TWI568853B - Method for genetic characteristics and individual identification in geese - Google Patents
Method for genetic characteristics and individual identification in geese Download PDFInfo
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本發明一般地涉及一鵝隻遺傳特性鑑定與個體鑑別的方法。具體而言,本發明涉及使用一微衛星標幟引子對組(microsatellite marker primer set)用於鵝遺傳特性鑑定與個體鑑別的方法。 The present invention generally relates to a method for genetic characterization and individual identification of a goose. In particular, the invention relates to a method for the identification and individual identification of goose genetic traits using a microsatellite marker primer set.
養鵝業為台灣重要農業之一,面對外來的競爭下,除了強化本身之飼養管理技術,亦須強化育種技能,以培育出更多符合市場需求之品系。在品系培育方面,隨著分子生物學不斷進步,DNA分子標記技術已廣泛作為遺傳分析與育種輔助之重要手段。 Goose industry is one of the important agriculture in Taiwan. In the face of external competition, in addition to strengthening its own breeding management technology, it is necessary to strengthen breeding skills to cultivate more products that meet market demand. In terms of line breeding, with the continuous advancement of molecular biology, DNA molecular marker technology has been widely used as an important means of genetic analysis and breeding assistance.
微衛星標幟(microsatellite marker)為分子標幟(molecular marker)的一種,已被應用在許多遺傳方面的研究,其包含基因圖譜建構、族群結構分析、個體鑑別與親子鑑別或生物入侵的溯源等。微衛星標幟為一DNA序列,由不同數目之重複片段(number of units)串連而成,重複之單位長度(unit size)可由單核苷酸(mononucleotide)、雙核苷酸(dinucleotide),或至五個核苷酸(pentanucleotide)所組成,並且廣泛分布於基因體中(Powell et al.,1996,Polymorphism revealed by simple sequence repeats.Trends Plant Sci.1:215-222.)。微衛星標幟廣泛存在於真核生物的細胞核內基因組中,其重複片段種類眾多,且重複次數多變,因此,具有高 度多態性。此外,微衛星標幟兩端的旁側區(flanking regions)具有同源(homologous)特性,序列保留性較高(Oliveira et al.,2006,Origin,evolution and genome distribution of microsatellites.Genet.Mol.Biol.29:294-307.),可應用於同屬(genus)物種的多態性分析上(Jia et al.,2013,Characterization and cross-species amplification of 11 polymorphic microsatellite loci in Liobagrus marginatoides.Conserv.Genet.Resour.5:1087-1089;Parine et al.,2013,Characterization and cross-species amplification of microsatellite markers in African Silverbill(Lonchura cantans).Genet.Mol.Res.12:5634-5639.),甚至僅只是同科(family)的物種(Kryger et al.,2002,Isolation and characterization of six polymorphic microsatellite loci in South African hares (Lepus saxatilis F.Cuvier,1823 and Lepus capensis Linnaeus,1758).Mol.Ecol.Notes 2:422-424.)。 The microsatellite marker is a molecular marker that has been applied in many genetic studies, including gene mapping construction, ethnic structure analysis, individual identification and paternity identification, or traceability of biological invasion. . The microsatellite marker is a DNA sequence which is formed by concatenating a different number of units, and the unit size of the repeat may be a mononucleotide, a dinucleotide, or It consists of five pentanucleotides and is widely distributed in the genome (Powell et al ., 1996, Polymorphism revealed by simple sequence repeats. Trends Plant Sci. 1:215-222.). Microsatellite markers are widely present in the genome of eukaryotic nuclei, and their repetitive fragments are numerous and variable in number of repeats. Therefore, they are highly polymorphic. In addition, the flanking regions at both ends of the microsatellite marker have homologous properties and high sequence retention (Oliveira et al ., 2006, Origin, evolution and genome distribution of microsatellites.Genet.Mol.Biol). .29:294-307.), applicable to polymorphism analysis of genus species (Jia et al ., 2013, Characterization and cross-species amplification of 11 polymorphic microsatellite loci in Liobagrus marginatoides. Conserv.Genet .Resour.5:1087-1089;Parine et al .,2013,Characterization and cross-species amplification of microsatellite markers in African Silverbill ( Lonchura cantans).Genet.Mol.Res.12:5634-5639.), even just Family species (Kryger et al ., 2002, Isolation and characterization of six polymorphic microsatellite loci in South African hares ( Lepus saxatilis F. Cuvier, 1823 and Lepus capensis Linnaeus, 1758). Mol. Ecol. Notes 2: 422-424.).
目前台灣尚無合適的鵝隻微衛星標幟商業套組以用於進行鵝隻繁殖場之個體遺傳分析或族群遺傳結構分析。因此,亟需開發適用於臺灣地區鵝隻族群之微衛星標幟。 At present, there is no suitable commercial set of goose microsatellite logos in Taiwan for individual genetic analysis or ethnic genetic structure analysis of goose breeding grounds. Therefore, there is an urgent need to develop microsatellite markers for the goose population in Taiwan.
本發明係提供一新穎微衛星標幟引子對組用於鵝隻遺傳特性鑑定與個體鑑別。該微衛星標幟引子對組所篩選出之微衛星標幟具有高多態性可被使用為不同族群鵝隻之遺傳特性分析,舉例來說,區分族群遺傳結構差異、個體鑑別率或親子排除率之分析。 The invention provides a novel microsatellite marker primer pair group for the genetic characterization and individual identification of goose. The microsatellite marker introduces a high polymorphism of the microsatellite markers selected by the group and can be used as an analysis of the genetic characteristics of different ethnic groups of geese, for example, distinguishing ethnic genetic structure differences, individual discrimination rates or parental exclusion Analysis of the rate.
在一方面,本發明提供一種用於鵝隻遺傳特性鑑定與個體鑑別的方法,該方法包括:(i)提供一待檢測鵝隻血液樣本,並萃取該血液樣本中之基因體DNA; (ii)以至少1組微衛星標幟引子對檢測該鵝隻之基因體DNA;其中該微衛星標幟引子對係選自由:(1)一分別具有SEQ ID NO:1及2之核苷酸序列的第一引子對;(2)一分別具有SEQ ID NO:3及4之核苷酸序列的第二引子對;(3)一分別具有SEQ ID NO:5及6之核苷酸序列的第三引子對;(4)一分別具有SEQ ID NO:7及8之核苷酸序列的第四引子對;(5)一分別具有SEQ ID NO:9及10之核苷酸序列的第五引子對;(6)一分別具有SEQ ID NO:11及12之核苷酸序列的第六引子對;(7)一分別具有SEQ ID NO:13及14之核苷酸序列的第七引子對;(8)一分別具有SEQ ID NO:15及16之核苷酸序列的第八引子對;(9)一分別具有SEQ ID NO:17及18之核苷酸序列的第九引子對;(10)一分別具有SEQ ID NO:19及20之核苷酸序列的第十引子對;(11)一分別具有SEQ ID NO:21及22之核苷酸序列的第十一引子對;(12)一分別具有SEQ ID NO:23及24之核苷酸序列的第十二引子對;(13)一分別具有SEQ ID NO:25及26之核苷酸序列的第十三引子對;(14)一分別具有SEQ ID NO:27及28之核苷酸序列的第十四引子對;及其組合所組成之群組;(iii)依步驟(ii)之檢測結果分析該鵝隻之基因型以評估其遺傳特性及個體鑑別。 In one aspect, the present invention provides a method for genetic characterization and individual identification of goose, the method comprising: (i) providing a blood sample of a goose to be detected, and extracting the genomic DNA in the blood sample; (ii) detecting the genomic DNA of the goose with at least one set of microsatellite primer primer pairs; wherein the microsatellite primer primer pair is selected from the group consisting of: (1) a nucleoside having SEQ ID NOS: 1 and 2, respectively a first primer pair of the acid sequence; (2) a second primer pair having the nucleotide sequences of SEQ ID NOS: 3 and 4, respectively; (3) a nucleotide sequence having SEQ ID NOS: 5 and 6, respectively a third primer pair; (4) a fourth primer pair having the nucleotide sequences of SEQ ID NOS: 7 and 8, respectively; (5) a nucleotide sequence having SEQ ID NOS: 9 and 10, respectively. a fifth primer pair; (6) a sixth primer pair having the nucleotide sequences of SEQ ID NOS: 11 and 12, respectively; (7) a seventh primer having the nucleotide sequences of SEQ ID NOS: 13 and 14, respectively. (8) an eighth primer pair having the nucleotide sequences of SEQ ID NOS: 15 and 16, respectively; (9) a ninth primer pair having the nucleotide sequences of SEQ ID NOS: 17 and 18, respectively; (10) a tenth primer pair having the nucleotide sequences of SEQ ID NOS: 19 and 20, respectively; (11) an eleventh pair of primers having the nucleotide sequences of SEQ ID NOS: 21 and 22, respectively; 12) A core having SEQ ID NOS: 23 and 24, respectively a twelfth primer pair of the acid sequence; (13) a thirteenth primer pair having the nucleotide sequences of SEQ ID NOS: 25 and 26, respectively; (14) a nucleoside having SEQ ID NOS: 27 and 28, respectively a fourteenth primer pair of the acid sequence; and a group consisting of the combinations; (iii) analyzing the genotype of the goose according to the test result of the step (ii) to evaluate the genetic characteristics and individual identification.
在本發明部分實施例中,其以至少8組微衛星標幟引子對檢測該鵝隻之基因體DNA。在一特定實施例中,其中該微衛星標幟引子對係第一引子對至第八引子對、第一引子對至第九引子對、或第一引子對至第 十四引子對。 In some embodiments of the invention, the genotype DNA of the goose is detected with at least 8 sets of microsatellite primer pairs. In a particular embodiment, wherein the microsatellite marker pair is from a first primer pair to an eighth primer pair, a first primer pair to a ninth primer pair, or a first primer pair Fourteen pairs of pairs.
在一方面,本發明提供一種用於鵝隻遺傳特性鑑定與個體鑑別的套組,其包括1組以上之微衛星標幟引子對檢測該鵝隻之基因體DNA,以取得該待檢測鵝隻之基因型;其中該微衛星標幟引子對係選自由第一引子對至第十四引子對,及其組合所組成之群。 In one aspect, the present invention provides a kit for genetic characterization and individual identification of goose, comprising one or more sets of microsatellite markers introduced to detect the genomic DNA of the goose to obtain the goose to be detected. a genotype; wherein the microsatellite primer pair is selected from the group consisting of a first primer pair to a fourteen primer pair, and a combination thereof.
在本發明一具體實施例中,該用於鵝隻遺傳特性鑑定與個體鑑別的套組之微衛星標幟引子對係第一引子對至第十四引子對。 In a specific embodiment of the present invention, the microsatellite marker primer pair for the genetic characterization and individual identification of the goose is a pair of first primer pairs to a fourteenth primer pair.
在另一方面,本發明提供一種用於鵝隻遺傳特性鑑定與個體鑑別的微衛星標幟引子對,其中該微衛星標幟引子對係第一引子對至第十四引子對。 In another aspect, the present invention provides a microsatellite marker primer pair for genetic identification and individual identification of a goose, wherein the microsatellite primer pair is a pair of first primer pairs to a fourteenth primer pair.
在部分具體實施例中,本發明之微衛星標幟引子對之微衛星標幟正向引子或微衛星標幟反向引子之5’端係連接有一螢光試劑。 In some embodiments, the microsatellite primer of the present invention has a fluorescent reagent attached to the 5' end of the microsatellite forward primer or the microsatellite reverse primer.
本發明之各個具體實例的細節說明如後。本發明之其他特徵將會經由以下各個具體實例中的詳細說明及申請專利範圍而清楚呈現。 Detailed descriptions of various specific examples of the invention are given below. Other features of the present invention will be apparent from the following detailed description and claims.
無須進一步的闡述,咸相信本發明所屬技術領域中具有通常知識者基於前述說明即可利用本發明至最廣的程度。因此,可以理解以下的說明僅僅是作為例示說明之用,而非以任何方式限制其餘的揭露內容。 Without further elaboration, it is believed that those of ordinary skill in the art of Therefore, it is to be understood that the following description is for illustrative purposes only and is not intended to limit the disclosure.
連同附圖閱讀能幫助瞭解前面的發明內容以及接下來的本發明詳細描述。在此所呈現之較佳圖式及具體實施例係以闡述本發明為目的。應理解的是,本發明並不侷限於所示之精確排列及方式。 The reading of the drawings together with the accompanying drawings will be able to aid the understanding of the foregoing invention and the detailed description of the invention. The preferred figures and embodiments presented herein are for the purpose of illustrating the invention. It should be understood that the invention is not limited to the precise arrangements and manner shown.
圖l顯示各鵝隻族群利用14組新微衛星標幟所得鄰位連接法(NJ)之親緣關 係樹。WR:白羅曼鵝;WC:白色華鵝;BC:褐色華鵝;H:雜交鵝;BS:黑天鵝。 Figure 1 shows the kinship of the ortho-joining method (NJ) obtained by each goose population using 14 sets of new microsatellite markers. Tie the tree. WR: White Roman Goose; WC: White Chinese Goose; BC: Brown Chinese Goose; H: Cross Goose; BS: Black Swan.
圖2顯示各鵝隻族群用14組新微衛星標幟繪製之主成分分析(PCA)3D圖。WR:白羅曼;WC:白色華鵝;BC:褐色華鵝;H:雜交鵝;BS:黑天鵝。 Figure 2 shows a principal component analysis (PCA) 3D plot of each goose population grouped with 14 new microsatellite markers. WR: White Roman; WC: White Chinese Goose; BC: Brown Chinese Goose; H: Cross Goose; BS: Black Swan.
圖3顯示各鵝隻個體利用14組新微衛星標幟所繪製之STRUCTURE群集分析圖(K=2~7)。K值為STRUCTURE分析中預設之群集數,以不同顏色表示不同群集。縱軸為個體的基因來源於該群集之比例,且每個圖條表示一個個體。1:白羅曼;2:白色華鵝;3:褐色華鵝;4:雜交鵝;5:黑天鵝。 Figure 3 shows a STRUCTURE cluster analysis (K=2~7) drawn by individual goose individuals using 14 new microsatellite markers. The K value is the number of clusters preset in the STRUCTURE analysis, representing different clusters in different colors. The vertical axis is the proportion of the individual's genes from the cluster, and each bar represents an individual. 1: White Roman; 2: White Chinese Goose; 3: Brown Chinese Goose; 4: Cross Goose; 5: Black Swan.
圖4顯示各鵝隻個體利用14組新微衛星標幟所得鄰位連接法(NJ)之親緣關係樹。●:白羅曼鵝;:雜交鵝;△:白色華鵝;:褐色華鵝;:黑天鵝。 Figure 4 shows the phylogenetic tree of the orthodontic linkage (NJ) obtained by each individual goose using 14 new microsatellite markers. ●: White Roman Goose; : hybrid goose; △: white Chinese goose; : brown Chinese goose; :Black Swan.
圖5顯示各品種鵝隻之個體鑑別率(P(ID))對微衛星標幟數目之折線圖。 Figure 5 shows a line graph of the individual identification rate (P (ID) ) of each species of goose on the number of microsatellite markers.
圖6顯示各品種鵝隻之近親個體鑑別率(P(ID)sib)對微衛星標幟數目之折線圖。 Figure 6 shows a line graph of the number of close relatives (P (ID) sib ) of various species of geese to the number of microsatellite markers.
圖7顯示各鵝隻個體利用5組微衛星標幟(5A26648、5A26681、5A5279、5A5397、5A265175)所得鄰位連接法(NJ)之親緣關係樹。●:白羅曼鵝;:雜交鵝;△:白色華鵝;:褐色華鵝;:黑天鵝。 Figure 7 shows the phylogenetic tree of the ortho-joining method (NJ) obtained by each individual goose using 5 sets of microsatellite markers (5A26648, 5A26681, 5A5279, 5A5397, 5A265175). ●: White Roman Goose; : hybrid goose; △: white Chinese goose; : brown Chinese goose; :Black Swan.
圖8顯示各鵝隻個體利用7組微衛星標幟(5A26648、5A26681、5A5279、5A5397、5A265175、5A26254、5A5305-1)所得鄰位連接法(NJ)之親緣關係樹。●:白羅曼鵝;:雜交鵝;:白色華鵝;:褐色華鵝;:黑天鵝。 Figure 8 shows the phylogenetic tree of the ortho-joining method (NJ) obtained by each individual goose individual using 7 sets of microsatellite markers (5A26648, 5A26681, 5A5279, 5A5397, 5A265175, 5A26254, 5A5305-1). ●: White Roman Goose; : hybrid goose; : White Chinese Goose; : brown Chinese goose; :Black Swan.
圖9顯示各鵝隻個體利用8組新微衛星標幟(5A26648、5A26681、5A5279、 5A5397、5A265175、5A26254、5A5305-1、5A265141)所得鄰位連接法(NJ)之親緣關係樹。●:白羅曼鵝;:雜交鵝;△:白色華鵝;:褐色華鵝;:黑天鵝。 Figure 9 shows the phylogenetic tree of the ortho-joining method (NJ) obtained by each individual goose individual using 8 sets of new microsatellite markers (5A26648, 5A26681, 5A5279, 5A5397, 5A265175, 5A26254, 5A5305-1, 5A265141). ●: White Roman Goose; : hybrid goose; △: white Chinese goose; : brown Chinese goose; :Black Swan.
圖10顯示各鵝隻個體利用9組新微衛星標幟(5A26648、5A26681、5A5279、5A5397、5A265175、5A26254、5A5305-1、5A265141、5A265151)所得鄰位連接法(NJ)之親緣關係樹。●:白羅曼鵝;:雜交鵝;△:白色華鵝;:褐色華鵝;:黑天鵝。 Figure 10 shows the phylogenetic tree of the ortho-ligation method (NJ) obtained by each individual goose individual using 9 sets of new microsatellite markers (5A26648, 5A26681, 5A5279, 5A5397, 5A265175, 5A26254, 5A5305-1, 5A265141, 5A265151). ●: White Roman Goose; : hybrid goose; △: white Chinese goose; : brown Chinese goose; :Black Swan.
除非另有定義,所有本文所用之技術性及科學性術語,對於屬於本發明領域之具有通常知識者而言,皆具有與其所習知者相同意義。 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as the ones of ordinary skill in the art.
除非文中有清楚指明者,於本文中所使用之單數形式「一」、「一種」、及「該」之涵義均為包括「至少一種」的複數形式。因此,例如,當提及「一成分」時,包括複數個該等成分及對該領域具有通常知識者所知之同等物。 The singular forms "a", "an", "the" and "the" are used in the plural. Thus, for example, reference to "a component" includes a plurality of such elements and equivalents to those of ordinary skill in the art.
本文所使用的「鵝隻」為一人類馴化之家禽,其源於野生的鴻雁(Anser anser)或灰雁(Anser cygnoides)。鵝(Anser anser domesticus)包括但不限於白羅曼鵝(White Roman)、白色華鵝(White Chinese)、褐色華鵝(Brown Chinese)、愛姆登鵝(Embdem goose)、土魯斯鵝(Toulouse goose)、獅頭鵝(Shi Tou goose)、克里模鵝(Grimaud geese)及雜交鵝。 The "goose" used in this article is a human domesticated poultry, which originates from the wild Anser anser or Anser cygnoides . Anser anser domesticus includes, but is not limited to, White Roman, White Chinese, Brown Chinese, Embdem goose, Toulouse goose ), Shi Tou goose, Grimaud geese and hybrid geese.
台灣鵝隻之個體遺傳分析或族群遺傳結構分析的微衛星標幟尚未完全闡明,在本發明中,新穎微衛星標幟被用於分析鵝隻之基因型多態性,包含白羅曼鵝、白色華鵝、褐色華鵝、雜交鵝和黑天鵝之遺傳變 異性、遺傳距離與分群、個體鑑別率等遺傳特性。 The microsatellite markers for individual genetic analysis or ethnic genetic structure analysis of Taiwanese geese have not yet been fully elucidated. In the present invention, novel microsatellite markers are used to analyze genotype polymorphisms of goose, including white roman goose, white Genetic variation of Chinese geese, brown geese, hybrid geese and black swan Genetic characteristics such as heterosexuality, genetic distance, grouping, and individual discrimination rate.
在本發明一具體實施例中,該等微衛星標幟遺傳變異性分析結果之平均期望異質度、觀測異質度與多態性訊息含量,皆屬於高多態性範圍,顯示該等微衛星標幟於分析鵝隻族群具高度適用性。而在本發明另一具體實施例中,該等微衛星標幟遺傳距離分析結果明確將鵝品種進行分群,顯示該等微衛星標幟於鵝品種差異檢測具有良好分辨性。此外,本發明所提供之微衛星標幟亦具有高個體鑑別率。 In a specific embodiment of the present invention, the average expected heterogeneity, the observed heterogeneity, and the polymorphism information content of the genetic variation analysis results of the microsatellite markers all belong to a high polymorphism range, and the microsatellite markers are displayed. The analysis of the goose population is highly applicable. In another embodiment of the present invention, the genetic distance analysis results of the microsatellite markers clearly group the goose breeds, indicating that the microsatellite markers have good resolution in the difference detection of goose breeds. In addition, the microsatellite marker provided by the present invention also has a high individual discrimination rate.
因此,本發明提供一種用於鵝隻遺傳特性鑑定與個體鑑別的方法,該方法包括:(i)提供一待檢測鵝隻血液樣本,並萃取該血液樣本中之基因體DNA;(ii)以至少1組微衛星標幟引子對檢測該鵝隻之基因體DNA;其中該微衛星標幟引子對係選自由:(1)一分別具有SEQ ID NO:1及2之核苷酸序列的第一引子對;(2)一分別具有SEQ ID NO:3及4之核苷酸序列的第二引子對;(3)一分別具有SEQ ID NO:5及6之核苷酸序列的第三引子對;(4)一分別具有SEQ ID NO:7及8之核苷酸序列的第四引子對;(5)一分別具有SEQ ID NO:9及10之核苷酸序列的第五引子對;(6)一分別具有SEQ ID NO:11及12之核苷酸序列的第六引子對;(7)一分別具有SEQ ID NO:13及14之核苷酸序列的第七引子對;(8)一分別具有SEQ ID NO:15及16之核苷酸序列的第八引子對;(9)一分別具有SEQ ID NO:17及18之核苷酸序列的第九引子對;(10)一分別具有SEQ ID NO:19及20之核苷酸序列的第十引子對; (11)一分別具有SEQ ID NO:21及22之核苷酸序列的第十一引子對;(12)一分別具有SEQ ID NO:23及24之核苷酸序列的第十二引子對;(13)一分別具有SEQ ID NO:25及26之核苷酸序列的第十三引子對;(14)一分別具有SEQ ID NO:27及28之核苷酸序列的第十四引子對;及其組合所組成之群組;(iii)依步驟(ii)之檢測結果分析該鵝隻之基因型以評估其遺傳特性及個體鑑別。 Accordingly, the present invention provides a method for genetic characterization and individual identification of goose, the method comprising: (i) providing a blood sample of a goose to be detected, and extracting the genomic DNA in the blood sample; (ii) At least one set of microsatellite primer pairs detects the genomic DNA of the goose; wherein the microsatellite primer pair is selected from the group consisting of: (1) a nucleotide sequence having SEQ ID NOS: 1 and 2, respectively a pair of primers; (2) a second primer pair having the nucleotide sequences of SEQ ID NOS: 3 and 4, respectively; (3) a third primer having the nucleotide sequences of SEQ ID NOS: 5 and 6, respectively (4) a fourth primer pair having the nucleotide sequences of SEQ ID NOS: 7 and 8, respectively; (5) a fifth primer pair having the nucleotide sequences of SEQ ID NOS: 9 and 10, respectively; (6) a sixth primer pair having the nucleotide sequences of SEQ ID NOS: 11 and 12, respectively; (7) a seventh primer pair having the nucleotide sequences of SEQ ID NOS: 13 and 14, respectively; a ninth primer pair having the nucleotide sequences of SEQ ID NOS: 15 and 16, respectively; (9) a ninth primer pair having the nucleotide sequences of SEQ ID NOS: 17 and 18, respectively; (10) Minute Having SEQ ID NO: 20 and a tenth of the 19 nucleotide primer sequence pair; (11) an eleventh pair of primers having the nucleotide sequences of SEQ ID NOS: 21 and 22, respectively; (12) a twelfth pair of primers having the nucleotide sequences of SEQ ID NOS: 23 and 24, respectively; (13) a thirteenth primer pair having the nucleotide sequences of SEQ ID NOS: 25 and 26, respectively; (14) a fourteenth primer pair having the nucleotide sequences of SEQ ID NOS: 27 and 28, respectively; And a combination of the combinations; (iii) analyzing the genotype of the goose according to the test result of step (ii) to evaluate its genetic characteristics and individual identification.
在本發明部分具體實施例中,其係以至少8組微衛星標幟引子對檢測該鵝隻之基因體DNA。在一特定具體實施例中,該微衛星標幟引子對係第一引子對至第八引子對、第一引子對至第九引子對、或第一引子對至第十四引子對。 In some embodiments of the invention, the genomic DNA of the goose is detected by at least 8 sets of microsatellite primer pairs. In a particular embodiment, the microsatellite marker pair is a pair of first to eighth, a first to a ninth, or a first to a fourteen.
此外,本發明提供一種用於鵝隻遺傳特性鑑定與個體鑑別的套組,其包括1組以上之微衛星標幟引子對檢測該鵝隻之基因體DNA,以取得該待檢測鵝隻之基因型;其中該微衛星標幟引子對係選自由第一引子對至第十四引子對,及其組合所組成之群。在一特定實施例中,該微衛星標幟引子對係第一引子對至第十四引子對。 In addition, the present invention provides a kit for genetic characterization and individual identification of goose, comprising one or more sets of microsatellite markers introduced to detect the genomic DNA of the goose to obtain the gene of the goose to be detected. And wherein the microsatellite marker primer pair is selected from the group consisting of a first primer pair to a fourteenth primer pair, and a combination thereof. In a particular embodiment, the microsatellite marker primer pair is a first primer pair to a fourteenth primer pair.
另外,本發明提供之微衛星標幟引子對係包括一微衛星標幟正向引子、以及一微衛星標幟反向引子,其中該微衛星標幟正向引子或反向引子之5’端係連接有一螢光試劑。 In addition, the microsatellite index introduction pair provided by the present invention includes a microsatellite flag forward primer and a microsatellite flag reverse primer, wherein the microsatellite flag forward or backward reference 5' end A fluorescent reagent is attached to the system.
在另一方面,本發明提供一種用於鵝隻遺傳特性鑑定與個體鑑別的微衛星標幟引子對,其中該微衛星標幟引子對係第一引子對至第十四引子對。 In another aspect, the present invention provides a microsatellite marker primer pair for genetic identification and individual identification of a goose, wherein the microsatellite primer pair is a pair of first primer pairs to a fourteenth primer pair.
本發明以下列實施例進一步說明,其在任何方面不應被解釋為限制。所有引用文獻之內容(包括文獻參考文獻、已授權的專利和公開的專利如本申請通篇引用)在此明確通過引用併入。 The invention is further illustrated by the following examples, which are not to be construed as limiting in any respect. The contents of all cited documents (including literature references, issued patents and published patents, which are hereby incorporated by reference in their entireties in the entireties in
實施例1 微衛星標幟之篩選及其引子對設計 Example 1 Screening of Microsatellite Signs and Design of Its Primer Pairs
1.試驗動物 Test animal
挑選自彰化種畜繁殖場白羅曼品種,一公一母兩鵝隻個體,場內編號分別為5A5及5A26。採集其血液樣本,並用Genomic DNA Isolation Reagent(GenePure Tech.Co.,LTD.,Taiwan)依照套組說明自全血抽取基因組DNA(genomic DNA,gDNA)。所得之gDNA濃度則利用NanoDrop 2000c(Thermo Fisher Scientific Inc.,USA)檢測,確認OD260/280值均達到1.8至2.0後,保存於-20℃冰箱備用。 The white roman varieties of Changhua breeding farms were selected, and one male, one female and two geese were single, and the numbers were 5A5 and 5A26 respectively. Blood samples were collected and genomic DNA (gDNA) was extracted from whole blood using Genomic DNA Isolation Reagent (GenePure Tech. Co., LTD., Taiwan) according to the kit instructions. The obtained gDNA concentration was measured by NanoDrop 2000c (Thermo Fisher Scientific Inc., USA), and it was confirmed that the OD260/280 values were all 1.8 to 2.0, and stored in a refrigerator at -20 ° C for use.
2.限制性內切酶分解(restriction enzyme digestion) 2. Restriction enzyme digestion
取5A5及5A26兩鵝隻個體gDNA各10μL(100ng/μL),利用Rsa I(catalog #R0167S,NEB,USA)與Xmn I(catalog #R0194S,NEB,USA)限制性內切酶進行分解,反應總體積為25μL,包含1X NEB接合酶緩衝液(ligase buffer)(1 X BSA、50mM NaCl、Xmn I與Rsa I各1μL),於37℃水浴槽反應1小時後,以1%瓊脂醣凝膠電泳確認酶切產物是否具有介於300至1000bp之最適DNA片段長度帶狀區域。 Take 10 μL (100 ng/μL) of each gDNA of 5A5 and 5A26, and decompose with Rsa I (catalog #R0167S, NEB, USA) and Xmn I (catalog #R0194S, NEB, USA) restriction enzymes. The total volume was 25 μL, including 1X NEB ligase buffer (1 X BSA, 50 mM NaCl, Xmn I and Rsa I 1 μL each), and reacted in a water bath at 37 ° C for 1 hour, using a 1% agarose gel. It was confirmed by electrophoresis whether the digested product has a band length region of an optimum DNA fragment length of 300 to 1000 bp.
3. DNA片段與連接子(linkers)之接合 3. Engagement of DNA fragments with linkers
連接子之製備係取等體積Super SNX24正向引子(5’GTTTAAGGCCTAGCTAGCAGAATC3’)與Super SNX24+4P反向引子(5’pGATTCTGCTAGCTAGGCCTTAAACAAAA3’)加入NaCl溶液調整至 最終濃度為100mM,並將此混合物預先加熱至95℃後緩慢冷卻至室溫以形成雙股連接子(ds Super SNX linkers)。接著製備總體積為10μL的接合反應溶液,其包含7μM雙股連接子、1X接合緩衝液、800單位(units)T4 DNA接合酶(catalog #M0202S,NEB,USA)。 The preparation of the linker was carried out by adding an equal volume of Super SNX24 forward primer (5'GTTTAAGGCCTAGCTAGCAGAATC3') and Super SNX24+4P reverse primer (5'pGATTCTGCTAGCTAGGCCTTAAACAAAA3') to the NaCl solution. The final concentration was 100 mM and the mixture was preheated to 95 ° C and then slowly cooled to room temperature to form double-stranded linkers (ds Super SNX linkers). Next, a total volume of 10 μL of the ligation reaction solution containing 7 μM double stranded linker, 1X ligation buffer, and 800 units of T4 DNA ligase (catalog #M0202S, NEB, USA) was prepared.
將接合反應溶液與酶切產物混合,置於16℃反應12小時以上,並以單股Super SNX24正向引子進行PCR反應,以確認接合反應是否完全。PCR反應總體積為25μL,包括1X PCR緩衝液、25μg/mL BSA、0.5μM Super SNX24正向引子、2.0mM MgCl2、0.15mM dNTP、1U Taq DNA聚合酶(DNA polymerase)(TAKARA Co.,Japan)及接合反應產物。利用Veriti® 96-孔熱循環儀(96-well Thermal Cycler)(ABI PRISM,USA)進行PCR反應,反應溫度條件為95℃變性2分鐘;95℃變性20秒、60℃鏈合20秒、72℃延伸1.5分鐘,重覆循環20次,最後冷卻至15℃。取PCR產物以1%瓊脂醣膠體進行電泳,檢測PCR產物是否成功增幅。 The ligation reaction solution was mixed with the enzyme-cut product, and reacted at 16 ° C for 12 hours or more, and subjected to a PCR reaction using a single-strand Super SNX24 forward primer to confirm whether the ligation reaction was complete. The total volume of the PCR reaction was 25 μL, including 1X PCR buffer, 25 μg/mL BSA, 0.5 μM Super SNX24 forward primer, 2.0 mM MgCl 2 , 0.15 mM dNTP, 1 U Taq DNA polymerase (TAKARA Co., Japan). And joining reaction products. The PCR reaction was carried out using a Veriti® 96-well Thermal Cycler (ABI PRISM, USA) at a reaction temperature of 95 ° C for 2 minutes; at 95 ° C for 20 seconds, at 60 ° C for 20 seconds, 72 The °C extension was 1.5 minutes, the cycle was repeated 20 times, and finally cooled to 15 °C. The PCR product was electrophoresed on a 1% agarose gel to detect whether the PCR product was successfully increased.
4.磁珠豐富化微衛星DNA片段(dynabead enrichment for microsatellite DNA fragments) 4. Magnetic beads enriched microsatellite DNA fragments (dynabead enrichment for microsatellite DNA fragments)
豐富化係利用標定生物素之重複序列探針(biotinylated-oligos)與基因庫中DNA片段進行雜合(hybridization);再利用卵白素(streptavidin)與生物素間具有親和性之特性,以帶有卵白素的磁珠分離出與標定生物素之重複序列探針雜合之DNA片段,便可利用磁力分離出含重複序列的片段,建立富含二至四核苷酸重複序列DNA片段之基因庫(library)。本發明根據Glenn等人(Glenn,et al.,2005,Isolating microsatellite DNA loci.Meth.Enzymol.395:202-222)所建議之重複序列種 類設計探針,依據相似鏈合溫度調整成三種組合,內各含四種探針,分別為 The enrichment system utilizes biotinylated-oligos to hybridize with DNA fragments in the gene bank; and reuses the affinity between streptavidin and biotin to carry The magnetic beads of the avidin separate the DNA fragment which is hybridized with the repeating sequence probe of the labeled biotin, and the fragment containing the repeat sequence can be separated by magnetic force to establish a gene bank rich in the DNA fragment of the two to four nucleotide repeats. (library). The present invention is based on the repeat species suggested by Glenn et al. (Glenn, et al., 2005, Isolating microsatellite DNA loci. Meth. Enzymol. 395: 202-222) Class design probes, adjusted according to similar chain temperature into three combinations, each containing four probes, respectively
探針組合一:(TG)12、(ACT)12、(ACTG)6、(ACAG)6。 Probe combination one: (TG) 12 , (ACT) 12 , (ACTG) 6 , (ACAG) 6 .
探針組合二:(AG)12、(ACAT)8、(AACT)8、(AAGT)8。 Probe combination two: (AG) 12 , (ACAT) 8 , (AACT) 8 , (AAGT) 8 .
探針組合三:(AAG)8、(AAAC)6、(AATC)6、(AGAT)6。 Probe combination three: (AAG) 8 , (AAAC) 6 , (AATC) 6 , (AGAT) 6 .
(1)探針雜合 (1) Probe hybridization
取10μL含有接合子之gDNA片段,加入反應溶液使總體積變成50μL,反應溶液中包括25μL 2X雜合溶液(12 X SSC及0.2% SDS)及上述任一探針組合,每種探針濃度為1μM。接著使用Veriti® 96-well Thermal Cycler(ABI PRISM,USA)進行雜合反應,先加熱至95℃、5分鐘,隨後自70℃起每5秒調降0.2℃直至50℃,再於50℃停留反應10分鐘,接著每5秒調降0.5℃直至40℃,最後冷卻至15℃。 Take 10 μL of the gDNA fragment containing the zygote, add the reaction solution to make the total volume 50 μL, and include 25 μL of 2X hybrid solution (12 X SSC and 0.2% SDS) and any combination of the above probes, each probe concentration is 1 μM. The hybrid reaction was then carried out using a Veriti® 96-well Thermal Cycler (ABI PRISM, USA), first heated to 95 ° C for 5 minutes, then 0.2 ° C to 50 ° C every 5 seconds from 70 ° C, and then stopped at 50 ° C. The reaction was carried out for 10 minutes, then down to 0.5 ° C every 5 seconds until 40 ° C, and finally cooled to 15 ° C.
(2)清洗精製磁珠 (2) cleaning and polishing magnetic beads
在進行雜合反應的同時,準備清洗並精製磁珠(Dynabeads® M-280 Streptavidin,catalog #11205D,InvitrogenTM,CA,USA)。取50μL磁珠並加入250μL TE緩衝液(10mM Tris pH8,2mM EDTA),混合均勻後以磁鐵隔著微量離心管吸附磁珠並去除上清液,再以250μL TE緩衝液及250μL 1X雜合溶液(6X SSC,0.1% SDS)清洗精製磁珠,最後以150μL 1X雜合溶液重新懸浮磁珠。 In parallel hybrid reaction, washed and purified to prepare magnetic beads (Dynabeads® M-280 Streptavidin, catalog # 11205D, Invitrogen TM, CA, USA). Take 50 μL of magnetic beads and add 250 μL of TE buffer (10 mM Tris pH8, 2 mM EDTA), mix well, then adsorb the magnetic beads with a magnet through a microcentrifuge tube and remove the supernatant, then add 250 μL of TE buffer and 250 μL of 1X hybrid solution. (6X SSC, 0.1% SDS) The purified magnetic beads were washed, and finally the magnetic beads were resuspended in 150 μL of 1X hybrid solution.
(3)含重複序列之gDNA片段純化 (3) Purification of gDNA fragments containing repeat sequences
將全部雜合產物加入精製後之磁珠,以振盪器於室溫中慢速混勻45分鐘,使磁珠與含有探針之gDNA片段結合。接著,利用磁鐵吸附 磁珠,再去除上清液。以400μL清洗溶液2(2 X SSC及0.1% SDS)與400μL清洗溶液1(1 X SSC,0.1% SDS)清洗磁珠,以將未與探針進行雜合的gDNA片段及多餘的探針洗除。最後加入200μLTLE緩衝液(0.01M Tris-HCl及0.2mM EDTA),以95℃加熱5分鐘後,取出上清液,並於上清液中加入22μL NaOAc/EDTA溶液(1.5M NaOAc,0.25M EDTA),再加入444μL 95%乙醇後混勻並置於-20℃下15分鐘,再以16,000g離心10分鐘以去除上清液。接著以500μL之70% ethanol清洗產物,待雜合產物完全風乾後,以25μL去離子水回溶,再以前述接合反應之PCR條件確認增幅含有重複序列之gDNA片段以進行後續試驗。 All the hybrid products were added to the purified magnetic beads, and the mixture was slowly mixed at room temperature for 45 minutes with a shaker to bind the magnetic beads to the gDNA fragment containing the probe. Then, using magnet adsorption Magnetic beads, and then remove the supernatant. Wash the magnetic beads with 400 μL of Wash Solution 2 (2 X SSC and 0.1% SDS) and 400 μL of Wash Solution 1 (1 X SSC, 0.1% SDS) to wash the gDNA fragments and excess probes that are not hybridized with the probe. except. Finally, 200 μL of TLE buffer (0.01 M Tris-HCl and 0.2 mM EDTA) was added, and after heating at 95 ° C for 5 minutes, the supernatant was taken out, and 22 μL of NaOAc/EDTA solution (1.5 M NaOAc, 0.25 M EDTA) was added to the supernatant. After adding 444 μL of 95% ethanol, the mixture was mixed and placed at -20 ° C for 15 minutes, and then centrifuged at 16,000 g for 10 minutes to remove the supernatant. Next, the product was washed with 500 μL of 70% ethanol, and after the hybrid product was completely air-dried, it was re-dissolved in 25 μL of deionized water, and the amplified gDNA fragment containing the repeat sequence was confirmed by the PCR conditions of the above-mentioned ligation reaction for subsequent experiments.
4.豐富化DNA片段與質體之接合 4. Enrich the junction of DNA fragments and plastids
為選殖含重複序列之DNA片段,將經豐富化與增幅的DNA片段嵌入pGEM-T載體(pGEM®-T Easy Vector system,Promega,USA),DNA片段與載體係以3:1之比例進行接合反應,反應總體積為10μL,其包含50ng pGEM-T載體、3單位T4 DNA接合酶、1X快速接合緩衝液及經豐富化的增幅DNA片段,於4℃下反應12小時以上。 In order to select a DNA fragment containing the repeat sequence, the enriched and amplified DNA fragment was inserted into the pGEM-T vector (pGEM®-T Easy Vector system, Promega, USA), and the DNA fragment and the vector were carried out in a ratio of 3:1. The ligation reaction, the total volume of the reaction was 10 μL, and contained 50 ng of pGEM-T vector, 3 units of T4 DNA ligase, 1X rapid ligation buffer, and enriched amplified DNA fragment, and reacted at 4 ° C for 12 hours or more.
5.質體轉形(plasmid transformation)及陽性菌落選殖(positive colonies selection) 5. plasmid transformation and positive colonies selection
將10μL完成接合反應之重組載體加入100μL HIT勝任細胞TM(Competent CellsTM)-DH5α(Real Biotech Co.,Taiwan),於冰上反應1至10分鐘。將菌液均分成四等份,每等份皆混合8μL IPTG與40μL 2% X-Gal並將菌液塗佈於含胺苄青黴素(ampicillin)(100μg/mL)之LB(Luria-Broth)固體培養基,並於37℃下培養16至18小時後挑取白色菌落, 並以上述確認接合反應之PCR條件,確認DNA片段是否成功與載體接合。根據PCR之結果挑出只含單一片段之陽性菌落共230個,轉培養至含有胺苄青黴素(100μg/mL)之3mL LB培養液的離心管中,接著於37℃下搖晃培養16至18小時。 The reaction of the recombinant vector added 10μL 100μL HIT complete engagement competent cells TM (Competent Cells TM) -DH5α ( Real Biotech Co., Taiwan), the reaction on ice for 1-10 minutes. The bacterial solution was divided into four equal portions, and 8 μL of IPTG and 40 μL of 2% X-Gal were mixed for each aliquot and the bacterial solution was applied to an LB (Luria-Broth) solid containing ampicillin (100 μg/mL). The medium was cultured at 37 ° C for 16 to 18 hours, and white colonies were picked, and the PCR conditions of the ligation reaction were confirmed as described above to confirm whether or not the DNA fragment was successfully ligated to the carrier. According to the results of the PCR, a total of 230 positive colonies containing only a single fragment were picked and transferred to a centrifuge tube containing 3 mL of LB medium containing ampicillin (100 μg/mL), followed by shaking at 37 ° C for 16 to 18 hours. .
7.質體定序與引子設計(sequencing and primer design) 7. Sequencing and primer design
以鹼處理法(alkaline lysis)抽取質體DNA(Green and Sambrook,2012),並利用T7引子以ABI 3730XL DNA分析儀(ABI PRISM,USA)進行核酸定序。篩選序列片段中二重複序列重複數8次以上或四重複序列重複數6次以上者,做為可能存在多態性之最低標準。定序的結果中共有65個菌落,接著利用Primer 3 Plus程式(Rozen and Skaletsky,2000)針對其重複單位兩側之序列進行引子設計,並在正向(forward)引子之5’端加上CAG標記(5’CAGTCGGGCGTATCA3’),並利用已標定FAM(ABI PRISM,USA)螢光之CAG標記做為所有引子對的第二正向引子,以避免所有引子皆需標定螢光的情況。 The plastid DNA was extracted by alkaline lysis (Green and Sambrook, 2012), and nucleic acid sequencing was performed using an A7 3730XL DNA analyzer (ABI PRISM, USA) using a T7 primer. In the screening sequence fragment, the number of repeats of the two repeats is more than 8 or the number of repeats of the four repeats is 6 or more, as the minimum standard for possible polymorphism. A total of 65 colonies were found in the sequenced results, followed by primer design for the sequences on both sides of the repeat unit using the Primer 3 Plus program (Rozen and Skaletsky, 2000), and CAG at the 5' end of the forward primer. Mark (5'CAGTCGGGCGTATCA3') and use the CAG marker that has been calibrated by FAM (ABI PRISM, USA) as the second forward primer for all primer pairs to avoid the need to calibrate all primers.
實施例2 微衛星標幟之PCR增幅與各品種族群多態性檢測 Example 2 PCR amplification of microsatellite markers and polymorphism detection of various ethnic groups
1.試驗動物 Test animal
挑選16隻白羅曼鵝進行初步PCR檢測以挑選具多態性之微衛星標幟;隨後以彰化種畜繁殖場之白羅曼鵝、白色華鵝、褐色華鵝、雜交鵝及做為外族群(outgroup)之黑天鵝,總共254隻鵝、5種族群進行基因型分析,其中99隻白羅曼鵝、40隻白色華鵝、63隻褐色華鵝、36隻雜交鵝及16隻黑天鵝(表1)。進行試驗之254隻鵝隻血液樣本,皆依前述萃取及純化gDNA之方法,進行gDNA萃取。 Sixteen white roman geese were selected for preliminary PCR detection to select polymorphic microsatellite markers. Subsequently, white roman geese, white geese, brown geese, hybrid geese and foreign groups (outgroup) were used in Changhua breeding farms. The black swan, a total of 254 geese, 5 ethnic groups for genotypic analysis, including 99 white Romanes, 40 white geese, 63 brown Chinese geese, 36 hybrid geese and 16 black swan (Table 1) . The blood samples of 254 goose tested were subjected to gDNA extraction according to the method of extracting and purifying gDNA.
2.微衛星標幟之增幅與各品種族群多態性檢測 2. The increase of microsatellite markers and the polymorphism detection of various ethnic groups
利用PCR檢測微衛星標幟引子對是否可成功增幅微衛星標幟,其反應總體積為20μL,其中包含30ng gDNA、0.04μM正向引子、0.2μM反向(reverse)引子、0.16μM CAG標記、1X PCR緩衝液、0.2mM dNTP及0.025U Taq DNA聚合酶。反應條件為95℃變性4分鐘;95℃變性50秒、60℃鏈合50秒、72℃延伸1分鐘,循環35次;最後再以72℃延伸7分鐘。反應完成後以1%瓊脂醣膠體進行電泳,確認是否成功增幅。 PCR can be used to detect whether the microsatellite marker primer can successfully increase the microsatellite marker, and the total reaction volume is 20 μL, including 30 ng gDNA, 0.04 μM forward primer, 0.2 μM reverse primer, 0.16 μM CAG marker, 1X PCR buffer, 0.2 mM dNTP and 0.025 U Taq DNA polymerase. The reaction conditions were denaturation at 95 ° C for 4 minutes; denaturation at 95 ° C for 50 seconds, 60 ° C for 50 seconds, 72 ° C for 1 minute, 35 cycles; and finally 72 ° C for 7 minutes. After the completion of the reaction, electrophoresis was carried out with a 1% agarose colloid to confirm whether or not the growth was successful.
樣品盤的配製係使用高密度甲氨(Hi-Di formamide)及600 Liz分子量標準品(GeneScan Size Standard GeneScan-600 LIZ)(Applied Biosystems,Foster City,CA,USA)以120:1比例混合後,取10μL加入96孔樣品盤,再加入稀釋之PCR產物1μL,以ABI 3730 DNA分析儀(ABI PRISM,USA)進行毛細管電泳分析,測定其增幅DNA的片段大小,所得片 段大小之結果再以Peak scanner 1.0(ABI PRISM,USA)軟體進行基因型分析。 The sample tray was prepared by mixing Hi-Di formamide and 600 Liz molecular weight standard (GeneScan Size Standard GeneScan-600 LIZ) (Applied Biosystems, Foster City, CA, USA) in a ratio of 120:1. 10 μL was added to a 96-well sample plate, and 1 μL of the diluted PCR product was added, and analyzed by capillary electrophoresis on an ABI 3730 DNA analyzer (ABI PRISM, USA) to determine the fragment size of the amplified DNA. The results of the segment size were then genotyped using the Peak scanner 1.0 (ABI PRISM, USA) software.
經多態性測試後,選出交替基因數大於等於2之14組微衛星標幟引子對進行鵝隻個體之基因型檢測。區分不同微衛星標幟產物片段大小,再利用不同螢光進行標記上述多態性測試所得之14組微衛星標幟引子對,其基因座(locus)、基序(motif)、及引子對序列如表2所示。 After the polymorphism test, 14 groups of microsatellite markers with the number of alternating genes greater than or equal to 2 were selected to carry out genotype detection of goose individuals. Differentiating the size of different microsatellite product fragments, and using different fluorescence to label the 14 sets of microsatellite primer pairs obtained by the above polymorphism test, the locus, motif, and primer pair sequence As shown in table 2.
該等微衛星標幟引子對包含14對微衛星標幟引子對,其中包含:(1)一分別具有SEQ ID NO:1及2之核苷酸序列的第一引子對;(2)一分別具有SEQ ID NO:3及4之核苷酸序列的第二引子對;(3)一分別具有SEQ ID NO:5及6之核苷酸序列的第三引子對;(4)一分別具有SEQ ID NO:7及8之核苷酸序列的第四引子對;(5)一分別具有SEQ ID NO:9及10之核苷酸序列的第五引子對;(6)一分別具有SEQ ID NO:11及12之核苷酸序列的第六引子對;(7)一分別具有SEQ ID NO:13及14之核苷酸序列的第七引子對;(8)一分別具有SEQ ID NO:15及16之核苷酸序列的第八引子對。(9)一分別具有SEQ ID NO:17及18之核苷酸序列的第九引子對;(10)一分別具有SEQ ID NO:19及20之核苷酸序列的第十引子對;(11)一分別具有SEQ ID NO:21及22之核苷酸序列的第十一引子對; (12)一分別具有SEQ ID NO:23及24之核苷酸序列的第十二引子對;(13)一分別具有SEQ ID NO:25及26之核苷酸序列的第十三引子對;(14)一分別具有SEQ ID NO:27及28之核苷酸序列的第十四引子對。 The microsatellite marker primer pair comprises 14 pairs of microsatellite marker primer pairs, comprising: (1) a first primer pair having the nucleotide sequences of SEQ ID NO: 1 and 2, respectively; (2) a difference a second primer pair having the nucleotide sequences of SEQ ID NOS: 3 and 4; (3) a third primer pair having the nucleotide sequences of SEQ ID NOS: 5 and 6, respectively; (4) one having SEQ, respectively ID NO: a fourth primer pair of the nucleotide sequences of 7 and 8; (5) a fifth primer pair having the nucleotide sequences of SEQ ID NOS: 9 and 10, respectively; (6) one having SEQ ID NO, respectively a sixth primer pair of nucleotide sequences of 11 and 12; (7) a seventh primer pair having the nucleotide sequences of SEQ ID NOS: 13 and 14, respectively; (8) one having SEQ ID NO: 15 respectively And an eighth primer pair of the nucleotide sequence of 16. (9) a ninth primer pair having the nucleotide sequences of SEQ ID NOS: 17 and 18, respectively; (10) a tenth primer pair having the nucleotide sequences of SEQ ID NOS: 19 and 20, respectively; An eleventh pair of primers having the nucleotide sequences of SEQ ID NOS: 21 and 22, respectively; (12) a twelfth primer pair having the nucleotide sequences of SEQ ID NOS: 23 and 24, respectively; (13) a thirteenth primer pair having the nucleotide sequences of SEQ ID NOS: 25 and 26, respectively; (14) A fourteenth primer pair having the nucleotide sequences of SEQ ID NOS: 27 and 28, respectively.
實施例3 遺傳特性分析 Example 3 Analysis of genetic characteristics
1.微衛星標幟適用性評估 1. Microsatellite flag applicability assessment
微衛星標幟適用性評估係將試驗所得結果利用微軟工具套件(Microsoft Toolkit)(Park,2001,Trypanotolerance in West African cattle and the population genetic effects of selection,博士論文)計算交替基因數(Na)、交替基因頻率、觀測異質度(HO)、期望異質度(HE)、及多態性訊息含量(PIC),其說明如下: The microsatellite suitability evaluation system uses the Microsoft Toolkit (Park, 2001, Trypanotolerance in West African cattle and the population genetic effects of selection) to calculate the number of alternating genes (N a ), Alternating gene frequency, observed heterogeneity (H O ), expected heterogeneity (H E ), and polymorphism message content (PIC) are described as follows:
1.交替基因數(number of alleles,Na):每個基因座的平均交替基因數,即族群內所有基因座上交替基因數目總合和基因座總數的比值。 1. number of alleles (N a ): the average number of alternate genes per locus, ie the ratio of the number of alternating genes at all loci in the population to the total number of loci.
其中n i :基因座i上所有交替基因的個數。 Where n i : the number of all altering genes on locus i .
m:所有基因座的個數。 m : number of all loci.
2.觀測異質度(observed heterozygosity,HO):每個基因座的觀測異質度,代表雜合子個體在族群中的實際比例。 2. Observed heterozygosity (H O ): The observed heterogeneity of each locus, representing the actual proportion of heterozygous individuals in the population.
其中p ij :基因座上基因型A i A j 之頻率。 Where p ij : the frequency of the genotype A i A j at the locus.
n:基因座上所有交替基因的個數。 n : number of all altering genes at the locus.
A i 、A j 分別為基因座上第i個與j個交替基因,且i不等於j。 A i and A j are the i- th and j -alternating genes at the locus, respectively, and i is not equal to j .
3.期望異質度(expected heterozygosity,HE):每個基因座的期望異質度,依據哈溫定律計算族群中異質度的期望值。 3. Expected heterozygosity (H E ): The expected heterogeneity of each locus, and the expected value of heterogeneity in the population is calculated according to Harmon's law.
其中p i :基因座上交替基因A i 之頻率。 Where p i : the frequency of the alternating gene A i at the locus.
n:基因座上所有交替基因的個數。 n : number of all altering genes at the locus.
A i 為基因座上第i個交替基因。 A i is the i- th alternating gene at the locus.
而當族群個體數目小於50時,期望異質度方程式須做修正為:
其中N:族群整體的個體數。 Where N : the number of individuals in the group as a whole.
4.多態性訊息含量(polymorphic information content,PIC):為每個基因座的多態性程度。 4. Polymorphic information content (PIC): The degree of polymorphism at each locus.
其中p i :基因座上交替基因A i 之頻率。 Where p i : the frequency of the alternating gene A i at the locus.
p j :基因座上交替基因A j 之頻率。 p j : the frequency of the alternating gene A j at the locus.
n:基因座上所有交替基因的個數。 n : number of all altering genes at the locus.
A i 、A j 分別為基因座上第i個與j個交替基因,且i不等於j。 A i and A j are the i- th and j -alternating genes at the locus, respectively, and i is not equal to j .
另以GENEPOP 4.0(Rousset,2007;GENEPOP’007:a complete re-implementation of the GENEPOP software for Windows and Linux,Mol.Ecol.Res,8:103-106)分析哈溫平衡檢定(HWE test)及賴特固定指數(Wright’s F-statistics),其說明如下: Another analysis of the HWE test and the GENEPOP 4.0 (Rousset, 2007; GENEPOP'007: a complete re-implementation of the GENEPOP software for Windows and Linux, Mol. Ecol. Res, 8: 103-106) Wright's F-statistics, which is described as follows:
1.哈溫平衡檢定(Hardy-Weinberg equilibrium test,HWE test):以馬可夫鏈 蒙地卡羅法(Markov Chain Monte Carlo,MCMC)進行費雪精確檢定(Fisher’s exact test)(Guo及Thompson,1992,Performing the exact test of Hardy-Weinberg proportion for multiple alleles.Biometrics 48:361-372)。 1. Hardy-Weinberg equilibrium test (HWE test): with Markov chain The Fisher's exact test was performed by Markov Chain Monte Carlo (MCMC) (Guo and Thompson, 1992, Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 48: 361-372) .
2.賴特固定指數(Wright’s F-statistics):係將固定指數延伸應用於探討整體族群內含有次族群(subpopulation)的結構。以單一基因座含雙交替基因A p 及A q 的情形舉例,必須先建立三種異質度,包括HI、HS及HT,其中:HI:為所有次族群之觀測異質度的平均,也代表任一個體為雜合子的機率。 2. Wright's F-statistics: A fixed exponential extension is applied to explore the structure of subpopulations in the overall population. For example, in the case of a single locus containing the double alternation genes A p and A q , three heterogeneities must be established, including H I , H S and H T , where: H I : is the average of the observed heterogeneity of all subgroups, Also represents the probability that any individual is heterozygous.
其中:次族群i內的觀測異質度。 among them : The observed heterogeneity within the subgroup i .
n:所有次族群的個數。 n : The number of all sub-groups.
HS:指在次族群均為哈溫平衡的假設下,所有次族群期望異質度的平均。 H S : refers to the average of the expected heterogeneity of all sub-groups under the assumption that the sub-groups are all Harvard equilibrium.
其中p i :次族群i內交替基因A p 之頻率。 Where p i : the frequency of the alternating gene A p in the subgroup i .
q i :次族群i內交替基因A q 之頻率。 q i : the frequency of the alternating gene A q in the subgroup i .
n:所有次族群的個數。 n : The number of all sub-groups.
HT:等同於結合所有次族群的交替基因A p 及A q 形成一個大族群後,再計算基因頻率p及q而得到的整體族群期望異質度。 H T: equivalent to the binding alternately Gene A p A q and all sub-groups to form a large group, then p and q allele frequency obtained the desired degree of heterogeneity in the overall group.
其中:所有次族群交替基因A p 頻率的平均。 among them : All sub-groups alternating frequency average gene A p.
:所有次族群交替基因A q 頻率的平均。 : Average of the frequency of all sub-group alternation genes A q .
當考慮非逢機交配與次族群之間不同的交替基因頻率時,則需要以上述三種異質度(HI、HS及HT)對固定指數進行相對應的調整,其 調整如下: When considering the different alternate gene frequencies between non-family mate and sub-group, the corresponding adjustments should be made to the fixed index by the above three heterogeneities (H I , H S and H T ), which are adjusted as follows:
1. F IS:比較所有次族群內個體觀測異質度的平均,和預期所有次族群符合哈溫平衡之期望異質度的平均;也就是以次族群的平均值取代原始固定指數公式中的各項參數。 1. F IS : Compare the average of the individual observed heterogeneity in all sub-groups and the expected heterogeneity of all sub-groups in accordance with the Harvard equilibrium; that is, replace the items in the original fixed-index formula with the average of the sub-group parameter.
F IS=(HS-HI)/HS F IS =(H S -H I )/H S
I及S分別代表個體(individuals)和次族群(subpopulations)。F IS為正值表示雜合子個體在各次族群的比例少於逢機交配環境的預期比例。 I and S represent individuals and subpopulations, respectively. F IS heterozygous for the positive value indicates the proportion of individuals in each sub-population of fewer than expected proportion of every machine mating environment.
2. F ST:比較預期所有次族群符合哈溫平衡之期望異質度的平均,和整體族群的期望異質度;視為全部次族群分化程度的估計值。 2. F ST : Compares the average expected heterogeneity of all sub-groups in accordance with the Harvard equilibrium, and the expected heterogeneity of the overall population; an estimate of the degree of differentiation of all sub-groups.
F ST=(HT-HS)/HT F ST =(H T -H S )/H T
S及T分別代表著次族群(subpopulations)和整體族群(total population)。F ST為正值表示因為次族群之間交替基因頻率差異所造成的異質度下降,雜合子個體缺少的情況。 S and T represent subpopulations and total population, respectively. A positive value of F ST indicates a decrease in heterogeneity due to a difference in frequency of alternate genes between subgroups, and a heterozygous individual is absent.
3. F IT:比較所有次族群內觀測異質度的平均和整體族群的期望異質度,綜合評估次族群內的非逢機交配與次族群間不同基因頻率,兩者對哈溫平衡的期望基因頻率造成的偏差。 3. F IT : Compare the average heterogeneity of observations in all sub-groups with the expected heterogeneity of the overall population, and comprehensively evaluate the different gene frequencies between the non-family mate and the sub-group within the sub-group, and the expected genes for the Harvard equilibrium The deviation caused by the frequency.
F IT=HT-HI/HT F IT =H T -H I /H T
F IT為正值表示整體族群的純合子個體比例高於一個理想的逢機交配,且具有相同交替基因頻率的族群。 A positive value of F IT indicates that the proportion of homozygous individuals in the overall population is higher than that of an ideal random mating, and has the same alternate gene frequency.
此外,使用POPGENE 1.32(Yeh等人,1999,POPGENE,version 1.32:the user friendly software for population genetic analysis)分析有效交替基因數(Ne)及連鎖不平衡(linkage disequilibrium,LD)檢定。其中,Ne為每個基因座上交替基因分布平均程度,意指在所有交替基因之頻率相同的情況下,能夠提供相同異質度的交替基因數目。 In addition, effective alternating gene number (N e ) and linkage disequilibrium (LD) assays were analyzed using POPGENE 1.32 (Yeh et al., 1999, POPGENE, version 1.32: the user friendly software for population genetic analysis). Among them, N e is the average degree of alternation gene distribution at each locus, which means the number of alternation genes that can provide the same heterogeneity when the frequencies of all the alternate genes are the same.
其中p i :基因座上交替基因A i 之頻率。 Where p i : the frequency of the alternating gene A i at the locus.
n:基因座上所有交替基因的個數。 n : number of all altering genes at the locus.
2.遺傳距離之計算與親緣關係樹之繪製 2. Calculation of genetic distance and mapping of kinship trees
使用Microsatellite Analyser(MSA)(Dieringer及Schlötterer,2003,MICROSATELLITE ANALYSER(MSA):a platform independent analysis tool for large microsatellite data sets.Mol.Ecol.Notes 3:167-169)計算個體間遺傳距離(Saitou及Nei,1987,The Neighbor-joining method:a method for reconstructing phylogenetic trees.Mol.Biol.Evol.4:406-425.),並且利用PHYLIP套裝軟體(Felsenstein,2002,Phylogeny Inference Package(PHYLIP))以鄰位連接法(Neighbor Joining,NJ)和未加權算術平均對群法(Unweighted Pair Group Method with Arithmetic Mean,UPGMA)繪製親緣關係樹。另以STRUCTURE 2.3.1軟體進行模擬,利用馬可夫鏈蒙地卡羅法(Markov Chain Monte Carlo,MCMC)分析分群數機率,依計算5,000次後的50,000次模擬可能分群(K=2~7),每個K值重複20次再繪製群集分析圖;並計算△K值(Evanno等人,2005,Detecting the number of clusters of individuals using the software STRUCTURE:a simulation study.Mol.Ecol.14:2611-2620)與L(K)值(Rosenberg等人,2001,Empirical evaluation of genetic clustering methods using multilocus genotypes from 20 chicken breeds.Genetics 159:699-713)。另以GeneAlEx(Peakall及Smouse,2012,GeneAlEx ver6.5:genetic analysis in Excel.Population genetic software for teaching and research-an update.Bioinformatics 28:2537-2539)所繪製的主座標分析(Principal Coordinate Analysis,PCoA)3D圖確認試驗所檢測彰化 種畜繁殖場五個鵝隻族群之分群情形與族群親緣關係。 Calculation of genetic distance between individuals using Microsatellite Analyser (MSA) (Dieringer and Schlötterer, 2003, MICROSELLITE ANALYSER (MSA): a platform independent analysis tool for large microsatellite data sets. Mol. Ecol. Notes 3: 167-169) (Saitou and Nei , 1987, The Neighbor-joining method: a method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.), and using the PHYLIP suite software (Felsenstein, 2002, Phylogeny Inference Package (PHYLIP)) in the ortho position Neighbor Joining (NJ) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) draw a phylogenetic tree. In addition, the STRUCTURE 2.3.1 software is used for simulation, and Markov Chain Monte Carlo (MCMC) is used to analyze the probability of grouping. According to the calculation of 50,000 simulations after 5,000 times, it is possible to group (K=2~7). Each K value is repeated 20 times and then the cluster analysis map is drawn; and the ΔK value is calculated (Evanno et al., 2005, Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14:2611-2620 And L(K) values (Rosenberg et al., 2001, Empirical evaluation of genetic clustering methods using multilocus genotypes from 20 chicken breeds. Genetics 159: 699-713). Principal Coordinate Analysis (PCoA), also drawn by GeneAlEx (Peakall and Smoothe, 2012, Gene AlEx ver 6.5: genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics 28: 2537-2539) The 3D map confirmation experiment detected the grouping situation of the five goose populations in the Changhua breeding farm and the ethnic relationship.
3.個體鑑別率與親子排除率 3. Individual identification rate and parental exclusion rate
利用族群遺傳多態性之分析結果,計算個體鑑別率(P(ID))(Paetkau及Strobeck,1994,Microsatellite analysis of genetic variation in black bear population.Mol.Ecol.3:489-495)與近親個體鑑別率(P(ID)sib)(Evett及Weir,1998,Interpreting DNA evidence:statistical genetics for forensic scientists.1st ed),並依據結合不同數目之微衛星標幟基因座所產生之綜合個體鑑別率繪製折線圖,其說明如下: Using the results of analysis of ethnic genetic polymorphisms, the individual discrimination rate (P (ID) ) was calculated (Paetkau and Strobeck, 1994, Microsatellite analysis of genetic variation in black bear population. Mol. Ecol. 3: 489-495) and close relatives. Identification rate (P (ID) sib ) (Evett and Weir, 1998, Interpreting DNA evidence: statistical genetics for forensic scientists. 1st ed), and based on the combined individual identification rate generated by combining different numbers of microsatellite loci A line chart, which is described as follows:
1.個體鑑別率(probability of identity,P(ID)):為隨機從族群中選擇兩個個體,兩者在單一基因座的基因型完全相同的機率。 1. probability of identity (P (ID) ): is the probability that two individuals are randomly selected from a population, and the genotypes of the two are identical at a single locus.
其中p i :基因座上交替基因A i 之頻率。 Where p i : the frequency of the alternating gene A i at the locus.
p j :基因座上交替基因A j 之頻率。 p j : the frequency of the alternating gene A j at the locus.
n:基因座上所有交替基因的個數。 n : number of all altering genes at the locus.
2.近親個體鑑別率(probability of identity among sibs,P(ID)sib):鑑別的個體之間具有密切的親屬關係時使用之。 2. Probability of identity among sibs, P (ID) sib : used when the identified individuals have close kinship.
其中p j :基因座上交替基因A j 之頻率。 Where p j : the frequency of the alternating gene A j at the locus.
n:基因座上所有交替基因的個數。 n : number of all altering genes at the locus.
親子排除率(power of exclusion,PE)之計算亦是依據Jamieson(Jamieson,1965,The genetics of transferrin in cattle.Heredity 20:419-441)所提及之公式加以計算,其中,親子排除率係指三人組親子鑑別 中母子與非父親之基因型與機率。 The calculation of the power of exclusion (PE) is also calculated according to the formula mentioned by Jamieson (Jamieson, 1965, The genetics of transferrin in cattle. Heredity 20: 419-441), wherein the parent-child exclusion rate refers to Three-person parent-child identification The genotype and probability of mother and child and non-father.
其中p i :基因座上交替基因A i 之頻率。 Where p i : the frequency of the alternating gene A i at the locus.
p j :基因座上交替基因A j 之頻率。 p j : the frequency of the alternating gene A j at the locus.
n:基因座上所有交替基因的個數。 n : number of all altering genes at the locus.
結果result
1.微衛星標幟與遺傳變異性1. Microsatellite markers and genetic variability
本發明利用14組新穎微衛星標幟引子對針對彰化種畜繁殖場五個鵝族群-白羅曼鵝、白色華鵝、褐色華鵝、雜交鵝和黑天鵝,共254隻鵝隻樣本進行基因型分析。除了黑天鵝外,以所得基因型分析數據進行全體鵝隻族群之遺傳檢測結果列於表3。如表3所示,每個基因座的交替基因數(Na)範圍在4(5A265164)至38(5A26681)之間,而Na平均值為12。有效交替基因數(Ne)之範圍介在1.746(5A266113)與9.155(5A26648)之間,且Ne平均值為3.625。此14組微衛星標幟引子對之期望異質度(HE)範圍在0.384(5A266113)至0.895(5A26648)之間,其平均值為0.613。另一方面,觀測異質度(HO)範圍在0.336(5A265164)至0.908(5A26648)之間,其平均值為0.609。多態性訊息含量(PIC)範圍在0.351(5A266113)至0.876(5A26648)之間,而其平均值為0.613(表3)。族群遺傳結構分析方面,F IS之範圍在-0.116(5A265151)至0.32(5A265164)之間,平均值為0.015,由此平均值為正值可知整體鵝隻族群中之次族群(subpopulation),亦即品種內雜合子比例少於預期。哈溫平衡檢定(HWE test)中,共有9個基因座顯著地偏離哈溫平衡(P<0.01),分別為基因座5A5111、5A5397、5A26216、5A26261、5A265151、5A265164、 5A265175、5A26648及5A26681。 The invention utilizes 14 sets of novel microsatellite markers to carry out genotypic analysis on a total of 254 goose samples for five goose groups - white roman goose, white goose, brown goose, hybrid goose and black swan in Changhua breeding farm. . In addition to the black swan, the genetic test results of the entire goose population using the obtained genotypic analysis data are shown in Table 3. As Table 3 shows the number of genes alternately each locus (N a) in the range between 4 (5A265164) to 38 (5A26681), and the average value of 12 N a. The effective number of alternate genes (N e ) is between 1.746 (5A266113) and 9.155 (5A26648), and the average value of N e is 3.625. The expected heterogeneity (H E ) of the 14 sets of microsatellite markers is between 0.384 (5A266113) and 0.895 (5A26648) with an average of 0.613. On the other hand, the observed heterogeneity (H O ) ranged from 0.336 (5A265164) to 0.908 (5A26648) with an average of 0.609. The polymorphism message content (PIC) ranged from 0.351 (5A266113) to 0.876 (5A26648) with an average of 0.613 (Table 3). In terms of genetic structure analysis, F IS ranged from -0.116 (5A265151) to 0.32 (5A265164) with an average of 0.015. The average value is positive, and the subpopulation in the whole goose population is also known. That is, the proportion of heterozygotes within the variety is less than expected. In the HWE test, a total of 9 loci significantly deviated from the Hawar equilibrium ( P <0.01), loci 5A5111, 5A5397, 5A26216, 5A26261, 5A265151, 5A265164, 5A265175, 5A26648, and 5A26681, respectively.
2.各品系鵝隻族群內遺傳變異性之評估結果2. Evaluation results of genetic variability within each goose population
試驗中白羅曼鵝、白色華鵝、褐色華鵝及雜交鵝四個鵝族 群,利用14組新微衛星標幟檢測之遺傳變異性分別列於表4至表7。四種品系鵝隻之各項遺傳變異性之平均值皆以雜交鵝之數值最高,白色華鵝與褐色華鵝之數值較低,白羅曼鵝之數值則介於兩者之間。各品種的Na平均值範圍在5.714至7.286之間,Ne平均值範圍在3.035到4.633之間(表8)。HE與HO之最高平均值皆出現在雜交鵝,分別為0.735與0.829,顯示具有較高遺傳變異性。HE與HO之最低平均值則是在褐色華鵝與白色華鵝,分別為0.543與0.518,因此遺傳變異性程度較低。白羅曼鵝之HE與HO平均值則介於兩者之間,分別為0.622與0.591。PIC之平均值方面,白羅曼鵝之平均值為0.569;白色華鵝之平均值為0.491;褐色華鵝之平均值為0.5;雜交鵝之平均值則為0.683。在F IS平均值方面,白羅曼鵝、白色華鵝、褐色華鵝與雜交鵝之數值,分別為0.045、0.065、0.052與-0.142。白羅曼鵝與雜交鵝偏離哈溫平衡的基因座分別有六個與八個,而白色華鵝與褐色華鵝偏離哈溫平衡的基因座分別有二個與一個。 In the experiment, the four geese of white roman geese, white geese, brown geese and hybrid geese were tested in 14 groups of new microsatellite markers. The genetic variability is shown in Tables 4 to 7. The average value of the genetic variability of the four species of geese was the highest among the hybrid geese. The values of white geese and brown geese were lower, and the value of white roman geese was between the two. The average value of N a for each variety ranged from 5.714 to 7.286, and the average value of N e ranged from 3.035 to 4.633 (Table 8). The highest average values of H E and H O were found in hybrid geese, 0.735 and 0.829, respectively, indicating high genetic variability. The lowest average values of H E and H O are in brown geese and white geese, 0.543 and 0.518, respectively, so the degree of genetic variability is low. The average value of H E and H O of white roman goose is between the two, 0.622 and 0.591 respectively. In terms of the average value of PIC, the average value of white roman goose is 0.569; the average value of white Chinese goose is 0.491; the average value of brown Chinese goose is 0.5; the average value of hybrid goose is 0.683. In terms of mean value F IS, Roman white geese, Chinese white geese, brown Chinese goose value hybridization goose, 0.045,0.065,0.052 and -0.142 respectively. There are six or eight loci in which the white roman goose and the hybrid goose deviate from the Harvard equilibrium, respectively, while the white Chinese goose and the brown Chinese goose have two or one loci that deviate from the Harvard equilibrium.
3.遺傳距離與分群3. Genetic distance and grouping
以微衛星標幟做族群遺傳分析主要目的為品種分析與個體鑑別,品種分析希望對不同品種的鵝在分子層次做出區分,避免雜交鵝做為種鵝,使種鵝品質不一致。本發明以核心種鵝場彰化種畜繁殖場之白羅曼鵝、白色華鵝、褐色華鵝與雜交鵝做為基準,由14組微衛星標幟檢測所有鵝隻各基因座之交替基因頻率結果,進一步計算族群之間的遺傳距離與族群間之F ST(表9)。由表9所示,除了黑天鵝外,白羅曼族群與褐色華鵝族群有最大的遺傳距離(0.703),而白色華鵝族群與褐色華鵝族群有最小的 遺傳距離(0.184),與族群分化指標F ST值結果相似,分別對應到最大值(0.362)與最小值(0.089)。進一步以遺傳距離利用鄰位連接法(NJ)繪製鵝隻族群親緣關係樹中(圖1),遺傳距離最近的白色華鵝與褐色華鵝形成一個群組,雜交鵝則在華鵝與白羅曼鵝之間,與預期相符。 The main purpose of genetic analysis of microsatellite markers is cultivar analysis and individual identification. Variety analysis hopes to distinguish the different species of geese at the molecular level, avoiding hybrid geese as breeding geese, and making the quality of the geese inconsistent. The invention uses the white roman goose, the white Chinese goose, the brown Chinese goose and the hybrid goose as the reference in the core species of the goose farm Changhua breeding farm, and the 14 sets of microsatellite markers detect the alternating gene frequency results of all the loci of each goose. Further calculate the genetic distance between the ethnic groups and the F ST between the ethnic groups (Table 9). As shown in Table 9, in addition to the black swan, the white Romance group and the brown Chinese goose group have the largest genetic distance (0.703), while the white Chinese goose group and the brown Chinese goose group have the smallest genetic distance (0.184), and ethnic differentiation. The results of the index F ST values are similar, corresponding to the maximum (0.362) and the minimum (0.089), respectively. Furthermore, the genetic distance was used to map the geese tree in the goose population (Fig. 1). The closest white geese and brown geese formed a group, and the hybrid geese were in Chinese goose and white roman. Between the geese, as expected.
利用主座標分析(PCoA)法繪製3D立體圖(圖2),其結果顯示第一座標軸(PC1)、第二座標軸(PC2)及第三座標軸(PC3)此三者可分別解釋61.06、32.62及4.66%的遺傳變異。藉由此三個主座標軸之變異量區分族群之間的遺傳距離,同樣可發現黑天鵝於第一主成分(PC1)時就可明顯與其他鵝群分開,而白色華鵝與褐色華鵝要到第三成分(PC3)時才可分開,雜交鵝也在華鵝與白羅曼鵝之間。 Using the main coordinate analysis (PCoA) method to draw a 3D perspective (Fig. 2), the results show that the first coordinate axis (PC1), the second coordinate axis (PC2) and the third coordinate axis (PC3) can explain 61.06, 32.62 and 4.66 respectively. % of genetic variation. By the variation of the three main coordinate axes to distinguish the genetic distance between the ethnic groups, it can also be found that the black swan can be clearly separated from other geese in the first principal component (PC1), while the white geese and brown geese are It can be separated when it comes to the third component (PC3). The hybrid goose is also between the Chinese goose and the white roman goose.
另外使用STRUCTURE軟體分析族群結構(圖3),在各個假設可能分群的K值(K=2~7)中,當K=2時,主要分成白羅曼鵝族群與其他族群,雜交鵝此時可明顯由兩成分構成;當K=3時,黑天鵝族群被分離,當K=4時,白色與褐色華鵝被分開;當K=5時,雜交鵝被定成一族群,剛好是試驗的五族群;當K=6~7時,只有雜交再被細分。 In addition, using the STRUCTURE software analysis group structure (Fig. 3), in the K values (K=2~7) of possible hypotheses, when K=2, it is mainly divided into white roman goose group and other ethnic groups. It is obviously composed of two components; when K=3, the black swan group is separated. When K=4, the white and brown geese are separated; when K=5, the hybrid geese are grouped into a group, which is just the test five. Ethnic group; when K=6~7, only hybridization is subdivided.
根據14組新的與其他已知的微衛星標幟之基因型,利用 Saitou及Nei(1987)之估算式計算出個體間的遺傳距離,再利用鄰位連接法(NJ)繪製鵝隻個體之親緣關係樹,如圖4所示。結果顯示,五個族群的個體在此圖可完全被分開成五個群聚,將來在檢驗其他族群時,族群群聚的落點即可判定此族群的個體是否有經過雜交。 Based on 14 new genotypes with other known microsatellite markers Saitou and Nei (1987) calculated the genetic distance between individuals, and then used the ortho-joining method (NJ) to map the phylogenetic tree of the goose individual, as shown in Figure 4. The results show that the individuals of the five ethnic groups can be completely divided into five clusters in this picture. In the future, when testing other ethnic groups, the population points of the ethnic group can determine whether the individuals of this ethnic group have been crossed.
4.鵝隻個體鑑別率之評估4. Evaluation of individual identification rate of goose
個體鑑別之分析分成各別四鵝隻品種與全體族群,分別計算14組新微衛星標幟之個體鑑別率(P(ID))、近親個體鑑別率(P(ID)sib)及綜合全部14組新微衛星標幟之P(ID)與P(ID)sib,其試驗結果列於表10及表11。如表10所示,白羅曼鵝於此14組新微衛星基因座之P(ID)範圍在0.019(5A26681)至0.419(5A26261),且綜合P(ID)為1.2×10-11。白色華鵝之P(ID)範圍在0.028(5A5279)至0.538(5A5111),綜合P(ID)為4.3×10-10。褐色華鵝之P(ID)範圍則在0.024(5A26648)至0.606(5A265141),綜合P(ID)為1.81×10-10,雜交鵝之P(ID)範圍則在0.012(5A26648)至0.293(5A5111),綜合P(ID)為2.73×10-15。 The analysis of individual identification is divided into four geese species and all ethnic groups, and the individual identification rate (P (ID) ) of 14 new microsatellite markers, the identification rate of close relatives (P (ID) sib ) and comprehensive all 14 are calculated respectively. The P (ID) and P (ID) sib of the new microsatellite set are shown in Table 10 and Table 11. As shown in Table 10, the P (ID) of the 14 groups of new microsatellite loci in the white roman geese ranged from 0.019 (5A26681) to 0.419 (5A26261), and the integrated P (ID) was 1.2×10 -11 . The P (ID) range of White Hua Goose ranges from 0.028 (5A5279) to 0.538 (5A5111), and the integrated P (ID) is 4.3×10 -10 . The P (ID) range of brown geese is 0.024 (5A26648) to 0.606 (5A265141), the integrated P (ID) is 1.81×10 -10 , and the P (ID) range of hybrid goose is 0.012 (5A26648) to 0.293 ( 5A5111), the integrated P (ID) is 2.73 × 10 -15 .
另當欲探討之族群個體有近親關係,如全同胞(full-sib)或半同胞(half-sib),則在計算個體鑑別率時,需修正以P(ID)sib進行評估較為適當,因此,本試驗亦同時計算當假設所檢測族群具有近親關係時之P(ID)sib。如表11所示,白羅曼鵝於此14組新微衛星基因座之P(ID)sib範圍在0.306(5A26681)至0.668(5A266113),且綜合P(ID)sib為3.79×10-5。白色華鵝之P(ID)sib範圍在0.32(5A5279)至743(5A5111),且綜合P(ID)sib為1.51×10-4。褐色華鵝之P(ID)sib範圍則在0.313(5A26648)至0.808(5A266113),且綜合P(ID)sib為1.39×10-4。雜交鵝之P(ID)sib範圍則在0.293(5A26648)至0.64(5A266113),且綜合P(ID)sib為1.39×10-6。此外,各品種鵝隻及全體族群中,不同數目新微衛星標幟之綜合P(ID)與綜合P(ID)sib也分別被計算並繪製成折線圖(圖5、6)。從不同數目新微衛星標幟之綜合P(ID)與綜合P(ID)sib所繪製成的折線圖中,也可推估在確知鵝隻族群數量的情況下,若恰好涵蓋所有族群個體數,其所需新微衛星標幟之最低數量。 In addition, when the ethnic groups to be explored have close relatives, such as full-sib or half-sib, it is more appropriate to correct the P (ID) sib when calculating the individual discrimination rate. This test also calculates P (ID) sib when it is assumed that the detected population has a close relationship. As shown in Table 11, the P (ID) sib of the 14 groups of new microsatellite loci in the white roman geese ranged from 0.306 (5A26681) to 0.668 (5A266113), and the integrated P (ID) sib was 3.79 × 10 -5 . The P (ID) sib of the white Chinese goose ranges from 0.32 (5A5279) to 743 (5A5111), and the integrated P (ID) sib is 1.51×10 -4 . The P (ID) sib range of the brown geese is 0.313 (5A26648) to 0.808 (5A266113), and the integrated P (ID) sib is 1.39 × 10 -4 . The P (ID) sib range of the hybrid goose ranged from 0.293 (5A26648) to 0.64 (5A266113), and the integrated P (ID) sib was 1.39 × 10 -6 . In addition, among the various geese and all ethnic groups, the integrated P (ID ) and integrated P (ID) sib of different numbers of new microsatellite markers were also calculated and plotted as line charts (Figures 5 and 6). From the line graphs drawn by the composite P (ID) and the integrated P (ID) sib of different numbers of new microsatellites, it is also possible to estimate the number of individuals in all ethnic groups if the number of goose populations is known. , the minimum number of new microsatellite markers required.
5.有效分群與個體鑑別之最低微衛星標幟數5. The minimum number of microsatellite markers for effective grouping and individual identification
由以上結果可知該14組微衛星標幟成功地在個體親緣關係樹中對台灣主要鵝種分群,並在個體鑑別率上遠遠超過台灣每年平均飼養鵝隻數1.95×106或屠宰鵝隻數5.16×106(行政院農委會農業統計年報,2013)。因此,理論上少於14組微衛星標幟即可獲得效果,為了找出最少的有效微衛星標幟數,分別隨機挑出五組、七組、八組、九組微衛星標幟,計算出個體親緣關係樹(圖7、8、9和10)。結果可發現,使用五組微衛星標幟(5A26648、5A26681、5A5279、5A5397、5A265175)時,除了白羅曼鵝外,其他三群均無法分開(圖7)。使用七組微衛星標幟時(5A26648、5A26681、5A5279、5A5397、5A265175、5A26254、5A5305-1),只有少數褐色華鵝無法與其他族群分開(圖8)。使用八組微衛星標幟(5A26648、5A26681、5A5279、5A5397、5A265175、5A26254、5A5305-1、5A265141)以上,其分群效果即可達到(圖9、10)。因此,以本發明之微衛星標幟針 對台灣主要鵝群進行種原鑑別時,僅需八組即能達到分群之功效,每增加一個微衛星標幟做檢測,其分群效果則越準確。 It can be seen from the above results that the 14 sets of microsatellite markers successfully clustered the main geese in Taiwan in the individual kinship tree, and the individual identification rate far exceeds the average annual number of geese feeding in Taiwan 1.95×10 6 or slaughtering geese The number is 5.16×10 6 (Agricultural Statistics Annual Report of the Executive Yuan Agricultural Committee, 2013). Therefore, theoretically less than 14 sets of microsatellite markers can be obtained. In order to find the minimum number of effective microsatellite markers, five groups, seven groups, eight groups, and nine groups of microsatellite markers are randomly selected and calculated. An individual kinship tree (Figures 7, 8, 9 and 10). As a result, it was found that when five sets of microsatellite markers (5A26648, 5A26681, 5A5279, 5A5397, 5A265175) were used, the other three groups could not be separated except for the white roman goose (Fig. 7). When using seven sets of microsatellite markers (5A26648, 5A26681, 5A5279, 5A5397, 5A265175, 5A26254, 5A5305-1), only a few brown geese could not be separated from other ethnic groups (Figure 8). Using eight sets of microsatellite markers (5A26648, 5A26681, 5A5279, 5A5397, 5A265175, 5A26254, 5A5305-1, 5A265141) above, the grouping effect can be achieved (Figures 9, 10). Therefore, when the microsatellite marker of the present invention is used for the original identification of the main geese in Taiwan, only eight groups can achieve the effect of grouping, and each additional microsatellite marker is detected, and the grouping effect is more accurate.
另外,以上述8組微衛星標幟(5A26648、5A26681、5A5279、5A5397、5A265175、5A26254、5A5305-1、5A265141)分別計算個體鑑別率(P(ID))、近親個體鑑別率(P(ID)sib)及綜合全部8組微衛星標幟之P(ID)與P(ID)sib。如表12及表13所示,白羅曼鵝於此8組新微衛星基因座之綜合P(ID)為1.24×10-8。白色華鵝之綜合P(ID)為7.05×10-8。褐色華鵝之綜合P(ID)為4.89×10-8,雜交鵝之綜合P(ID)為5.12×10-11。每品種之綜合個體鑑別率均超過上述台灣每年平均飼養鵝隻數1.95×106或屠宰鵝隻數5.16×106隻。因此,本發明之微衛星標幟針對應用於台灣鵝隻個體鑑別時,僅需八組即能達到個體鑑別之功效,每增加一組微衛星標幟進行檢測,其個體鑑別效果越好。 In addition, the individual discrimination rates (P (ID) ) and the identification rate of close relatives (P (ID) ) were calculated using the above 8 sets of microsatellite markers (5A26648, 5A26681, 5A5279, 5A5397, 5A265175, 5A26254, 5A5305-1, 5A265141) . Sib ) and P (ID) and P (ID) sib of all 8 sets of microsatellite markers. As shown in Table 12 and Table 13, the comprehensive P (ID) of the 8 groups of new microsatellite loci in the white roman geese is 1.24 × 10 -8 . The integrated P (ID) of White Hua Goose is 7.05×10 -8 . The integrated P (ID) of brown geese is 4.89×10 -8 , and the integrated P (ID) of hybrid geese is 5.12×10 -11 . The comprehensive individual identification rate of each variety exceeded the above average number of geese in the above-mentioned Taiwan, 1.95×10 6 or the number of slaughtered geese 5.16×10 6 . Therefore, the microsatellite marker of the present invention can be used for the identification of individual geese in Taiwan, and only needs eight groups to achieve the effect of individual identification. Each additional micro-satellite marker is detected, and the individual identification effect is better.
總而言之,本發明所提供之14組微衛星標幟於分析臺灣鵝隻族群時,其結果顯示,其平均期望異質度、觀測異質度與多態性訊息含量,皆屬於高多態性範圍,表示該等微衛星標幟於分析族群具高度適用性;而由鄰位連接法繪製之鵝隻個體親緣關係樹中,可觀察到其族群分布可明確地區分,此結果顯示該等微衛星標幟應用於鵝隻品種差異檢測具有良好分辨性。 In summary, the 14 sets of microsatellite markers provided by the present invention analyze the results of the Taiwanese goose population, and the results show that the average expected heterogeneity, the observed heterogeneity and the polymorphism information content are all in a high polymorphism range, indicating The microsatellite markers are highly applicable to the analysis population; and the individual phylogenetic tree of the goose drawn by the ortho-connect method can be observed that the ethnic distribution can be clearly distinguished, and the results show that the microsatellite markers It is applied to the difference detection of goose varieties and has good resolution.
此外,試驗結果顯示本發明所提供之14組微衛星標幟其鵝隻綜合個體鑑別率或綜合近親個體鑑別率之效力可涵蓋台灣每年平均飼養鵝隻數或屠宰鵝隻數。 In addition, the test results show that the 14 sets of microsatellite markers provided by the present invention have the effect of comprehensive individual identification rate of goose or comprehensive identification rate of close relatives, which may cover the average number of geese or slaughter geese in Taiwan per year.
<110> 王,佩華 <110> Wang, Peihua
<120> 鵝隻遺傳特性鑑定與個體鑑別方法 <120> Genetic identification and individual identification methods of goose
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Andres K. et al., "Applicability of anatid and galliform microsatellite markers to the genetic diversity studies of domestic geese (Anser anser domesticus) through the genotyping of the endangered zatorska breed", BMC Research Notes, 2011, 4:65, p.1-10, 2011/03/16 * |
Huang Yinhua et al., "Characterization of 35 novel microsatellite DNA markers from the duck (Anas platyrhynchos) genome and cross-amplification in other birds", Genetics Selection Evolution, vol.37, p.455-472, 2005/07/05 * |
Tu YJ et al., "Genetic Diversity of 14 Indigenous Grey Goose Breeds in China Based on Microsatellite Markers", Asian-Australasian Journal of Animal Sciences, vol.19, no.1, p.1-6, 2005/12/02 * |
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