‘抛橘’的遗传鉴定及其无核机理初步研究

发布时间：2019-07-02 09:31

【摘要】：柑橘是世界上重要的水果,柑橘的无核及晚熟性状一直以来都是柑橘育种工作者重点关注内容。最近本课题组在福建省发现一个名为‘抛橘’的无核柑橘地方资源,其在当地具有近百年的种植历史,但文献未有记载。‘抛橘’具有果肉似橙,果皮有褶皱且易剥离,果实大小与葡萄柚相类似的特点,果实成熟期一般在次年一月中旬左右,可挂果至次年4月中旬,单果均重349.8g,可食率平均为45.9%,可溶性固形物含量为10.4%,可滴定酸含量为1.62%。综上,‘抛橘’具有较高的研究和开发价值。本课题研究以‘抛橘’为研究对象,从雌雄配子体育性等方面初步研究了‘抛橘’无核的原因;利用形态学观察和SSR标记对‘抛橘’进行遗传鉴定。取得的主要研究结果如下:一、‘抛橘’的雌雄配子体发育正常1.连续两年,观察花粉育性指数。使用醋酸洋红染色观察‘抛橘’的花粉染色活力,结果显示正常率均在80%以上;利用培养基上培养‘抛橘’花粉,结果显示萌芽率均在75%以上;利用血球计数板统计其每花药花粉量为2×104粒左右。2.解剖学结果表明:‘抛橘’雄配子体发育正常。扫描电镜结果显示‘抛橘’花粉形态正常,并且正常率可达到80%以上。3.石蜡切片观察:观察了‘抛橘’雌配子体的四个发育时期,未发现败育及畸形发育的情况;对‘抛橘’雄配子体的发育进行了观察,其发育正常。综上,‘抛橘’的无核原因可能为自交不亲和或胚胎败育。二、‘抛橘’的遗传鉴定1.7对cp SSR引物和13对nSSR引物分别对24和22个柑橘属及其近缘属植物进行了UPGMA聚类分析,推测:‘抛橘’是以柚类(Citrus maxima)×酸橙类(citrus aurantium)杂交而来。2.基因重测序数据分析显示:?基因重测序数据分析显示:‘抛橘’与酸橙的基因组序列相似度在85%以上,推测‘抛橘’可能属于酸橙的一种;尽管叶片形态、果实外形以及果肉质地和色泽与温州蜜柑相近,但与温州蜜柑基因组序列相似度只有63.49%,不能发现存在直接亲缘关系的证据。3.13对nSSR引物对38个柑橘属及其近缘属植物进行了UPGMA聚类分析,结果显示:‘抛橘’和代代酸橙有较密切的亲缘关系。推测:代代酸橙可能是‘抛橘’的一种类型或者是杂交亲本之一。三、q PCR检测4个与SI相关基因在‘抛橘’不同组织中的表达量结果显示:Cg Rns6、Cg Rns9、Cg Rns L在‘抛橘’不同组织中的表达没有规律,而Cg RNS3在‘抛橘’的雌蕊中特异性表达,表明‘抛橘’无核的原因可能是自交不亲和,而Cg RNS3可能是其自交不亲和的候选基因。
[Abstract]:Citrus is an important fruit in the world. The seedless and late maturing traits of citrus have always been the focus of citrus breeders. Recently, our research group discovered a seedless citrus local resource called 'throwing orange' in Fujian Province, which has a planting history of nearly a hundred years, but it has not been recorded in the literature. 'throwing orange' has the characteristics of orange flesh, fold and easy peeling, and the fruit size is similar to that of grapefruit. The fruit ripening period is generally in the middle of January of the following year. The average weight of single fruit is 349.8g, the average edible rate is 45.9%, the soluble solid content is 10.4%, and the titratable acid content is 1.62%. In summary, 'throwing orange' has a high research and development value. In this study, the reason of seedlessness of 'throwing orange' was studied from the aspects of male and female gametes, and the genetic identification of 'throwing orange' was carried out by morphological observation and SSR markers. The main results are as follows: first, the male and female gametophyte development of 'throwing orange' is normal. The pollen fertility index was observed for two years in a row. The pollen staining activity of 'throwing orange' was observed by magnitine acetate staining, the results showed that the normal rate was above 80%, the germination rate of 'throwing orange' pollen was above 75%, and the amount of anther powder per anther was about 2 脳 104 grains by blood cell counting board. The anatomical results showed that the male gametophyte developed normally. The results of scanning electron microscope (SEM) showed that the pollen morphology of 'throwing orange' was normal, and the normal rate could reach more than 80%. Paraffin section observation: the four developmental stages of female gametophyte of 'throwing orange' were observed, and no abortion and abnormal development were found, and the development of male gametophyte of 'throwing orange' was observed, and the development of male gametophyte was normal. In summary, the seedless cause of orange throwing may be self-incompatibility or embryo abortion. 2. UPGMA cluster analysis of 24 and 22 citrus and their related plants was carried out with 1.7 pairs of cp SSR primers and 13 pairs of nSSR primers, respectively. it was inferred that 'throwing orange' was made by (citrus aurantium) hybrid of grapefruit (Citrus maxima) 脳 lime. 2. The analysis of gene resequencing data shows that:? The analysis of gene resequencing data showed that the similarity of genome sequence between 'throwing orange' and lime was more than 85%. It was speculated that 'throwing orange' might belong to a kind of lime. Although the leaf morphology, fruit shape, pulp texture and color were similar to those of Satsuma mandarin, the similarity of genome sequence with Satsuma mandarin was only 63.49%, and no evidence of direct genetic relationship could be found. 3.13 pairs of nSSR primers were used to analyze 38 citrus and their related plants by UPGMA cluster analysis. The results showed that there was a close genetic relationship between 'throwing orange' and generation lime. It is speculated that the generation lime may be a type of 'throwing orange' or one of the hybrid parents. The expression of four SI related genes in different tissues of 'throwing orange' was detected by three, q PCR. The results showed that the expression of Cg Rns6,Cg Rns9,Cg Rns L in different tissues of 'throwing orange' was irregular, while the specific expression of Cg RNS3 in the pistil of 'throwing orange' indicated that the reason for the lack of nucleus in 'throwing orange' may be self-incompatibility, while Cg RNS3 may be a candidate gene for self-incompatibility.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S666

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