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WO2013016879A1 - 一种批量基因克隆的方法 - Google Patents

一种批量基因克隆的方法 Download PDF

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WO2013016879A1
WO2013016879A1 PCT/CN2011/078358 CN2011078358W WO2013016879A1 WO 2013016879 A1 WO2013016879 A1 WO 2013016879A1 CN 2011078358 W CN2011078358 W CN 2011078358W WO 2013016879 A1 WO2013016879 A1 WO 2013016879A1
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recessive
target
pool
gene
dominant
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彭海
张静
章伟雄
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Jianghan University
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Jianghan University
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    • C12Q1/6869—Methods for sequencing

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  • the invention belongs to the field of genetics and discloses a method for batch gene cloning. Background technique
  • the classical gene mapping strategy stems from Morgan's linkage theory, that is, adjacent genes on the chromosome (ie, gene linkage) cannot be freely separated, but tend to be transmitted to the offspring as a whole, and the traits they control tend to appear at the same time. The closer the two are, the more likely they are to appear at the same time, and the fewer the recombination traits. Therefore, the distance between the two can be measured by the ratio of recombinant traits (gametes). If one of the genes is known to be on the chromosome (referred to as a marker gene), the location of the other gene can be inferred based on the rate of recombination.
  • a marker gene the location of the other gene can be inferred based on the rate of recombination.
  • a yellow round pea variety (genotype YYRR) is hybridized with a green wrinkle variety (genotype yyrr) (genotype YyRr), and F 1 selfing may produce four kinds of gametes, YR, Yr, yR and yr.
  • F 2 generation their proportion can be determined, and then the exchange rate can be calculated. If the exchange rate is 1%, and the Y gene controlling the color is known to be located on chromosome 3, then it can be speculated that the R gene controlling the shape of the pea kernel is located on chromosome 3 and is separated from the Y gene by lcM.
  • the seed shape is divided into pools, which is equivalent to the SSR17 pool. Therefore, the amplification products of the SSR17 between the two pools are different, and there is no difference. This identified the target gene on the same chromosome as SSR17. Then analyze the performance of each individual F 2 and calculate the genetic distance between R/r and SSR17 to further locate the specific position of R/r on the chromosome.
  • the object of the embodiments of the present invention is to provide a method for realizing rapid and accurate cloning of multiple genes by the same experiment in view of the above drawbacks of the prior art.
  • a method for batch gene cloning comprises the following steps: selecting a parent having different target traits as a parent, and the two parents are hybridized to construct an isolated population, and randomly selecting more than 100 genotypes as gene clones from the isolated population Group, genomic high-throughput sequencing with two parents and preliminary de novo (direct) assembly of the genome, alignment of the two parental genomes, obtaining differential alleles between parents, according to different target traits, clonal populations
  • the sequencing data is classified into different explicit pools and corresponding recessive pools. According to the exact matching method, the matching between the dominant and the recessive pools of the difference between the parental and the recessive pools is compared, and the separation ratio is calculated.
  • the alleles with the separation ratio of 3: 1 and 0: 1 in the dominant pool and the recessive pool are obtained, that is, the candidate sites of the target trait are obtained; the recessive pool group is expanded, and the PCR and sequencing are checked one by one.
  • Each candidate site in each recessive pool, or the recessive pool candidate site is re-sequenced after enrichment, and no dominant progeny in the recessive pool Site is the target locus, full length cloned by PCR amplification target genes by genetic complementation experiments target gene function.
  • a method of gene cloning comprising the following steps:
  • Target sites are determined in two ways. The first way: When there are more than 50 candidate sites, the candidate sites are enriched in the recessive pool by hybridization (such as Agilent SureSelect platform) or PCR, and then high-throughput sequencing is performed to detect unique candidate positions in dominant parents. Whether the point appears in the recessive pool, if it does not appear, it is the gene locus of the target trait; The second way: When the candidate locus is less than 50, ordinary PCR, real-time PCR,
  • each candidate site of each individual in the recessive pool is checked one by one, and the dominant locus site that does not appear is the target site.
  • the parental hybrid constructing the isolated population is constructed using a pure lineage parent with a close genetic distance.
  • the present invention relies on sequencing rather than molecular labeling and can be used in any biological species, greatly broadening the scope of gene cloning and utilization.
  • Direct cloning of the target gene itself, rather than the segment containing the target gene, is clear and there are no problems with no markers available. Most of the steps are guaranteed by probability, with judgment criteria, and the risk is greatly reduced. Experiments can be completed in a short period of time (such as weeks or months), and the speed is greatly accelerated.
  • FIG. 1 is a schematic flow chart of a method for cloning a plant batch gene according to Embodiment 1 of the present invention
  • Fig. 2 is a schematic view showing the flow of a method for simultaneously cloning rice high-bar, anti-breast, and rice blast resistance genes according to Example 2 of the present invention.
  • the F 2 population can be used as a gene cloning population.
  • the recombinant inbred line is continuously obtained as a clonal population, and the method is as follows. From the F 2 generation, no selection was made, and the "single-grain" method (one seed per plant) was self-crossed and formed into a progeny group until stable, and a recombinant inbred line was obtained. The self-crossing process is carried out under isolated conditions to avoid possible fly pollination and interfere with the results of the study. According to the distance of the genetic relationship of the hybrid parents, the length of self-stationary stability is different, and generally 9-10 generations can be stabilized. The obtained recombinant inbred lines are no longer separated and can be permanently stored and repeatedly observed for traits.
  • the Fragment library was constructed in a high-throughput sequencer (such as SOLiD 5500 or Illumina HiSeq 1000) operating manual, and each plant was subjected to high-throughput sequencing.
  • the depth of sequencing is preferably over 20 times the genome to facilitate assembly.
  • Preliminary de novo assembly of individual genomes in 2 parents and 100 clonal populations was performed using the genome assembly program ABySS (http: ⁇ www.bcgsc.ca/platform/bioinfo/software/abyss). Comparing the two parental genomes, an equipotential site with differences between the two parents was obtained.
  • the single-sequence sequencing data was divided into dominant pools and recessive pools. For example, suppose the target trait is plant height, with two relative traits, high and short, and high dominant. Then, among the 100 offspring, all the plant genome sequencing data that were high were classified into the dominant pool, and the genome sequencing data of all the plants that were short was classified as the recessive pool. If the target trait is pathogen resistant, the sequencing data of the 100 plants can be regrouped into disease-resistant pools and susceptible pools. According to the law of free combination, if 1 pair of genes is controlled, the ratio of dominant pool to recessive pool plant should be 3: 1, and the theoretical values in this example are 75 and 25 plants, respectively.
  • the difference site sequence in the parent is compared with the genomic data of each plant in the cloned population, and calculated The ratio of dominant and recessive loci in the dominant and recessive pools for each allele.
  • the statistical test separates the alleles in the dominant and recessive pools by 3:1 and 0:1 (dominant sequence: recessive sequence), defining this
  • the class site is a target trait candidate gene.
  • the determination of the target gene is only carried out by selecting recessive groups, but it is necessary to expand the number of recessive groups (for example, from 25 to 1000).
  • the number of candidate sites exceeds 50, submit a sequence of target gene candidate sites to Agilent, and design candidate sites according to Agilent's web-based probe design (https://earray.chem.agilent.com/earray/)
  • candidate sites were captured and sequenced in the Agilent SureSelect kit operating manual, followed by high-throughput sequencing.
  • PCR amplification detection is performed on each candidate site of each individual by PCR.
  • the PCR amplification detection is divided into a plurality of types, and can be selected according to different situations.
  • it can be detected by ordinary PCR plus agarose electrophoresis or PAGE gel electrophoresis.
  • the Tagman probe can be designed for real-time PCR detection.
  • high-throughput real-time PCR methods such as OpenArrary can be used for detection.
  • the dominant sequence of the alleles in the recessive pool can be counted. If there is no dominant sequence, it is the target site.
  • the full-length sequence of the target gene is obtained by comparing the parental genome, and the cloned target gene is PCR-amplified, and the function of the cloned gene is verified by a general genetic complementation experiment.
  • Example 2 The invention is further illustrated by a more specific embodiment: Example 2:
  • RH3 and RH5 are two rice parents selected for breeding. The similarity of these two parents is greater, but there are differences in plant height, bacterial leaf resistance and rice blast resistance. Among them, RH3 average plant height is 135 cm, resistance to C. albicans physiological race P6, resistance to rice blast physiological race S12 (preferred race from Hubei Enshi rice blast high-risk area); RH5 average plant height is 89 cm, sense Chlorophyceae physiological race P6, susceptible rice blast physiological race S12. RH3 and RH5 were positively reciprocated to produce high rods (average plant height 134.5 cm), resistance to C. albicans physiological race P6, and resistance to rice blast physiological race S12, and there was no significant difference in plant height between positive and negative crosses.
  • High rod, bacterial leaf resistance and rice blast resistance are dominant and not controlled by cytoplasmic genes. Planting? 2 A total of 20,000 strains, according to plant height > 135cm and ⁇ 90 ( ⁇ 11 group divided into two groups of high pole and dwarf rod, of which 13783 high rods and 4637 small rods. According to the method of leaf inoculation before rice heading Two weeks after inoculation of P6 stem races P6, according to lesions ⁇ 2 ( ⁇ 11 and >7 ( ⁇ 1, the population was divided into two groups: anti-breast and leucorrhea, among which the anti-disease group had 12017 There are 3,985 strains in the susceptible group.
  • the inoculation of rice blasts in the artificial greenhouse was carried out by spraying the rice blast race S12 (the spore concentration was adjusted to 1.5-2.0X10 5 Spores/ml), investigated the incidence after 10 days of inoculation, and identified typical fusiform lesions or dead plants as susceptible plants, and identified plants with no obvious symptoms as disease-resistant plants, and obtained resistance to rice blast and rice.
  • the rickets were 9228 and 3101.
  • the above three pairs of traits were tested by the chi-square test with a probability of 95%, which met the 3:1 separation ratio, indicating that the resistance to high pole, blight and rice blast was affected by one.
  • the rice genome is about 0.3 G.
  • a preliminary de novo assembly of the two parental RH3 and RH5 genomes was performed using the genome assembly program ABySS (http: ⁇ www.bcgsc.ca/platform/bioinfo/software/abyss). Comparing the differences between the two parental genomes, a total of 1895 alleles were obtained between the RH3 and RH5 genomes, and the corresponding sites in RH3 were named A1, A2... A1895, corresponding to
  • the corresponding sites in RH5 are named al, a2... al895.
  • the 1895 alleles were aligned with 27 dwarf plants in a perfectly matched manner, and the dominant parental sequences with 28 alleles were not found in these 27 recessive plants.
  • 32 and 25 dominant parent sequences were found to be absent in all recessive plants by comparing the plants with Phyllostachys praecox and Phytophthora sinensis.
  • To test the separation ratio of the dominant sequence sequence that did not appear in the recessive population and the corresponding recessive isotopic sequence in the dominant pool it was found that most of them met the separation ratio of 3:1, at 95%. Under the probability guarantee, none of the ratios of 1:1 are consistent, indicating that these loci should be the target sequence itself or the linkage locus, and they are respectively defined as candidate genes for plant height, resistance to bacterial blight and resistance to rice blast.
  • the number of recessive populations of dwarf, white leaf and rice blast was expanded to 960 (real-time PCR reaction was planned using AB's TaqMan® OpenArray® Genotyping Plates. The minimum price of the plate was 10, depending on the actual situation. , the number of sites X samples is 96x32 format). Submit a sequence of candidate alleles for all three traits to AB, and design the TagMan probes for these candidate alleles by AB and produce TaqMan® OpenArray®
  • Genotyping Plates genotyped candidate loci for all three target traits in an expanded recessive population according to the OpenArray assay.
  • the results of genotyping showed that the dominant parent sequence of each target trait except for one allele was not found in 960 recessive individuals, and the dominant sequence of the other candidate sites appeared at least twice. This indicates that these sites are not the target sites, but only the linkage sites of the target sites.
  • the amplified products of 4 sites with dominant sequence appearing less than or equal to 5 times were recovered and sent to Shanghai Shengong Biotechnology Co., Ltd. for sequencing. The sequencing results showed that these four sites were consistent with high-throughput sequencing. The sequence of sexual sites is consistent.
  • Target gene locus sequences controlling plant height, bacterial leaf resistance and rice blast resistance were obtained as follows: Target gene loci controlling plant height, bacterial leaf resistance and rice blast resistance were obtained.
  • the sequences are SEQ ID N0.1, SEQ ID NO. 2 and SEQ ID N0.3 in the Sequence Listing, respectively.
  • the target gene locus sequence for controlling resistance to Phyllostachys praecox was obtained as SEQ ID NO. 2 in the Sequence Listing; the target gene locus sequence for controlling rice blast resistance was obtained as SEQ ID N0.3 in the Sequence Listing.
  • the plant genome structure prediction program such as GeneSeqer@PlantGDB (http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi), according to the 10K sequence of each of the three loci in the RH3 genome.
  • the gene structure was predicted, and it was found that these three loci were located in the corresponding gene sequences, and each base was obtained according to the predicted structure.
  • the starting point and ending point of the cause According to the sequence of 500 bp before the start point of each gene and 500 bp after the end point, the predicted primers were designed according to Primer5 software and its default parameters. The primer design parameters were based on the software default parameters.
  • the PCR amplification system was carried out according to the kit DreamTaqTM Green PCR Master Mix (2X) (K1081, fermentas) recommended system, ie containing PCR mixture 50 ⁇ 1, RH23 template 15 ng, primer 2 ⁇ 1 (concentration: 0.5 ⁇ / ⁇ ), plus pure water The total volume is 100 ⁇ 1.
  • the amplification procedure was as follows: pre-denaturation at 94 ° C for 4 minutes; 94 ° C, 1 minute, 51 ° C to 54 ° C, 3 minutes, 72 ° C, 3 minutes, cycle 44 times; 72 ° C extension for 8 minutes.
  • the plant height gene amplification primer pair, the Sense primer is SEQ ID N0.4 in the sequence listing: ACTCACTCCCGCTCAAC A , reverse primer
  • Antisense primer is SEQ ID N0.5: CATTC ATCCGTCGTTCCA in the sequence listing, the annealing temperature is 54 °C; the Sense primer of the amplification primer pair of the bacterial leaf resistance gene is SEQ ID N0.6 in the sequence listing:
  • Antisense primer is SEQ ID N0.7 in the sequence listing:
  • annealing temperature is 51 °C; amplification primer pair of rice blast resistance gene, Sense primer is SEQ ID N0.8 in the sequence listing: AGTTCCTGGAGATCTGTAGC ACGTA, Antisense primer is SEQ ID N0 in the sequence listing. 9: TACGTGCTAC AGATCTCCAGGAACT, annealing temperature is 53.2 °C.
  • the amplified product was detected on a 2% agarose gel.
  • the PCR product was recovered and purified according to the agarose gel recovery kit (K0691, Fermentas) operating manual and sent to Shanghai Shengong Biotechnology Co., Ltd. for sequencing. The sequencing results were identical to the sequence assembled by RH3 high-throughput sequencing.
  • the full-length sequence of the plant height gene is SEQ ID NO. 10 in the sequence listing
  • the full-length sequence of the bacterial leaf resistance gene is SEQ ID NO. 11 in the sequence listing, rice blast resistance
  • the full-length gene sequence is SEQ ID N0.12 in the Sequence Listing.
  • the genes controlling high rod, resistance to white blight and rice blast resistance were transferred to the parental RH5 of Dwarf, B. leucocephala and susceptible blast, respectively, and the transgenic positive plants showed high pole.
  • Bacterial blight or resistance to rice blast demonstrates the correctness of the cloned genes of the examples of the present invention.
  • the invention is based on sequencing and can be used for any species, directly cloning the gene itself, and differently grouping the sequence data obtained in the same experiment, thereby achieving the purpose of cloning multiple genes, and realizing the full exploitation and utilization of the data.
  • the workload is greatly reduced, the efficiency and speed are greatly accelerated, and the risk is reduced.

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Abstract

提供了一种批量基因克隆的方法，其包括以下步骤：将具有多个目标性状差异的两个亲本杂交构建分离群；分离群体中随机选择的基因克隆群体和两个亲本一起进行基因组高通量测序；比对两个亲本基因组，获得亲本间差异等位位点；按不同目标性状，将克隆群体的测序数据归入不同的显性池与对应的隐性池，按照完全匹配的方式，比对亲本间差异等位位点序列在显性池与隐性池间的匹配情况，并计算分离比；通过统计检验，获得目标性状的候选基因位点；然后确定目标基因座位，通过PCR扩增克隆全长目标基因，并通过遗传互补实验验证目标基因的功能。该方法对同一次实验获得的测序数据进行不同的分组，对数据进行充分的发掘与利用，实现克隆多个基因的目的。

Description

说 明 书
一种批量基因克隆的方法
技术领域
本发明属于遗传学领域， 公开了一种批量基因克隆的方法。 背景技术
自然界物种性状丰富多样， 如株高、 抗病性、 产量等。 从孟德尔时代起， 就逐渐认识 到了性状是由遗传因子控制，摩尔根进一步将遗传因子明确为"基因"， 并指出基因位于染色 体上且呈线性排列。 确定基因在染色体上具体的位置 （基因定位） 是研究性状的基础， 也 是分离、 克隆并利用基因的前提。
经典的基因定位策略源于摩尔根的连锁理论， 即染色体上相邻基因 （即基因连锁） 不 能自由分离， 而是倾向于整体向后代传递， 它们所控制的性状也倾向于同时出现。 二者相 邻越近， 性状同时出现的可能性越大， 重组性状越少。 因此， 由重组性状 （配子） 的比例 可以度量二者间的距离。 若已知道其中一个基因在染色体上的位置 （称这样的基因为标记 基因）， 就可以根据重组率推断另一个基因的位置。 例如， 黄色圆粒豌豆品种 （基因型分别 为 YYRR) 与绿色皱粒品种 （基因型为 yyrr) 杂交产生 (基因型 YyRr)， F1自交可能产 生 YR、 Yr、 yR和 yr 4种配子， 根据 F2代的性状表现可以确定它们的比例， 进而计算交换 率。 若交换率为 1%， 且已知控制颜色的 Y基因位于第 3染色体上， 那么就可以推测， 控制 豌豆籽粒形状的 R基因位于第 3染色体且与 Y基因相距 lcM。 从以上的分析可以看出， 经 典连锁标记定位的实质是找到与目标性状连锁的已知分子标记， 并计算二者的交换率， 进 而推断目标基因的位置。 分离群体分池是基因定位的实际操作策略， 以上述实例进行说明 如下。 以籽粒形状为标准， 将 F2群体划分为两个部分（池）： 显性池（随机抽取的圆粒单株) 和隐性池 （随机抽取的皱粒单株）， 比较豌豆基因组上已知的 1000 个 SSR 标记 ( SSR1-SSR1000 ) 扩增产物在这两个池间的差异。 若标记 SSR17与籽粒形状 R/r连锁， 那 么对籽粒形状分池， 也就相当于对 SSR17分池， 因此， SSR17在两个池间的扩增产物是有 差异的， 相反就没有差异， 由此确定目标基因与 SSR17处于同一染色体。 再分析 F2各个单 株的表现， 计算 R/r与 SSR17的遗传距离， 即可进一步定位 R/r在该染色体上的具体位置。
分子标记连锁定位基因经过几十年的发展， 已成为基因定位的经典方法， 但该方法依 旧存在明显缺陷， 主要表现如下： ( 1 ) 经典分子标记定位克隆基因时， 一次杂交与分子标记实验只针对一个目标性状， 很难实现一次实验克隆多个基因的目的， 效率不高。
(2)大部分物种未开发分子标记， 基因克隆还很困难； 精细定位区间狭窄， 交换率低， 计算交换值的实验工作十分庞大； 常出现无标记可用， 克隆工作无法进行的情况； 仅找到 含目标基因的区段， 需要进行基因预测， 假阳性或假阴性的实验结果无法避免。
发明内容
本发明实施例的目的是针对上述现有技术的缺陷， 提供了一种利用同一次实验实现多 个基因的快速、 准确克隆的方法。
为了实现上述目的本发明采取的技术方案是：
一种批量基因克隆的方法， 包括以下步骤： 选用在多个目标性状上有差异的材料做亲 本， 将两个亲本杂交构建分离群体， 从分离群体中随机选择 100个以上的基因型作为基因 克隆群体， 与两个亲本一起基因组高通量测序并进行基因组初步 de novo (直接） 组装， 对 两个亲本基因组进行比对， 获得亲本间差异等位位点， 按不同目标性状， 将克隆群体的测 序数据归入不同的显性池与对应的隐性池， 按完全匹配的方式， 比对亲本间差异等位位点 序列在显性池与隐性池间的匹配情况， 并计算分离比， 通过统计检验， 获得显性池与隐性 池中分离比分别为 3: 1和 0: 1 的等位位点， 即获得目标性状的候选位点； 扩大隐性池群 体， PCR、测序逐个检查隐性池中每个个体的每个候选位点， 或隐性池候选位点富集后重测 序， 隐性池中完全没有出现的显性亲本位点即为目标基因座位， 通过 PCR扩增克隆全长目 标基因， 通过遗传互补实验验证目标基因功能。
本发明更具体的技术方案是：
一种基因克隆方法， 包括以下步骤：
( 1 ) 基因克隆群体的构建与遗传分析： 选择多个目标性状上有差异的材料做亲本， 通过杂交构建分离群体， 从分离群体中随机选择 100个以上的基因型作为基因克隆群体；
(2) 确定目标性状基因候选位点： 分别提取克隆群体中每个个体和 2个亲本的基因组 DNA, 按高通量测序流程分别构建文库， PCR并高通量测序； 按 de novo组装， 初步构建 克隆群体中每个个体和 2个亲本全基因组序列。 构建过程中， 忽略难以正确组装的重复序 列 （因为它们多数为非蛋白编码基因）。 按不同目标性状， 将克隆群体的测序数据归入不同 的显性池与对应的隐性池， 按完全匹配的方式， 比对亲本间差异等位位点序列在显性池与 隐性池间的匹配情况， 并计算分离比， 通过统计检验， 获得显性池与隐性池中分离比分别 为 3: 1和 0: 1的等位位点， 将它们定义为目标性状的候选位点。
(4) 目标位点的确定： 通过两个方式确定目标位点。 第一种方式： 候选位点超过 50 个时， 通过杂交 （如安捷伦 SureSelect平台） 或 PCR方式在隐性池中富集候选位点后再次 高通量测序， 检测显性亲本中特有的候选位点是否在隐性池中出现， 若没有出现， 则为目 标性状的基因位点； 第二种方式： 候选位点不足 50个时， 可采用普通 PCR、 实时 PCR、
SNaPshot (测序）或高通量的 OpenArray (定制芯片）检测方式， 逐个检查隐性池每个个体 的每个候选位点， 没有出现的显性座位位点即为目标位点。
(5) 基因克隆与功能验证： 通过比对亲本基因组的方式获得基因的全长序列， PCR扩 增克隆目标基因， 按通用的遗传互补实验验证所克隆基因的功能。
所述亲本杂交构建分离群体是利用遗传距离近的纯系亲本构建。
本发明实施例的有益效果是：
( 1 )利用本发明的方法， 只需要一次杂交与测序实验， 即可实现克隆多个基因的目的。
(2) 本发明依赖于测序而非分子标记， 可用于任何生物物种， 极大地拓宽了基因克隆 与利用范围。 直接克隆目标基因本身， 而非包含目标基因的区段， 结果明确， 也不存在无 标记可用的问题。 大部分步骤有概率保证， 具有判断标准， 风险大为降低。 实验可在较短 时间 （如数周或数月） 内完成， 速度大为加快。 附图说明
图 1是本发明实施例 1提供的植物批量基因克隆方法流程示意图；
图 2是本发明实施例 2提供的同时克隆水稻高杆、 抗白叶枯、 抗稻瘟病基因的方法流 程示意图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明， 但不作为对本发明的限定。
实施例 1 :
参见图 1， 一种植物批量基因克隆方法：
( 1 ) 构建分离群体
选择具有多个目标性状差异的两个亲本进行正反交 （除目标性状外， 其它性状差异越 小越好）， 通过分子标记或田间性状观察， 去除假杂种。根据 的表现判断控制性状的基因 座位是显性还是隐性。 根据正反交的性状是否有差异， 判断是否具有细胞质效应， 若没有 差异， 表明不受细胞质基因影响， 否则该性状与细胞质基因相关。 Fi 交形成 F2群体， 根 据相对性状在 F2群体中植株的比例， 判断控制目标性状的基因对数， 卡方检验若符合 3: 1 的分离比， 则为 1对基因控制， 否则为多对基因控制。 F2群体即可作为基因克隆群体， 为 了方便反复多次观察分离后代每个个体的表现型， 继续自交获得重组自交系作为克隆群体， 方法如下。 从 F2代起， 不进行任何选择， 采用"单粒传 "法 （每个单株收 1颗种子） 自交收 种并形成后代群体， 直至稳定， 获得重组自交系。 自交过程都在隔离条件下进行， 以避免 可能的飞花传粉， 干扰研究结果。 根据杂交亲本亲缘关系的远近， 自交稳定的时间长短有 一定差异， 一般 9-10代即可稳定。 获得的重组自交系不再分离， 可永久保存并反复观察性 状。
(2) 植株选择与 DNA提取。
从克隆群体中随机选择 100个单株与将 2个杂交亲本一起， 按植物 /种子基因组 DNA 提取试剂盒 （北京天漠科技开发有限公司， 货号： D6120) 操作手册提取 DNA。 通过分光 光度计测定并计算 A260/A280比值， 以判断 DNA质量与含量。
( 3 ) 高通量测序、 基因组组装、 比对与候选位点的获得。 按高通量测序仪 （如 SOLiD 5500或 Illumina HiSeq 1000) 操作手册构建 Fragment文库、 对每一单株进行高通量测序。 测序深度最好覆盖基因组 20倍以上， 以便于组装。 用基因组 组装程序 ABySS (http：〃 www.bcgsc.ca/platform/bioinfo/software/abyss)对 2个亲本和 100个克隆 群体中的个体基因组进行初步 de novo组装。 比对 2个亲本基因组， 获得 2个亲本之间具有差 异的等位位点。 根据所检测的 100个单株的性状表现和所要定位的性状， 将单株测序数据分为显性池 和隐性池。 例如， 假设目标性状为株高， 具有高和矮两个相对性状， 且高为显性。 那么， 将这 100个后代中， 表现为高的所有植株基因组测序数据归入显性池， 表现为矮的所有植 株的基因组测序数据归入为隐性池。 若目标性状为病原抗性， 又可将这 100个植株的测序 数据重新分组， 分为抗病池和感病池。 根据自由组合规律， 若为 1 对基因控制， 显性池与 隐性池植株的比例应为 3: 1， 本实施例中的理论值分别为 75和 25个植株。 按完全匹配的 比对原则， 将亲本中的差异位点序列与克隆群体中每一植株基因组数据进行比对， 并计算 各等位位点在显性池与隐性池中显性基因与隐性位点序列的比例。 在 95 %的概率保证下， 统计检验在显性池与隐性池中的等位位点分离比分别为 3: 1和 0: 1 (显性序列： 隐性序列） 的位点， 定义此类位点为目标性状候选基因。
设隐性混合池中个体数为 n，则与目标位点不连锁的位点的显性与隐性序列分离比也为 0： 1的概率为 0.52n， 当 n=25 (本实施例中的期望值） 时， 该概率为 0.52*25=0.00%， 为小概 率不可发生事件， 即通过测序， 可将所有与目标位点不连锁的位点区分开。 设某位点与目 标位点连锁， 只有当交换发生时， 才能将它与目标位点区分开， 设连锁距离为 m cM， 则发 生交换的概率为 1- ( l-m ) 2n。 当 n=25 (本实施例中的期望值） 且 m>5.51时， 发生交换 的概率大于 1- ( 1-5.51 % ) 2x25=94.1 o 即有 94.1 %的把握将与目标位点遗传距离大于 5.51 的位点区分开。
(4) 目标基因的确定
目标基因的确定只选择隐性群体进行， 但需要扩大隐性群体的个数 （如从 25个扩大为 1000个）。 有两种方式从候选基因中确定目标基因。 当候选位点超过 50个时， 向安捷伦公 司提交 目标基因候选位点的序列， 按安捷伦基于 Web 的探针设计方案 (https://earray.chem.agilent.com/earray/) 设计候选位点的杂交探针， 按安捷伦 SureSelect试 剂盒操作手册对候选位点进行捕获并建立测序文库， 之后进行高通量测序。 当候选位点不 不足 50个时按 PCR的方式对每一个体的每一候选位点进行 PCR扩增检测。 其中， PCR扩 增检测分为多种， 可根据不同的情况进行选择。 当位点很少且等位位点序列长度差异较大 时， 可采用普通 PCR加琼脂糖电泳或 PAGE胶电泳的方式进行检测。 当等位位点间序列差 异不大时， 可设计 Tagman探针进行实时 PCR检测。 当位点与群体个体都较多时， 可用高 通量的实时 PCR方法如 OpenArrary进行检测。
不论采用以上那种策略， 均可统计隐性池中是否出现等位位点的显性序列， 若无显性 序列，即为目标位点。本实施例中，当 0=1000且111>0.29时，发生交换的概率大于 1-( 1-0.29 % ) 2X 1QQQ=99.7 %。 即有 99.7 %的把握将与目标位点遗传距离大于 0.29的连锁位点区分开。在遗 传距离为 0.29范围之内且具有多态性的位点多数情况下已经没有 （因为要求亲本遗传距离 不要太大）。若偶尔还有，可采用继续扩大群体检测或采用基因生物信息学预测的方式排除， 最后再通过遗传互补实验进行验证。
( 5 ) 目标基因克隆与功能验证。
通过比对亲本基因组的方式获得目标基因的全长序列， PCR扩增克隆目标基因， 按通 用的遗传互补实验验证所克隆基因的功能。
下面通过一个更具体的实施例对本发明作进一步说明： 实施例 2:
参见图 2， 水稻高杆、 抗白叶枯、 抗稻瘟病基因克隆的方法：
( 1 ) 构建克隆群体
RH3与 RH5为育种选育的两个水稻亲本， 这两个亲本的相似性较大， 但在株高、 白叶 枯抗性和稻瘟病抗性上有差异。 其中， RH3平均株高为 135 cm、 抗白叶菌生理小种 P6、 抗 稻瘟病生理小种 S12 (从湖北恩施稻瘟病高发区分离的优势小种）； RH5平均株高为 89 cm、 感白叶菌生理小种 P6、 感稻瘟病生理小种 S12。 RH3与 RH5进行正反交产生 表现为 高杆 （平均株高 134.5 cm)、 抗白叶菌生理小种 P6、 抗稻瘟病生理小种 S12， 且正反交之间 株高没有显著差异， 表明高杆、 白叶枯抗性和稻瘟病抗性均为显性且不受细胞质基因控制。 种植？2共 20000株， 按株高 >135cm和<90(^11将群体分为高杆和矮杆两个群体， 其中高杆 13783株， 矮杆为 4637株。 按剪叶接种法于水稻抽穗前接种白叶枯生理小种 P6两周后， 按 病斑<2 (^11和>7 (^1， 将群体分为抗白叶枯和感白叶枯两个群体， 其中抗病群体有 12017株， 感病群体有 3985株。 为了减少稻瘟病小种接种难度， 于人工温室中在水稻苗三叶一心时采 用喷雾稻瘟病生理小种 S12方式接种 （孢子浓度调节为 1.5-2.0X105个孢子 /毫升）， 接种 10 天后调查发病情况， 将具有典型梭形病斑或死亡植株判为感病植株， 将没有明显感病症状 的植株确定为抗病植株， 共获得抗稻瘟病和感稻瘟病的植株分别为 9228和 3101株。 以上 3 对性状经卡方检验在 95%的概率保证下符合 3: 1的分离比， 表明高杆、 白叶枯抗性和稻瘟 病抗性均受一对显性核基因控制。从 F2群体中随机选择 100个植株作为克隆群体。在这 100 个植株中， 高杆与矮杆的植株数分别 73和 27株， 抗白叶枯和感白叶枯的植株分别为 72和 28株， 抗稻瘟病和感稻瘟病的植株数分别为 78和 22株。
(2) DNA提取
取上述 100个植株及亲本 RH3、 RH5叶片， 分别按植物 DNA提取试剂盒（DP305, 天 更， 北京） 操作手册分离纯化基因组 DNA， 利用 ND2000超微量分光光度计测定样品浓度 与纯度。
(3) 亲本测序与目标基因候选位点的确定
按 Illumina HiSeq 2000高通量测序技术规程构建两个亲本 RH3、 RH5和 100个分离后 代个体的基因组 Fragment文库、 PCR扩增后高通量测序。采用条形码对每份材料进行编码， 以示区分。 测序后， 所有 102份材料共获得 559.8 G Clean data数据量， 每个材料平均有 559.8/102=5.49 G数据量， 变异范围为 3.82〜7.03G。 水稻基因组大约为 0.3 G， 本次测序平 均覆盖基因组 5.49/0.3=18.30倍， 变异范围为 12.77〜23.43倍。
用基因组组装程序 ABySS (http：〃 www.bcgsc.ca/platform/bioinfo/software/abyss)对两个亲 本 RH3和 RH5基因组进行初步 de novo组装。比较两亲本基因组间的差异，在 RH3和 RH5 基因组间共获得 1895个等位位点， 将 RH3中相应的位点命名为 A1, A2...... A1895, 对应在
RH5中相应位点命名为 al、 a2...... al895。 按完全匹配的方式， 将这 1895对等位位点与 27 个矮杆植株进行比对， 发现有 28个等位位点的显性亲本序列没有在这 27个隐性植株中出 现。 按同样的方法， 通过比对感白叶枯和感稻瘟病的植株， 分别发现 32个和 25个显性亲 本序列在所有的隐性植株中都没有出现。 检验以上在隐性群体中没有出现的显性位点序列 与对应的隐性等位点序列在显性池中的分离比，发现绝大部分都符合 3: 1的分离比，在 95% 的概率保证下， 均不符合 1 : 1的分离比， 表明这些位点应该是目标序列本身或连锁位点， 将它们分别定义为株高、 抗白叶枯、 抗稻瘟病的候选基因。
(4) 目标基因的确定
将矮杆、 感白叶枯和感稻瘟病的隐性群体数量扩大为 960株 （拟采用 AB公司 TaqMan® OpenArray® Genotyping Plates进行实时 PCR反应检测， 该反应板最低订制 10张， 根据实际情 况， 位点 X样本数选择 96x32的格式）。 向 AB公司提交所有这三个性状的候选等位位点序列， 由 AB公司设计检测这些候选等位位点的 TagMan探针并生产 TaqMan® OpenArray®
Genotyping Plates,按照 OpenArray检测流程在扩大的隐性群体中对所有三个目标性状的候选 位点进行基因分型。 基因分型的结果表明： 每个目标性状除 1个等位位点中的显性亲本序列 在 960个隐性个体中均未出现外， 其余候选位点的显性序列至少出现了 2次， 表明这些位点 不是目标位点， 而仅为目标位点的连锁位点。 对显性序列出现小于等于 5次的 4个位点的扩 增产物进行回收， 并送交上海深工生物技术有限公司测序， 测序结果表明这 4个位点均与高 通量测序中的显性位点序列一致。 通过以上步骤， 获得了控制株高、 白叶枯抗性和稻瘟病 抗性的目标基因位点序列分别如下： 获得了控制株高、 白叶枯抗性和稻瘟病抗性的目标基 因位点序列分别为序列表中的 SEQ ID N0.1、 SEQ ID NO.2和 SEQ ID N0.3。获得了控制白叶 枯抗性的目标基因位点序列为序列表中的 SEQ ID NO.2; 获得了控制稻瘟病抗性的目标基因 位点序列为序列表中的 SEQ ID N0.3。
根据三个位点中每个位点在 RH3基因组中前后各 10K序列， 按植物基因组结构预测程 序， 如 GeneSeqer@PlantGDB ( http://www.plantgdb.org/cgi-bin/GeneSeqer.cgi ) ， 对基因结 构进行预测， 发现这三个位点分别位于相应的基因序列内， 根据预测的结构， 获得每个基 因的起始点和结束点。 根据每个基因起始点前 500bp和结束点后 500bp的序列按 Primer5软件 及其默认参数设计相应引物对预测获得的基因进行扩增， 引物设计的参数按软件默认的参 数进行。 PCR扩增体系按试剂盒 DreamTaq™ Green PCR Master Mix (2X) ( K1081 , fermentas)推荐体系进行，即含 PCR混合物 50μ1， RH23模板 15 ng，引物 2μ1(浓度： 0.5μΜ/μί)， 加纯水至总体积为 100μ1。 扩增程序如下： 94°C预变性 4分钟； 94°C， 1分钟， 51 °C〜54°C， 3 分钟， 72°C， 3分钟， 循环 44次； 72°C延伸 8分钟。 其中， 株高基因扩增引物对中， 正向引 物 （Sense primer) 为序列表中的 SEQ ID N0.4: ACTCACTCCCGCTCAAC A , 反向引物
(Antisense primer) 为序列表中的 SEQ ID N0.5: CATTC ATCCGTCGTTCCA , 退火温度为 54 °C ; 白叶枯抗性基因的扩增引物对的 Sense primer为序列表中的 SEQ ID N0.6:
GCTCCTATTCGCAACCTG , Antisense primer为序列表中的 SEQ ID N0.7:
TACCA AACAAAC AGAGGC , 退火温度为 51 °C ; 稻瘟病抗性基因的扩增引物对中， Sense primer为序列表中的 SEQ ID N0.8: AGTTCCTGGAGATCTGTAGC ACGTA , Antisense primer 为序列表中的 SEQ ID N0.9: TACGTGCTAC AGATCTCCAGGAACT , 退火温度为 53.2°C。 扩增产物用 2%的琼脂糖凝胶检测。 PCR产物按琼脂糖凝胶回收试剂盒（ K0691 , Fermentas ) 操作手册回收纯化扩增片段并送交上海深工生物技术有限公司测序，测序结果与 RH3高通量 测序后拼装的序列完全一致。 通过以上技术手段， 获得株高基因的全长序列为序列表中的 SEQ ID NO.10, 白叶枯抗性基因的全长序列为序列表中的 SEQ ID NO.l l， 稻瘟病抗性的全 长基因序列为序列表中的 SEQ ID N0.12。
(4)功能互补验证
利用根癌农杆菌 Ti质粒载体， 将控制高杆、 抗白叶枯和抗稻瘟病的基因分别转入矮杆、 感白叶枯和感稻瘟病的亲本 RH5中， 转基因阳性植株表现为高杆、抗白叶枯或抗稻瘟病， 证 明了本发明实施例所克隆基因的正确性。
本发明以测序为基础， 可用于任何物种， 直接克隆基因本身， 对同一次实验获得的测 序数据进行不同的分组， 即可实现克隆多个基因的目的， 实现了数据的充分发掘与利用。 采用本发明的方法， 工作量大为减少、 效率与速度大为加快、 而且降低了风险。
以上所述的实施例， 只是本发明较优选的具体实施方式的一种， 本领域的技术人员在 本发明技术方案范围内进行的通常变化和替换都应包含在本发明的保护范围内。

Claims

权 利 要 求 书
1. 一种批量基因克隆的方法， 其特征在于， 包括以下步骤： 选用在多个目标性状上 有差异的材料做亲本， 将两个亲本杂交构建分离群体， 从分离群体中随机选择 100个以上 的基因型作为基因克隆群体， 提取克隆群体中每个个体与两个亲本基因组一起高通量测序， 并进行基因组初步直接组装； 对两个亲本基因组进行比对， 获得亲本间差异等位位点； 按 不同目标性状， 将克隆群体的测序数据归入不同的显性池与对应的隐性池， 按完全匹配的 方式， 比对亲本间差异等位位点序列在显性池与隐性池间的匹配情况， 并计算分离比； 通 过统计检验， 获得显性池与隐性池中分离比分别为 3: 1和 0: 1 的等位位点， 即获得目标 性状的候选位点； 扩大隐性池群体， PCR逐个检查隐性池中每个个体的每个候选位点或隐 性池候选位点富集后重测序， 在隐性池中完全没有出现的显性亲本位点即为目标基因座位； 通过 PCR扩增克隆全长目标基因， 通过遗传互补实验验证目标基因功能。
2. 根据权利要求 1所述的批量基因克隆的方法， 其特征在于， 包括以下步骤：
( 1 ) 构建基因克隆群体： 选择多个目标性状上有差异的材料做亲本， 将两个亲本通 过杂交构建分离群体， 从分离群体中随机选择 100个以上的基因型作为基因克隆群体；
(2)确定候选位点： 分别提取克隆群体中每个个体和 2个亲本基因组 DNA， 按高通量 测序流程构建文库， PCR 并高通量测序； 按直接组装， 初步构建克隆群体中每个个体和 2 个亲本的全基因组序列； 按不同目标性状， 将克隆群体的测序数据归入不同的显性池与对 应的隐性池， 按完全匹配的方式， 比对亲本间差异等位位点序列在显性池与隐性池间的匹 配情况， 并计算分离比； 通过统计检验， 获得显性池与隐性池中分离比分别为 3: 1和 0: 1 的等位位点， 该等位位点为目标性状的候选位点；
(4) 目标位点的确定： 当候选位点超过 50个时， 通过杂交或 PCR方式在隐性池中富 集候选位点后再次高通量测序， 检测显性亲本中特有的候选位点是否在隐性池中出现， 若 没有出现，则为目标性状的基因位点； 当候选位点不足 50个时，采用普通 PCR、实时 PCR、 高通量 PCR或测序技术的检测方式， 逐个检查隐性池每个个体的每个候选位点， 没有出现 的显性座位的等位位点即为目标位点；
(5)基因克隆与功能验证： 通过比对亲本基因组的方式获得基因的全长序列， PCR扩 增克隆目标基因， 按通用的遗传互补实验验证所克隆基因的功能。
3. 根据权利要求 1或 2所述的批量基因克隆的方法， 其特征在于， 所述亲本 建分离群体是利用遗传距离近的纯系亲本。
PCT/CN2011/078358 2011-07-29 2011-08-12 一种批量基因克隆的方法 Ceased WO2013016879A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9073911B2 (en) 2011-06-09 2015-07-07 Hoffmann-La Roche Inc. Pyrazole derivatives

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103388025B (zh) * 2013-07-10 2015-04-29 华中农业大学 基于克隆dna混合池的全基因组测序方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006138696A2 (en) * 2005-06-17 2006-12-28 Genizon Biosciences, Inc. Genemap of the human genes associated with longevity
CN101045928A (zh) * 2007-02-12 2007-10-03 湖南西城杂交水稻基因科技有限公司 一种克隆野生稻新抗性基因的方法
CN101760462A (zh) * 2008-12-19 2010-06-30 李祥 植物抗病基因的克隆

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101544974A (zh) * 2009-05-04 2009-09-30 中国农业科学院油料作物研究所 一种高通量的数量性状调控基因分离的方法
CN101886132B (zh) * 2009-07-15 2013-09-18 北京百迈客生物科技有限公司 基于测序和bsa技术的性状相关的分子标记筛选方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006138696A2 (en) * 2005-06-17 2006-12-28 Genizon Biosciences, Inc. Genemap of the human genes associated with longevity
CN101045928A (zh) * 2007-02-12 2007-10-03 湖南西城杂交水稻基因科技有限公司 一种克隆野生稻新抗性基因的方法
CN101760462A (zh) * 2008-12-19 2010-06-30 李祥 植物抗病基因的克隆

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GUO, LIHONG ET AL.: "Batch cloning and a high throughput screening for virulent genes of Streptococcus mutans", JOURNAL OF MODERN STOMATOLOGY, vol. 19, no. 3, 31 May 2005 (2005-05-31), pages 266 - 269 *
LI, SHANWEN: "Study on Genetic Variation of Parents and Progenies and Their Molecular Basis in Populus", CHINESE SELECTED DOCTORAL DISSERTATIONS AND MASTER'S THESES FULL-TEXT DATABASES (DOCTOR), AGRICULTURE, no. 4, 15 August 2005 (2005-08-15), pages D049 - 24 *
WANG ZHONGDONG ET AL.: "Cloning and Characterization of Genes Differentially Expressed in Human Dental Pulp Cells and Gingival Fibroblasts", WEST CHINA JOURNAL OF STOMATOLOGY, vol. 5, no. 1, 28 February 2007 (2007-02-28), pages 75 - 78 *
XU, D.Q. ET AL.: "Identification of a differential gene HUMMLC2B between Fl hybrids Landrace x Yorkshire and their female parents Yorkshire", GENE, vol. 352, 6 June 2005 (2005-06-06), pages 118 - 126, XP004945819, DOI: doi:10.1016/j.gene.2005.04.010 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9073911B2 (en) 2011-06-09 2015-07-07 Hoffmann-La Roche Inc. Pyrazole derivatives

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