核桃重要種質(zhì)遺傳分析及主栽品種指紋鑒定
發(fā)布時(shí)間：2018-08-21 09:47
【摘要】：核桃重要種質(zhì)資源的遺傳分析是核桃育種中親本選擇的基礎(chǔ)工作,可為育種中種質(zhì)的創(chuàng)新、資源合理利用和遺傳改良提供科學(xué)支持。核桃種質(zhì)鑒定則是遺傳改良和知識(shí)產(chǎn)權(quán)保護(hù)的重要依據(jù)。傳統(tǒng)的形態(tài)學(xué)鑒定方法簡(jiǎn)單、直觀,但由于核桃育種親本利用的集中化,使選育的新品種在形態(tài)上愈加趨于相似,形態(tài)學(xué)方法已無法滿足眾多品種的鑒定和純度分析。分子標(biāo)記可直接反映個(gè)體間DNA水平的差異,能穩(wěn)定遺傳,信息量大,與形態(tài)學(xué)鑒定結(jié)合有效彌補(bǔ)了形態(tài)學(xué)鑒定的不足,可快捷、準(zhǔn)確、穩(wěn)定的進(jìn)行資源評(píng)價(jià)。 本研究從核桃BAC文庫末端序列(BES)中開發(fā)了SSR引物,將其用于核桃種質(zhì)遺傳分析與品種鑒定中。以自交的親本和子代為對(duì)象開展了形態(tài)和分子方面的研究,探討了核桃自交形成的遺傳學(xué)機(jī)制及其對(duì)表型的影響。同時(shí),以堅(jiān)果表型為基礎(chǔ),SSR標(biāo)記輔助建立了部分品種的指紋圖譜。獲得了以下主要結(jié)果： 1.從NCBI數(shù)據(jù)庫下載了核桃BAC末端序列22740條。應(yīng)用MISA軟件搜索獲得SSR位點(diǎn)4732個(gè),SSR出現(xiàn)頻率為1/2.8kb。其中,A/T、AT/AT、ATT/AAT分別為最豐富的單核苷酸、二核苷酸和三核苷酸重復(fù)。選擇具不同類型和重復(fù)次數(shù)核心基元的序列設(shè)計(jì)合成310對(duì)引物,篩選后獲得特異性較高的多態(tài)性引物116對(duì)。 選取其中19對(duì)SSR引物對(duì)核桃屬中6個(gè)種及1個(gè)種間雜交種的20個(gè)基因型進(jìn)行SSR分析,每對(duì)引物檢測(cè)到2-9個(gè)多態(tài)性位點(diǎn),平均多態(tài)性位點(diǎn)為5.4個(gè)。其中17個(gè)位點(diǎn)的多態(tài)信息含量高于0.5,總平均值為0.662,多態(tài)性較高。聚類分析顯示不同基因型依存種屬分類及地理起源分組。它們?cè)诤颂?J. regia L.)、泡核桃(J. sigillata Dode)、麻核桃(J. hopeiensis Hu)和核桃楸(J. mandshurica Maxim)中多態(tài)性的比例分別為92%(107對(duì))、71%(82對(duì))、49%(57對(duì))和41%(47對(duì))。 2.以晚實(shí)品種‘大泡核桃’1979-1982年自交產(chǎn)生的實(shí)生后代9個(gè)單株(SP1～SP9)為研究對(duì)象,觀察其植物表型并對(duì)親本和9個(gè)自交后代進(jìn)行了遺傳分析。自交后代的表型出現(xiàn)明顯差異,主要表現(xiàn)在童期長(zhǎng)短：SP1～SP4播種后2年開花結(jié)果,SP5～SP8為7-8年開花結(jié)果,而SP9自1980年播種至今未開花；所結(jié)的種子包括泡核桃(堅(jiān)果殼厚0.1-0.9mm)、夾棉核桃(殼厚1.0-1.5mm)和鐵核桃(殼厚1.6-2.0mm或超過2.0mm)三種類型；部分植株生長(zhǎng)量小,抗性差,樹齡超過30年樹高僅4.5-5.5m。用58個(gè)SSR標(biāo)記分析自交后代的遺傳組成,結(jié)果顯示在‘大泡核桃’后代表現(xiàn)一定程度的近交衰退。所有子代的基因組純合度增加,并且在SP9中表現(xiàn)最為明顯。SP5表型和遺傳組成都與親本最相似,相同SSR位點(diǎn)的比例為82.8%。 3.用28對(duì)SSR引物分析了我國(guó)62份核桃(J. regia)種質(zhì)的遺傳關(guān)系。每個(gè)引物擴(kuò)增的等位基因數(shù)為2-7個(gè),期望雜合度(He)范圍從0.382-0.821,平均為0.609；香農(nóng)信息指數(shù)從0.637(WJR291)到1.750(WJR265)不等,平均為1.122。以19對(duì)BES-SSR引物計(jì)算的62個(gè)品種間的遺傳相似系數(shù)從0.47-0.97,遺傳多樣性水平較高。聚類分析顯示62份種質(zhì)可分為趨向于地理起源分類的三組。本研究的結(jié)果可對(duì)品種分類和雜交親本選擇提供參考,雜交育種中用親緣關(guān)系較遠(yuǎn)的不同組的材料為親本進(jìn)行雜交,有利于培育出變異類型豐富的后代群體。 4.建立了堅(jiān)果表型與SSR標(biāo)記結(jié)合進(jìn)行核桃(J. reiga)品種鑒指紋鑒定的體系。用12個(gè)BES-SSR標(biāo)記輔助核桃屬新品種DUS測(cè)試指南中22個(gè)堅(jiān)果形狀特征,對(duì)我國(guó)35個(gè)主栽核桃品種進(jìn)行區(qū)分,最終選定4個(gè)果形特征[堅(jiān)果縱切面形狀——圓形、(闊)卵圓形、(闊)橢圓形和果頂形狀——尖]和4對(duì)引物(WJR031、WJR069、WJR265和WJR281)建立了35個(gè)品種的指紋圖譜。首先以果形特征將35個(gè)品種分為4組,每組中用最多2對(duì)引物就可區(qū)分組內(nèi)品種,每一品種均具唯一的果形特征和SSR標(biāo)記等位基因的組合。如果不考慮果形特征,用上述4對(duì)SSR引物也可將所分析的35個(gè)品種有效區(qū)分。
[Abstract]:Genetic analysis of important walnut germplasm resources is the basic work of parental selection in walnut breeding. It can provide scientific support for germplasm innovation, rational utilization and genetic improvement in walnut breeding. The centralized utilization of parents in peach breeding has made the morphological similarity of new peach varieties more and more similar. Morphological methods have been unable to satisfy the identification and purity analysis of many varieties. Foot can evaluate resources quickly, accurately and steadily.
In this study, SSR primers were developed from the BAC terminal sequence (BES) of walnut and used in genetic analysis of walnut germplasm and variety identification. SSR fingerprinting assisted the establishment of fingerprints of some cultivars. The following results were obtained:
1. 22740 BAC terminal sequences of walnut were downloaded from NCBI database. 4732 SSR loci and 1/2.8 KB SSR frequency were obtained by MISA software. Among them, A/T, AT/AT and ATT/AAT were the most abundant single nucleotide, dinucleotide and trinucleotide repeats, respectively. 310 pairs of primers with different types and repeats were designed and synthesized. After screening, 116 pairs of polymorphic primers with high specificity were obtained.
Nineteen pairs of SSR primers were selected to analyze 20 genotypes of six species and one interspecific hybrid of Walnut. Each pair of primers detected 2-9 polymorphic loci, with an average polymorphic locus of 5.4. The polymorphic information content of 17 loci was higher than 0.5, with a total average value of 0.662 and high polymorphism. Taxonomy and geographic origin of the genus Juglans. The percentage of polymorphism in walnut (J. regia L.), walnut (J. sigillata Dode), Juglans (J. hopeiensis Hu) and Juglans (J. mandshurica Maxim) was 92% (107), 71% (82), 49% (57) and 41% (47) respectively.
2. Nine seedling progenies (SP1-SP9) of late-bearing cultivar'Dahuo Walnut'from 1979 to 1982 were selected as the research objects. The phenotypes of the progenies were observed and their parents and 9 progenies were analyzed. It is 7-8 years flowering and fruiting, but SP9 has not flowered since 1980; the seeds include walnut (nut shell thickness 0.1-0.9 mm), cotton walnut (shell thickness 1.0-1.5 mm) and iron walnut (shell thickness 1.6-2.0 mm or more than 2.0 mm); some plants have small growth, poor resistance, tree height over 30 years old is only 4.5-5.5 mm. The results showed that the progenies of'Bubble Walnut'showed a certain degree of inbreeding decline. Genome homozygosity of all progenies increased and was most obvious in SP9. The phenotype and genetic composition of SP5 were most similar to those of parents, and the proportion of the same SSR locus was 82.8%.
3. The genetic relationship of 62 walnut (J. regia) Germplasms in China was analyzed with 28 pairs of SSR primers. The number of alleles amplified by each primer was 2-7, and the expected heterozygosity (He) ranged from 0.382-0.821, averaging 0.609; Shannon's information index ranged from 0.637 (WJR291) to 1.750 (WJR265), averaging 1.122. The genetic similarity coefficient ranged from 0.47 to 0.97, and the level of genetic diversity was high. Cluster analysis showed that 62 germplasms could be divided into three groups tending to geographical origin classification. The offspring group with abundant variation types.
4. A system of fingerprint identification of walnut (J. reiga) cultivars was established by combining the phenotype of walnut with SSR markers. Twelve BES-SSR markers were used to assist the identification of 22 nuts in the DUS test guidelines for new walnut cultivars. Thirty-five main Walnut Cultivars in China were distinguished and four fruit-shaped characteristics were selected. The fingerprints of 35 cultivars were established by using round, (broad) oval and apical shape-tip primers and four pairs of primers (WJR031, WJR069, WJR265 and WJR281). If the fruit shape characteristics are not considered, the 4 pairs of SSR primers can be used to distinguish the 35 varieties.
【學(xué)位授予單位】：北京林業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：S664.1
本文編號(hào)：2195326
[Abstract]:Genetic analysis of important walnut germplasm resources is the basic work of parental selection in walnut breeding. It can provide scientific support for germplasm innovation, rational utilization and genetic improvement in walnut breeding. The centralized utilization of parents in peach breeding has made the morphological similarity of new peach varieties more and more similar. Morphological methods have been unable to satisfy the identification and purity analysis of many varieties. Foot can evaluate resources quickly, accurately and steadily.
In this study, SSR primers were developed from the BAC terminal sequence (BES) of walnut and used in genetic analysis of walnut germplasm and variety identification. SSR fingerprinting assisted the establishment of fingerprints of some cultivars. The following results were obtained:
1. 22740 BAC terminal sequences of walnut were downloaded from NCBI database. 4732 SSR loci and 1/2.8 KB SSR frequency were obtained by MISA software. Among them, A/T, AT/AT and ATT/AAT were the most abundant single nucleotide, dinucleotide and trinucleotide repeats, respectively. 310 pairs of primers with different types and repeats were designed and synthesized. After screening, 116 pairs of polymorphic primers with high specificity were obtained.
Nineteen pairs of SSR primers were selected to analyze 20 genotypes of six species and one interspecific hybrid of Walnut. Each pair of primers detected 2-9 polymorphic loci, with an average polymorphic locus of 5.4. The polymorphic information content of 17 loci was higher than 0.5, with a total average value of 0.662 and high polymorphism. Taxonomy and geographic origin of the genus Juglans. The percentage of polymorphism in walnut (J. regia L.), walnut (J. sigillata Dode), Juglans (J. hopeiensis Hu) and Juglans (J. mandshurica Maxim) was 92% (107), 71% (82), 49% (57) and 41% (47) respectively.
2. Nine seedling progenies (SP1-SP9) of late-bearing cultivar'Dahuo Walnut'from 1979 to 1982 were selected as the research objects. The phenotypes of the progenies were observed and their parents and 9 progenies were analyzed. It is 7-8 years flowering and fruiting, but SP9 has not flowered since 1980; the seeds include walnut (nut shell thickness 0.1-0.9 mm), cotton walnut (shell thickness 1.0-1.5 mm) and iron walnut (shell thickness 1.6-2.0 mm or more than 2.0 mm); some plants have small growth, poor resistance, tree height over 30 years old is only 4.5-5.5 mm. The results showed that the progenies of'Bubble Walnut'showed a certain degree of inbreeding decline. Genome homozygosity of all progenies increased and was most obvious in SP9. The phenotype and genetic composition of SP5 were most similar to those of parents, and the proportion of the same SSR locus was 82.8%.
3. The genetic relationship of 62 walnut (J. regia) Germplasms in China was analyzed with 28 pairs of SSR primers. The number of alleles amplified by each primer was 2-7, and the expected heterozygosity (He) ranged from 0.382-0.821, averaging 0.609; Shannon's information index ranged from 0.637 (WJR291) to 1.750 (WJR265), averaging 1.122. The genetic similarity coefficient ranged from 0.47 to 0.97, and the level of genetic diversity was high. Cluster analysis showed that 62 germplasms could be divided into three groups tending to geographical origin classification. The offspring group with abundant variation types.
4. A system of fingerprint identification of walnut (J. reiga) cultivars was established by combining the phenotype of walnut with SSR markers. Twelve BES-SSR markers were used to assist the identification of 22 nuts in the DUS test guidelines for new walnut cultivars. Thirty-five main Walnut Cultivars in China were distinguished and four fruit-shaped characteristics were selected. The fingerprints of 35 cultivars were established by using round, (broad) oval and apical shape-tip primers and four pairs of primers (WJR031, WJR069, WJR265 and WJR281). If the fruit shape characteristics are not considered, the 4 pairs of SSR primers can be used to distinguish the 35 varieties.
【學(xué)位授予單位】：北京林業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：S664.1
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