离体雌核发育诱导洋葱单倍体与分子标记开发

发布时间：2018-04-20 03:02

本文选题：洋葱(Allium + cepa　；参考：《山东农业大学》2016年博士论文

【摘要】：洋葱(Allium cepa L.,染色体数2n=2x=16)是百合科(Liliaceae)葱属(Allium)二年生蔬菜,在世界范围内栽培广泛,其年生产量和生产面积居于世界蔬菜生产的第三位。我国种植洋葱的历史虽然较短,但是发展迅速,在全国各地广泛栽培,是重要的出口创汇蔬菜。洋葱是典型的异花授粉蔬菜,自交衰退严重。传统育种方法育成优良的自交系需要6~10年的时间,而通过单倍体途径在较短时间内就可以选育出整齐一致的纯系,明显提高选择效率。此外,单倍体加倍获得的双单倍体可作为重要性状的遗传分析、分子标记及数量性状研究的理想材料。本研究以不同来源的中日照型洋葱自交系、常规种、杂交种为材料,通过离体雌核发育途径诱导培养洋葱花蕾,对适宜培养基的筛选、培养程序优化、植株再生、染色体倍性鉴定、染色体加倍技术、分子标记鉴定等进行了研究。利用获得的洋葱不育双单倍体、可育双单倍体以及二者杂交获得的F1,采用SLAF-seq技术进行测序,获得多态性SLAF标记,并开发出大量特异性SNP位点。主要结果如下:1.以引自日本的3个中日照型洋葱杂交种的花蕾为外植体材料,研究了2,4-D和6-BA浓度及配比对单倍体诱导培养的影响。结果表明,含有1.5 mg·L-1 2,4-D+1.5 mg·L-1 6-BA和2.0 mg·L-1 2,4-D+2.0 mg·L-1 6-BA的B5培养基适于胚的诱导,诱导率最高达到4.00%。2.对洋葱花蕾的诱导培养程序进行了优化研究,结果表明不同花蕾大小对洋葱的离体雌核发育有很大影响。直径2.1~3.0 mm的花蕾离体诱导培养时成胚率明显低于其它直径的花蕾。直径3.1~4.0 mm的花蕾诱导成胚率最高。‘地球’的花蕾经低温预处理1 d后成胚率明显增高,预处理3 d后胚的诱导率开始明显下降。而‘大宝’的花蕾经低温预处理1 d后成胚率没有显著变化,预处理3 d后胚的诱导率明显下降。3.不同基因型洋葱雌核发育胚的诱导率存在很大差异。本试验供试的9个洋葱基因型中胚的诱导率最高的是杂交种‘地球’,诱导率为4.67%,其次为‘大宝’和‘阿盾’,诱导率分别为4.33%和4.00%,没有显著差异。2个常规种‘天正红玉’和‘天正黄金’分别诱导出了6枚和8枚胚,诱导率显著少于其他基因型。2个自交系503和217分别诱导出13枚和10枚胚,诱导率显著大于常规种,但显著少于5个杂交种。总的来说,杂交种的胚诱导率大于自交系,自交系的诱导率大于常规种。4.将所获得的雌核发育胚转移至含有30 g·L-1蔗糖的1/2B5+0.5mg·L-1NAA培养基中,5d后即可发育成正常植株。利用流式细胞仪对所获得的再生植株进行dna相对含量测定,结果显示正常二倍体的样品分离峰出现在140相对荧光强度中,据此断定分离峰出现在70荧光强度的为单倍体材料。为进一步验证再生植株的倍性,对通过流式细胞仪鉴定过的植株再进行根尖染色体计数分析,结果表明经鉴定为二倍体的植株染色体数为2n=2x=16,鉴定为单倍体的染色体数为n=x=8,与流式细胞仪的鉴定结果完全吻合。5.研究了秋水仙素不同浓度、不同处理时间对单倍体植株染色体加倍效果的影响。结果表明,在处理时间相同的情况下,随着秋水仙素浓度升高,再生植株的存活率降低,但二倍体率增加;在相同秋水仙素浓度下,随处理时间延长,植株的成活率下降,但二倍体率增加。浓度为200mg·l-1的秋水仙素处理48h,对两个供试品种的染色体加倍效果最好,成活率分别为61.11%、50.00%,加倍率分别为44.44%、38.89%。6.利用dnf-566、rns-357和acskp1标记对部分再生植株进行了检测。‘阿盾’的11株再生植株均为纯合的msms或msms,‘地球’的15株再生植株中在ms位点的基因型均为纯合的。‘大宝’的16株再生植株中,有13株在ms位点的基因型均为纯合的,3株是杂合的。7.生根良好的再生植株经驯化、炼苗后,移栽到试验基地网棚,绝大多数再生植株成活,形态正常,生长发育良好。洋葱单倍体植株矮小、细弱,开花期与二倍体洋葱植株基本一致,但抽生花薹矮小。整个花序较小,花蕾数少而且小。花药小,没有花粉。双单倍体植株性状表现符合二倍体特征,能正常开花。经分子标记鉴定为msms基因型的双单倍体植株育性正常,而鉴定为msms基因型的双单倍体植株表现为雄性不育。育性正常的双单倍体植株开花后套袋,放入苍蝇授粉自交,获得了发育饱满、发芽力强的自交种子。对不育双单倍体植株,利用其作为母本与可育双单倍体杂交,获得了f1代种子。8.以获得的可育双单倍体dh-17、不育双单倍体dh-1及部分f1代单株为材料,利用基于简化基因组深度测序的slaf-seq技术,选择洋葱转录组作为参考进行电子酶切预测,最终确定使用pvuii+scai酶切。将南芥的测序读长与参考基因组的比对结果显示双端比对效率为80.60%,大部分测序读长的插入片段长度都在范围之内,表明建库质量良好。通过测序共获得125.98m读长,各样品所获读长数量在29736~30825419范围内。所有样品的测序质量值q30均大于80%,说明测序碱基错误率较低,所获数据合格。测序获得平均gc含量为35.77%,而且含量比较平均,说明达到了测序要求。对满足质量要求的测序数据进行聚类分析,共开发出各类型标签294911个,其中多态性SLAF标签14776个,仅占SLAF标签总数的5%。根据基因型编码规则对筛选出的多态性标签进行基因型编码,共得到可编码8种基因型的标签6384个,其中5354个多态性标签符合aa×bb类型,占可编码标签的83.86%。利用多态性标签进一步进行SNP位点的开发,共得到36109个群体的SNP。样品亲缘关系鉴定结果表明异常标签数目所占比例均远远小于0.5%,由此可以判定样品F1-1、F1-2、F1-3、F1-4、F1-5均为DH-17和DH-1的子代。
[Abstract]:Onion (Allium CEPA L., chromosome number 2n=2x=16) is a biennial vegetable of Liliaceae (Liliaceae) (Allium), which is widely cultivated worldwide. Its annual production and production area are third in the world's vegetable production. The history of planting onions in China is short, but it develops rapidly and is widely cultivated throughout the country. It is an important export. The onion is a typical non flower pollinated vegetable, which has a serious self breeding decline. The traditional breeding method will take 6~10 years to develop a fine inbred line, while the haploid way can produce a uniform pure line in a short time, which can obviously improve the selection efficiency. In addition, the haploid haploid can be used as an important factor. Genetic analysis, molecular markers and quantitative characters for the study of molecular markers and quantitative traits. In this study, onion inbred lines, conventional species, and hybrids from different sources were used to induce the culture of onion buds, the selection of suitable medium, optimization of culture program, plant regeneration, chromosome ploidy identification, and dyeing. The double haploid of the onions, the fertile double haploid and the F1 obtained by the two hybrids were obtained. The polymorphic SLAF markers were sequenced by SLAF-seq technique, and a large number of specific SNP loci were developed. The main results are as follows: 1. from Japan's 3 medium sunshine onions The effects of the concentration and ratio of 2,4-D and 6-BA on the induction and culture of haploid were studied. The results showed that the culture medium containing 1.5 mg. L-1 2,4-D+1.5 mg L-1 6-BA and 2 mg L-1 2,4-D+2.0 mg. The results showed that the size of different buds had a great effect on the development of onions in vitro. The rate of bud formation in the bud of 2.1~3.0 mm in vitro was significantly lower than that of other buds in vitro. The bud induction rate of 3.1~4.0 mm in diameter was the highest. The rate of embryo of 'earth' bud was significantly higher after 1 D at low temperature. The induction rate of embryo began to decrease obviously after 3 D pretreatment, but the embryo rate of the bud of 'Dabao' was not significantly changed after 1 D pretreated at low temperature. The induction rate of embryo was obviously decreased after the pretreatment of 3 D, and the induction rate of the embryo of different genotypes of onion was very different. The induction rate of embryo in the 9 onions genotypes was the highest in this test. The induction rate of the hybrid species' earth 'was 4.67%, followed by' Dabao 'and' ADUN ', the induction rates were 4.33% and 4%, respectively. There was no significant difference in the induction of 6 and 8 embryos from.2 conventional species' Tian Zheng Hongyu' and 'Tian Zheng gold' respectively. The induction rate was significantly less than that of the.2 inbred lines of 503 and 217, respectively. The induction rate of the 10 embryos was significantly greater than that of the conventional species, but significantly less than 5 hybrids. In general, the embryo induction rate of the hybrids was greater than that of the inbred lines. The induction rate of the inbred lines was greater than that of the conventional species.4. and transferred to the 1/2B5+0.5mg L-1NAA medium containing 30 g. L-1 sucrose. The normal plants could be developed into normal plants after 5D. DNA relative content of the regenerated plants was measured by flow cytometry. The results showed that the peak of sample separation of normal diploid appeared in 140 relative fluorescence intensity. Accordingly, it was concluded that the peak of the separation peak appeared at 70 fluorescence intensity as haploid material. The chromosome number of the root apex was analyzed. The results showed that the number of chromosomes of the plants identified as diploid was 2n=2x=16, and the number of chromosomes of the haploid was n=x=8. It was identical with the identification results of the flow cytometry. The effects of different concentration of colchicine on the chromosome doubling of haploid plants were studied by.5.. The results showed that with the same treatment time, the survival rate of regenerated plants decreased with the increase of colchicine concentration, but the diploid rate increased. With the same colchicine concentration, the survival rate of the plant decreased with the treatment time, but the diploid rate increased. The concentration of 200mg L-1 colchicine treated 48h and the dyeing of two tested varieties. The redoubled effect was best, the survival rate was 61.11%, 50%, and the doubling rate was 44.44% respectively. 38.89%.6. used dnf-566, rns-357 and acskp1 markers to detect some regenerated plants. The 11 plantlets of ADUN were all homozygous MSMS or MSMS, and the genotype of MS loci in the 15 plantlets of 'earth' were homozygous. Of the 16 regenerated plants of Dabao, 13 plants were homozygous at the MS locus, and the 3 of the heterozygous.7. plants were domesticated, and then transplanted into the test base net shed. The vast majority of the regenerated plants survived, the form was normal and the growth and development were good. The haploid plants of onions were small and weak, flowering and diploid oceans. The plants of the onion are basically the same, but the sucker stems are small. The whole flower inflorescence is smaller, the number of flower buds is small and small. The anthers are small and no pollen. The character of the double haploid plants conforms to the diploid characteristics and can blossom normally. The double haploid plants of the MSMS genotype identified by molecular markers are normal, and the identification of the double haploid plants of the MSMS genotype is the performance of the double haploid plant. For male sterile, the double haploid plant with normal fertility was put into the bag after blooming and put into the self crossing of the fly pollination. The self bred seed with full development and strong germination was obtained. The sterile double haploid plant was used as the mother parent with the fertile double haploid. The fertile double haploid dh-17 and the sterile double haploid DH-1 were obtained by the F1 generation. Using the slaf-seq technology based on simplified genome depth sequencing, using the simplified genomic depth sequencing, the onion transcriptional group was selected as a reference for the electronic enzyme cutting prediction, and the pvuii+scai enzyme cut was finally determined. The comparison between the sequence reading length of the southern mustard and the reference genome showed that the efficiency of the double end alignment was 80.60%, most of the sequencing read long inserts were inserted. The length of the fragment was within the range, indicating that the quality of the building was good. The length of the 125.98m reading length was obtained by sequencing. The number of reading lengths of each sample was within the range of 29736~30825419. The sequencing quality value of all samples was more than 80%, indicating that the error rate of the sequence base was lower and the data were qualified. The average GC content was 35.77%, and the content ratio was compared. According to the average, it has reached the requirement of sequencing. In the cluster analysis of the sequencing data satisfying the quality requirements, 294911 different types of labels were developed, of which 14776 were polymorphic SLAF tags, and only the 5%. of the total number of SLAF tags was encoded by genotyping according to the genetic code rules, and 8 genes were encoded. There are 6384 types of tags, of which 5354 polymorphic tags conform to the AA x BB type, and the 83.86%. using polymorphic tags for the encoded labels further develop the SNP loci. A total of 36109 groups of SNP. samples show that the number of abnormal tags is far less than 0.5%, thus the sample F1-1, F1-2 can be determined. F1-3, F1-4, and F1-5 are all progeny of DH-17 and DH-1.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S633.2

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