南方紅豆杉EST-SSR標(biāo)記的開發(fā)及其在父本分析中的應(yīng)用
發(fā)布時間：2018-09-06 17:27
【摘要】：南方紅豆杉(Taxus chinensis var.mairei)是我國特有珍稀瀕危樹種,其基因組信息匱乏,分子遺傳學(xué)研究相對滯后。目前,南方紅豆杉可有效利用的分子標(biāo)記數(shù)量極其有限,遠不能滿足遺傳學(xué)和育種的研究要求。本研究以NCBI數(shù)據(jù)庫中的4種紅豆杉轉(zhuǎn)錄組序列為基礎(chǔ),利用生物信息學(xué)軟件,開發(fā)南方紅豆杉EST-SSR分子標(biāo)記,檢驗多態(tài)性位點在紅豆杉種間擴增情況,并將其應(yīng)用于南方紅豆杉父本分析。相關(guān)研究結(jié)果對紅豆杉屬物種保護和分子育種具有重要意義。主要研究結(jié)果如下:(1)分析了 4種紅豆杉轉(zhuǎn)錄組序列特征。以NCBI數(shù)據(jù)庫中4種紅豆杉轉(zhuǎn)錄組序列為基礎(chǔ),利用生物信息學(xué)軟件對轉(zhuǎn)錄組序列進行分析,分別得到南方紅豆杉(Taxus chinensis var.mairei)、東北紅豆杉(Taxus cuspidata)、歐洲紅豆杉(Taxus baccata)和曼地亞紅豆杉(Taxus media)SSR位點2 344、2 361、1 357和2 312個。在2-6bp重復(fù)單元的微衛(wèi)星中,南方紅豆杉和曼地亞紅豆杉以六核苷酸重復(fù)基元為主,其次為三核苷酸;東北紅豆杉和歐洲紅豆杉以三核苷酸為主,其次為六核苷酸。4種紅豆杉,二核苷酸、三核苷酸、六核苷酸優(yōu)勢重復(fù)基序類型均為AG/CT、AAG/CTT、AAGAGG/CCTCTT。(2)開發(fā)得到112個紅豆杉屬多態(tài)性位點。以4種紅豆杉位點信息為基礎(chǔ),選擇重復(fù)單元數(shù)大于或等于9的位點合成150對引物。基因分型結(jié)果表明,112對引物具有多態(tài)性,多態(tài)性比率達到74.67%。其中,南方紅豆杉、東北紅豆杉、歐洲紅豆杉和曼地亞紅豆杉中各開發(fā)得到59、17、13和23個多態(tài)性位點。(3)開發(fā)得到103個南方紅豆杉多態(tài)性位點。利用16個單株樣本對103個EST-SSR位點進行遺傳參數(shù)分析,共檢測到462個等位基因,每位點等位基因數(shù)(Na)變化范圍為2-11,平均每個位點的等位基因數(shù)為4.41。各位點觀測雜合度(Ho)的變化區(qū)間為0-1,平均值為0.459;期望雜合度(He)的變化區(qū)間為0.0625-0.891,平均值為0.513;近交系數(shù)(FIS)的變化區(qū)間為-0.654-1,平均值為0.085。多態(tài)性信息含量(PIC)變化區(qū)間為0.059-0.849,平均多態(tài)性信息含量為0.453。14個位點存在無效等位基因(Oosterhout值),頻率變化區(qū)間為0.135-0.470;22個位點偏離哈代-溫伯格平衡(Hardy-Weinberg equilibrium)。(4)分析了 107個位點在5種紅豆杉中的種間擴增情況。其平均擴增率為96.07%。其中,只有一個位點(Ma-25497-83A)沒有擴增,4個位點(C-52641-193B,Ma-1402-721B,Ma-186-234D,Ma-6383-968B)的擴增率小于 50%,100 個位點的種間擴增率達到100%。(5)檢驗了開發(fā)位點的有效性和準(zhǔn)確性。48株紅豆杉樣本的主成分分析(PCA)結(jié)果與種的分類相一致,6種紅豆杉屬的聚類分析結(jié)果與其它標(biāo)記結(jié)果基本類似。(6)篩選10個多態(tài)性豐富的位點對三個南方紅豆杉群體進行父本分析。CERVUS軟件在95%的置信度可確定祁陽群體、攸縣群體、八大公山群體的自由授粉父本各12個(占總子代的37.50%)、21個(占總子代的84.00%)、7個(占總子代的18.91%),三個群體候選父本的累積排除率分別達到了 98.32%,93.80%和97.40%。父本分析結(jié)果表明南方紅豆杉有效花粉散布距離為10-25m。
[Abstract]:Taxus chinensis var. mairei is a rare and endangered tree species in China. Its genome information is scarce and molecular genetics research is relatively lagging behind. At present, the number of molecular markers available in Taxus chinensis var. mairei is extremely limited, far from meeting the requirements of genetics and breeding. In this study, four species of Taxus chinensis var. mairei in NCBI database were used. Based on the transcriptome sequence of Taxus chinensis, EST-SSR molecular markers were developed by using bioinformatics software to test the amplification of polymorphic loci in Taxus chinensis, and applied to the analysis of male parents of Taxus chinensis. (1) The transcriptome sequence characteristics of four Taxus species were analyzed. Based on the transcriptome sequences of four Taxus species in NCBI database, the transcriptome sequences were analyzed by bioinformatics software. The transcriptome sequences were obtained from Taxus chinensis var. mairei, Taxus cuspidata, Taxus baccata and Taxus mandiana, respectively. There were 2 344,2 361,1 357 and 2 312 SSR loci in Taxus media. Among the 2-6 BP repeat units, Taxus chinensis var. mairei and Taxus mandii were dominated by hexanucleotide repeat units, followed by trinucleotides; Taxus chinensis var. mairei and Taxus chinensis var. mairei were dominated by trinucleotides, followed by hexanucleotides. The dominant repeat motifs of acid and hexanucleotide were AG/CT, AAG/CTT, AAGAGG/CCTCTT. (2) 112 polymorphic loci of Taxus were developed. Based on the information of four Taxus loci, 150 pairs of primers were synthesized by selecting loci with repeat units greater than or equal to 9. Among them, 59, 17, 13 and 23 polymorphic loci were developed respectively in Taxus chinensis var. mairei, Taxus cuspidata var. mairei, Taxus cuspidata var. mairei and Taxus mandii. (3) 103 polymorphic loci were developed. A total of 462 alleles were detected in 103 EST-SSR loci using 16 single plant samples. The variation range of allele number (Na) was 2-11, and the average number of alleles per locus was 4.41. The variation range of observed heterozygosity (Ho) was 0-1, with an average value of 0.459; the variation range of expected heterozygosity (He) was 0.0625-0.891, with an average value of 0.513; the variation range of inbreeding coefficient (FIS) was - 0.654-1, with an average value of 0.085. The variation range of PIC was 0.059-0.849, the average polymorphism information content was 0.453.14 loci had invalid alleles (Oosterhout value), the frequency range was 0.135-0.470; 22 loci deviated from Hardy-Weinberg equilibrium. (4) The amplification of 107 loci among 5 Taxus species was analyzed. The amplification rate was 96.07%. Only one locus (Ma-25497-83A) was not amplified. The amplification rate of four loci (C-52641-193B, Ma-1402-721B, Ma-186-234D, Ma-6383-968B) was less than 50%. The inter-species amplification rate of 100 loci was 100%. (5) The validity and accuracy of the developed loci were tested. The results of cluster analysis of 6 taxa were similar to those of other markers. (6) 10 polymorphic loci were screened for paternity analysis of three populations of Taxus chinensis var. mairei. The cumulative exclusion rates of the three male candidates were 98.32%, 93.80% and 97.40% respectively. The results of the paternal analysis showed that the effective pollen dispersal distance of Taxus chinensis var. mairei was 10-25m.
【學(xué)位授予單位】：中南林業(yè)科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S791.49
，
本文編號：2227043
[Abstract]:Taxus chinensis var. mairei is a rare and endangered tree species in China. Its genome information is scarce and molecular genetics research is relatively lagging behind. At present, the number of molecular markers available in Taxus chinensis var. mairei is extremely limited, far from meeting the requirements of genetics and breeding. In this study, four species of Taxus chinensis var. mairei in NCBI database were used. Based on the transcriptome sequence of Taxus chinensis, EST-SSR molecular markers were developed by using bioinformatics software to test the amplification of polymorphic loci in Taxus chinensis, and applied to the analysis of male parents of Taxus chinensis. (1) The transcriptome sequence characteristics of four Taxus species were analyzed. Based on the transcriptome sequences of four Taxus species in NCBI database, the transcriptome sequences were analyzed by bioinformatics software. The transcriptome sequences were obtained from Taxus chinensis var. mairei, Taxus cuspidata, Taxus baccata and Taxus mandiana, respectively. There were 2 344,2 361,1 357 and 2 312 SSR loci in Taxus media. Among the 2-6 BP repeat units, Taxus chinensis var. mairei and Taxus mandii were dominated by hexanucleotide repeat units, followed by trinucleotides; Taxus chinensis var. mairei and Taxus chinensis var. mairei were dominated by trinucleotides, followed by hexanucleotides. The dominant repeat motifs of acid and hexanucleotide were AG/CT, AAG/CTT, AAGAGG/CCTCTT. (2) 112 polymorphic loci of Taxus were developed. Based on the information of four Taxus loci, 150 pairs of primers were synthesized by selecting loci with repeat units greater than or equal to 9. Among them, 59, 17, 13 and 23 polymorphic loci were developed respectively in Taxus chinensis var. mairei, Taxus cuspidata var. mairei, Taxus cuspidata var. mairei and Taxus mandii. (3) 103 polymorphic loci were developed. A total of 462 alleles were detected in 103 EST-SSR loci using 16 single plant samples. The variation range of allele number (Na) was 2-11, and the average number of alleles per locus was 4.41. The variation range of observed heterozygosity (Ho) was 0-1, with an average value of 0.459; the variation range of expected heterozygosity (He) was 0.0625-0.891, with an average value of 0.513; the variation range of inbreeding coefficient (FIS) was - 0.654-1, with an average value of 0.085. The variation range of PIC was 0.059-0.849, the average polymorphism information content was 0.453.14 loci had invalid alleles (Oosterhout value), the frequency range was 0.135-0.470; 22 loci deviated from Hardy-Weinberg equilibrium. (4) The amplification of 107 loci among 5 Taxus species was analyzed. The amplification rate was 96.07%. Only one locus (Ma-25497-83A) was not amplified. The amplification rate of four loci (C-52641-193B, Ma-1402-721B, Ma-186-234D, Ma-6383-968B) was less than 50%. The inter-species amplification rate of 100 loci was 100%. (5) The validity and accuracy of the developed loci were tested. The results of cluster analysis of 6 taxa were similar to those of other markers. (6) 10 polymorphic loci were screened for paternity analysis of three populations of Taxus chinensis var. mairei. The cumulative exclusion rates of the three male candidates were 98.32%, 93.80% and 97.40% respectively. The results of the paternal analysis showed that the effective pollen dispersal distance of Taxus chinensis var. mairei was 10-25m.
【學(xué)位授予單位】：中南林業(yè)科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S791.49
，
本文編號：2227043
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