發(fā)布時間：2018-04-20 03:02

  本文選題：洋蔥(Allium + cepa�。� 參考：《山東農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：洋蔥(Allium cepa L.,染色體數(shù)2n=2x=16)是百合科(Liliaceae)蔥屬(Allium)二年生蔬菜,在世界范圍內(nèi)栽培廣泛,其年生產(chǎn)量和生產(chǎn)面積居于世界蔬菜生產(chǎn)的第三位。我國種植洋蔥的歷史雖然較短,但是發(fā)展迅速,在全國各地廣泛栽培,是重要的出口創(chuàng)匯蔬菜。洋蔥是典型的異花授粉蔬菜,自交衰退嚴重。傳統(tǒng)育種方法育成優(yōu)良的自交系需要6~10年的時間,而通過單倍體途徑在較短時間內(nèi)就可以選育出整齊一致的純系,明顯提高選擇效率。此外,單倍體加倍獲得的雙單倍體可作為重要性狀的遺傳分析、分子標記及數(shù)量性狀研究的理想材料。本研究以不同來源的中日照型洋蔥自交系、常規(guī)種、雜交種為材料,通過離體雌核發(fā)育途徑誘導(dǎo)培養(yǎng)洋蔥花蕾,對適宜培養(yǎng)基的篩選、培養(yǎng)程序優(yōu)化、植株再生、染色體倍性鑒定、染色體加倍技術(shù)、分子標記鑒定等進行了研究。利用獲得的洋蔥不育雙單倍體、可育雙單倍體以及二者雜交獲得的F1,采用SLAF-seq技術(shù)進行測序,獲得多態(tài)性SLAF標記,并開發(fā)出大量特異性SNP位點。主要結(jié)果如下:1.以引自日本的3個中日照型洋蔥雜交種的花蕾為外植體材料,研究了2,4-D和6-BA濃度及配比對單倍體誘導(dǎo)培養(yǎng)的影響。結(jié)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培養(yǎng)基適于胚的誘導(dǎo),誘導(dǎo)率最高達到4.00%。2.對洋蔥花蕾的誘導(dǎo)培養(yǎng)程序進行了優(yōu)化研究,結(jié)果表明不同花蕾大小對洋蔥的離體雌核發(fā)育有很大影響。直徑2.1~3.0 mm的花蕾離體誘導(dǎo)培養(yǎng)時成胚率明顯低于其它直徑的花蕾。直徑3.1~4.0 mm的花蕾誘導(dǎo)成胚率最高�！�地球’的花蕾經(jīng)低溫預(yù)處理1 d后成胚率明顯增高,預(yù)處理3 d后胚的誘導(dǎo)率開始明顯下降。而‘大寶’的花蕾經(jīng)低溫預(yù)處理1 d后成胚率沒有顯著變化,預(yù)處理3 d后胚的誘導(dǎo)率明顯下降。3.不同基因型洋蔥雌核發(fā)育胚的誘導(dǎo)率存在很大差異。本試驗供試的9個洋蔥基因型中胚的誘導(dǎo)率最高的是雜交種‘地球’,誘導(dǎo)率為4.67%,其次為‘大寶’和‘阿盾’,誘導(dǎo)率分別為4.33%和4.00%,沒有顯著差異。2個常規(guī)種‘天正紅玉’和‘天正黃金’分別誘導(dǎo)出了6枚和8枚胚,誘導(dǎo)率顯著少于其他基因型。2個自交系503和217分別誘導(dǎo)出13枚和10枚胚,誘導(dǎo)率顯著大于常規(guī)種,但顯著少于5個雜交種�？偟膩碚f,雜交種的胚誘導(dǎo)率大于自交系,自交系的誘導(dǎo)率大于常規(guī)種。4.將所獲得的雌核發(fā)育胚轉(zhuǎn)移至含有30 g·L-1蔗糖的1/2B5+0.5mg·L-1NAA培養(yǎng)基中,5d后即可發(fā)育成正常植株。利用流式細胞儀對所獲得的再生植株進行dna相對含量測定,結(jié)果顯示正常二倍體的樣品分離峰出現(xiàn)在140相對熒光強度中,據(jù)此斷定分離峰出現(xiàn)在70熒光強度的為單倍體材料。為進一步驗證再生植株的倍性,對通過流式細胞儀鑒定過的植株再進行根尖染色體計數(shù)分析,結(jié)果表明經(jīng)鑒定為二倍體的植株染色體數(shù)為2n=2x=16,鑒定為單倍體的染色體數(shù)為n=x=8,與流式細胞儀的鑒定結(jié)果完全吻合。5.研究了秋水仙素不同濃度、不同處理時間對單倍體植株染色體加倍效果的影響。結(jié)果表明,在處理時間相同的情況下,隨著秋水仙素濃度升高,再生植株的存活率降低,但二倍體率增加;在相同秋水仙素濃度下,隨處理時間延長,植株的成活率下降,但二倍體率增加。濃度為200mg·l-1的秋水仙素處理48h,對兩個供試品種的染色體加倍效果最好,成活率分別為61.11%、50.00%,加倍率分別為44.44%、38.89%。6.利用dnf-566、rns-357和acskp1標記對部分再生植株進行了檢測�！�阿盾’的11株再生植株均為純合的msms或msms,‘地球’的15株再生植株中在ms位點的基因型均為純合的。‘大寶’的16株再生植株中,有13株在ms位點的基因型均為純合的,3株是雜合的。7.生根良好的再生植株經(jīng)馴化、煉苗后,移栽到試驗基地網(wǎng)棚,絕大多數(shù)再生植株成活,形態(tài)正常,生長發(fā)育良好。洋蔥單倍體植株矮小、細弱,開花期與二倍體洋蔥植株基本一致,但抽生花薹矮小。整個花序較小,花蕾數(shù)少而且小�；ㄋ幮�,沒有花粉。雙單倍體植株性狀表現(xiàn)符合二倍體特征,能正常開花。經(jīng)分子標記鑒定為msms基因型的雙單倍體植株育性正常,而鑒定為msms基因型的雙單倍體植株表現(xiàn)為雄性不育。育性正常的雙單倍體植株開花后套袋,放入蒼蠅授粉自交,獲得了發(fā)育飽滿、發(fā)芽力強的自交種子。對不育雙單倍體植株,利用其作為母本與可育雙單倍體雜交,獲得了f1代種子。8.以獲得的可育雙單倍體dh-17、不育雙單倍體dh-1及部分f1代單株為材料,利用基于簡化基因組深度測序的slaf-seq技術(shù),選擇洋蔥轉(zhuǎn)錄組作為參考進行電子酶切預(yù)測,最終確定使用pvuii+scai酶切。將南芥的測序讀長與參考基因組的比對結(jié)果顯示雙端比對效率為80.60%,大部分測序讀長的插入片段長度都在范圍之內(nèi),表明建庫質(zhì)量良好。通過測序共獲得125.98m讀長,各樣品所獲讀長數(shù)量在29736~30825419范圍內(nèi)。所有樣品的測序質(zhì)量值q30均大于80%,說明測序堿基錯誤率較低,所獲數(shù)據(jù)合格。測序獲得平均gc含量為35.77%,而且含量比較平均,說明達到了測序要求。對滿足質(zhì)量要求的測序數(shù)據(jù)進行聚類分析,共開發(fā)出各類型標簽294911個,其中多態(tài)性SLAF標簽14776個,僅占SLAF標簽總數(shù)的5%。根據(jù)基因型編碼規(guī)則對篩選出的多態(tài)性標簽進行基因型編碼,共得到可編碼8種基因型的標簽6384個,其中5354個多態(tài)性標簽符合aa×bb類型,占可編碼標簽的83.86%。利用多態(tài)性標簽進一步進行SNP位點的開發(fā),共得到36109個群體的SNP。樣品親緣關(guān)系鑒定結(jié)果表明異常標簽數(shù)目所占比例均遠遠小于0.5%,由此可以判定樣品F1-1、F1-2、F1-3、F1-4、F1-5均為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.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S633.2

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