符合3∶1分离比，因此认为源于异常棉的致死性状在此群体中是由一对隐性基因控制的遗传规律。
表5不同表型材料的基因型
Figure PCTCN2021097861-appb-000007
1：苏8269基因型；2：异常棉基因型；3：杂合基因型
2.4致死性状的表型鉴定
致死性状调查在大田分单株进行，从移栽前的幼苗期开始至成熟期，每隔两星期调查一次。表型性状划分为致死型和正常型两种。其致死性状具体表现为：首先植株大约长至7-8果枝时，顶端叶片变为红色(图2b)，逐渐蔓延整个植株，随后整株叶片枯萎脱落，同时植株顶端坏死，最后植株呈光杆状(图2d)；有少部分植株顶端在坏死后从侧面新发出两个侧端，能正常开花、成铃吐絮，但成铃较少。凡叶片正反 面表现红色，并出现致死症状的单株归为致死型，而整个植株具有正常绿色叶片的单株归为正常型(图2a、图2c)。
虽然，上文中已经用一般性说明及具体实施例对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (9)

  1. 能引起陆地棉致死的异常棉染色体片段A11-9，其特征在于，所述染色体片段A11-9来自于异常棉，位于棉花染色体组的第11号染色体，通过6对SSR标记：NAU5192、A11_175、JAAS3191、A11_243、JAAS3310、A11_193标记，以异常棉的DNA为模板，使用6对SSR标记同时对异常棉的DNA扩增，同时含有6对SSR标记的目的条带的染色体片段即为异常棉的染色体片段A11-9；所述6个SSR标记引物序列及其扩增片段长度如下：
    NAU5192：正向引物序列为SEQ ID NO.1，反向引物序列为SEQ ID NO.2，扩增目的片段长度为280bp；
    A11_175：正向引物序列为SEQ ID NO.3，反向引物序列为SEQ ID NO.4，扩增目的片段长度为210bp；
    JAAS3191：正向引物序列为SEQ ID NO.5，反向引物序列为SEQ ID NO.6，扩增目的片段长度为270bp；
    A11_243：正向引物序列为SEQ ID NO.7，反向引物序列为SEQ ID NO.8，扩增目的片段长度为280bp；
    JAAS3310：正向引物序列为SEQ ID NO.9，反向引物序列为SEQ ID NO.10，扩增目的片段长度为250bp；
    A11_193：正向引物序列为SEQ ID NO.11，反向引物序列为SEQ ID NO.12，扩增目的片段长度为250bp。
  2. 异常棉染色体片段A11-9的分子标记，其特征在于，所述分子标记由NAU5192、A11_175、JAAS3191、A11_243、JAAS3310、A11_193组成，各分子标记的引物序列及其扩增目的目标条带的长度如下：
    NAU5192：正向引物序列为SEQ ID NO.1，反向引物序列为SEQ ID NO.2，在异常棉基因组中扩增目的片段长度为280bp；
    A11_175：正向引物序列为SEQ ID NO.3，反向引物序列为SEQ ID NO.4，，在异常棉基因组中扩增目的片段长度为210bp；
    JAAS3191：正向引物序列为SEQ ID NO.5，反向引物序列为SEQ ID NO.6，在异常棉基因组中扩增目的片段长度为270bp；
    A11_243：正向引物序列为SEQ ID NO.7，反向引物序列为SEQ ID NO.8， 在异常棉基因组中扩增目的片段长度为280bp；
    JAAS3310：正向引物序列为SEQ ID NO.9，反向引物序列为SEQ ID NO.10，在异常棉基因组中扩增目的片段长度为250bp；
    A11_193：正向引物序列为SEQ ID NO.11，反向引物序列为SEQ ID NO.12，在异常棉基因组中扩增目的片段长度为250bp。
  3. 异常棉染色体片段A11-9上的SSR标记引物，其特征在于：所述标记引物的序列为：
    NAU5192：正向引物序列为SEQ ID NO.1，反向引物序列为SEQ ID NO.2；
    A11_175：正向引物序列为SEQ ID NO.3，反向引物序列为SEQ ID NO.4；
    JAAS3191：正向引物序列为SEQ ID NO.5，反向引物序列为SEQ ID NO.6；
    A11_243：正向引物序列为SEQ ID NO.7，反向引物序列为SEQ ID NO.8；
    JAAS3310：正向引物序列为SEQ ID NO.9，反向引物序列为SEQ ID NO.10；
    A11_193：正向引物序列为SEQ ID NO.11，反向引物序列为SEQ ID NO.12。
  4. 权利要求2所述的分子标记或权利要求3所述的分子标记引物在异常棉致死基因定位和棉花分子育种中的应用。
  5. 根据权利要求4所述的应用，其特征在于，所述分子标记或分子标记引物用于鉴定或辅助鉴定棉花植株致死性状。
  6. 根据权利要求5所述的应用，其特征在于，所述致死性状具体表现为：植株长至7-8果枝时，顶端叶片变为红色，逐渐蔓延整个植株，随后整株叶片枯萎脱落，同时植株顶端坏死，最后植株呈光杆状；或
    植株顶端在坏死后从侧面新发出两个侧端，能正常开花、成铃吐絮，但成铃少。
  7. 包含权利要求3所述标记引物的试剂盒。
  8. 权利要求7所述试剂盒在鉴定棉花植株致死性状中的应用。
  9. 用于检测权利要求2所述分子标记是否存在的试剂在棉花致死基因定位和/或鉴定棉花致死性状中的应用。
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Publication number Priority date Publication date Assignee Title
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005059146A1 (en) * 2003-12-15 2005-06-30 Vib Vzw Avehicle for plant transformation
CN105624289A (zh) * 2016-01-07 2016-06-01 中国农业科学院棉花研究所 引物组及其应用、利用该引物组进行棉花种质资源遗传多样性分析的方法
CN111763757A (zh) * 2020-06-24 2020-10-13 江苏省农业科学院 能引起陆地棉致死的异常棉染色体片段及其分子标记和应用

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8940485B2 (en) * 2008-11-12 2015-01-27 Apdn (B.V.I.) Inc. Methods for genotyping mature cotton fibers and textiles
CN102229979B (zh) * 2011-05-17 2012-11-14 南京农业大学 一种利用海岛棉染色体片段导入系改良棉花纤维长度的分子育种方法
CN103255139B (zh) * 2013-05-07 2015-04-15 南京农业大学 一个棉花高强纤维主效qtl及其分子标记和应用
CN103571852B (zh) * 2013-09-05 2016-01-13 南京农业大学 一个能显著增加陆地棉衣分的海岛棉染色体片段及其ssr标记
CN103602743B (zh) * 2013-11-20 2014-10-15 中国农业科学院农产品加工研究所 一种用于分子标记辅助育种检测棉花植株对黄萎病抗性的方法
CN104488695A (zh) * 2014-12-01 2015-04-08 中国农业科学院棉花研究所 一种培育特早熟、高衣分新品系中213的方法
CN110129475B (zh) * 2019-05-09 2022-07-12 江苏省农业科学院 棉花高衣分相关的ssr核酸序列及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005059146A1 (en) * 2003-12-15 2005-06-30 Vib Vzw Avehicle for plant transformation
CN105624289A (zh) * 2016-01-07 2016-06-01 中国农业科学院棉花研究所 引物组及其应用、利用该引物组进行棉花种质资源遗传多样性分析的方法
CN111763757A (zh) * 2020-06-24 2020-10-13 江苏省农业科学院 能引起陆地棉致死的异常棉染色体片段及其分子标记和应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DENG JIEQIONG, FANG LEI, ZHU XIEFEI, ZHOU BAOLIANG, ZHANG TIANZHEN: "A CC-NBS-LRR gene induces hybrid lethality in cotton", JOURNAL OF EXPERIMENTAL BOTANY, OXFORD UNIVERSITY PRESS, GB, vol. 70, no. 19, 15 October 2019 (2019-10-15), GB , pages 5145 - 5156, XP055882845, ISSN: 0022-0957, DOI: 10.1093/jxb/erz312 *
LI SONG ; WANGZHEN GUO ; TIANZHEN ZHANG: "Interaction of novel Dobzhansky–Muller type genes for the induction of hybrid lethality between Gossypium hirsutum and G. barbadense cv. Coastland R4-4", THEORETICAL AND APPLIED GENETICS ; INTERNATIONAL JOURNAL OF PLANT BREEDING RESEARCH, SPRINGER, BERLIN, DE, vol. 119, no. 1, 28 March 2009 (2009-03-28), Berlin, DE , pages 33 - 41, XP019698381, ISSN: 1432-2242 *
P. J. SAMORA, D. M. STELLY, R. J. KOHEL: "Localization and Mapping of the Le1 and Gl2 Loci of Cotton (Gossypium hirsutum L.)", THE JOURNAL OF HEREDITY, vol. 85, no. 2, March 1994 (1994-03-01), pages 152 - 157, XP009532923, ISSN: 0022-1503, DOI: 10.1093/oxfordjournals.jhered.a111418 *
蔡小彦 等 (CAI, XIAOYAN ET AL.): "棉花种间远缘杂交及F1真假杂种的SSR鉴定方法研究 (Identification of Cotton Interspecific Hybrids with SSR Markers)", 中国棉花 (CHINA COTTON), vol. 45, no. 11, 31 December 2018 (2018-12-31), XP55882851 *

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