AFLP analysis of genetic diversity and relationship among some Chinese domestic ducks and wild ducks

The amplified fragment length polymorphic (AFLP) technique was used to analyze the genome DNA polymorphism among 8 breeds of domestic ducks and 2 species of wild ducks. Nine of the 17 selected primers pairs gave reproducible polymorphic DNA amplification bands. The amplified bands ranged from 44 to 83 per primer pair. Of the 513 AFLP markers obtained, 498 were polymorphic. The proportion of polymorphic loci was 97.1%. The genetic distance (D) and similarity coefficients (GS) were calculated based on the polymorphic data. Between domestic ducks D ranged from 0.331 to 0.589, while between domestic ducks and the wild ducks, it ranged from 0.298 to 0.520 (vs. Anas Platyrhynchos) and from 0.316 to 0.522 (vs. A. Poecilorhyncha), respectively. The variance analysis showed no significant difference between the two groups of data, which indicated that both mallard and spot-billed ducks made contributions to domestic duck evolution. A dendrogram was constructed according to the D value. __________ Translated from Journal of Xiamen University (Natural Science), 2005, 44(5): 729–733 [译自: 厦门大学学报 (自然科学版), 2005, 44(5): 729–733]     

Genetic signature of hybridization between Chinese spot-billed ducks and domesticated ducks

In this study, we analyzed 93 whole genomes from Chinese spot-billed ducks (CSB), meat-type ducks (MET), and egg and dual purpose-type ducks (EDT) to characterize the genetic material flowing between the CSB and modern ducks. Using a frequency of shared identical-by-descent method, approximately 10.9 Mb introgression segments containing 140 genes were identified showing the signatures of introgression between CSB and EDT. Meanwhile, nearly 10.6 M introgression regions containing 149 genes were identified between CSB and MET. Based on the haplotypes tree of each segment, we found that the introgression between CSB and domesticated ducks was asymmetric with a high level of gene flow from domestic to CSB and a low level of migration in the opposite direction. Moreover, we identified several genes that were introgressions from CSB and showed the signature of positive selection, which may contribute to the breeding of modern ducks. Our results provide new insight into the evolution and breeding history of domestic ducks and may be useful for the future management of wild and domestic duck populations.     

Comparative Evaluation of RAPD and AFLP Based Genetic Diversity in Brinjal (Solanum melongena)

The present investigation was carried out with an objective of evaluating genetic diversity in brinjal (Solanum melongena) using DNA markers. A total of 38 brinjal accessions including one wild-species, Solanum sisymbrifolium were characterized using random amplified polymorphic DNA (RAP D) and amplified fragment length polymorphism (AFLP) techniques. Out of 45 primers employed to generate RAPD profiles, reproducible patterns were obtained with 32 primers and 30 (93.7%) of these detected polymorphism. A total of 149 bands were obtained, out of which 108 (72.4%) were polymorphic. AFLP analysis was carried out using four primer combinations. Each of these primers was highly polymorphic. Out of 253 fragments amplified from these four primer combinations, 237 (93.6%) were polymorphic. The extent of pair-wise similarity ranged from 0.264 to 0.946 with a mean of 0.787 in RAPD, in contrast to a range of 0.103 to 0.847 with a mean of 0.434 in AFLP. The wild species clustered separately from the brinjal genotypes. In the dendrogram constructed separately using RAPD and AFLP markers, the brinjal genotypes were grouped into clusters and sub-clusters, and the varieties released by IARI remained together on both the dendrograms. All the 30 RAPD primers in combination and each of the four primer pairs in AFLP could distinguish the brinjal accessions from each other. AFLP was thus found to be more efficient than RAPD in estimation of genetic diversity and differentiation of varieties in brinjal.     

Origin and domestication history of Peking ducks deltermined through microsatellite and mitochondrial marker analysis

In order to elucidate the domestication history of Peking ducks, 190 blood samples from six Chinese indigenous duck breeds were collected with186 individualsgenotyped by 15 microsatellite markers. Both the F_ST and Nei’s standard genetic distances (D_s) from the microsatellite data indicated high genetic differentiation between Peking duck and other Chinese indigenous breeds. The haplotype network with mtDNA data showed that most of the Peking duck haplotypes were distinctly different from those of other domestic breeds. Although the H01 haplotype was shared by all domesticated duck breeds, Peking ducks displayed 12 specific domestic duck haplotypes, including four similar haplotypes H02, H04, H08 and H22, that formed a single haplogroup (A). Both H02 and H22 haplotypes were also shared by mallard and Peking ducks, indicating that Peking ducks originated from wild mallard ducks.     

Genetic structure of Eurasian and North American mallard ducks based on mtDNA data

To elucidate the origin and genetic structure of the domesticated duck in Eurasia and North America, we sequenced 114 duck D-loop sequences and retrieved 489 D-loop sequences from GenBank. In total, 603 ducks including 50 duck breeds/populations from eight countries (China, France, Russia, India, Kazakhstan, Mongolia, Thailand and USA) were used in this study. One hundred and thirty-four haplotypes and 81 variable sites were detected. H49 was the predominant haplotype, which was considered to be the same dominant haplotype found in the previous studies, and was found in 309 birds. The smallest values for both genetic differentiation index (F(ST), 0.04156) and the number of the net nucleotide substitutions between two populations (D(A), 0.00018) were observed between Eurasian domestic ducks and Eurasian mallards. No geography, breed or population clusters were observed in the Eurasian domestic ducks and mallards. Five haplotypes were shared by USA mallards and Eurasian domestic duck/Eurasian mallards. Only one haplotype (H49) was shared by Eurasian domestic ducks and China spot-billed ducks. By combining phylogenetic analyses, haplotype network profile, genetic distances and shared haplotypes, we can draw two major conclusions: (i) Eurasian and North American mallards show a clear geographic distribution pattern; (ii) Eurasian domestic ducks are derived from the Eurasian mallards, not from the spot-billed ducks.     

Genetic diversity of tufted ducks (<Emphasis Type="Italic">Aythya fuligula</Emphasis>, Anatidae) in Eastern Europe

The tufted duck (Aythya fuligula, Anatidae) is widespread in the Palaearctic across Northern Eurasia. Birds breeding in Northern and Eastern Europe are highly migratory, while populations from Western Europe are partially migratory or resident. The aim of this study is to explore genetic variation within and between ducks breeding in Latvia and migrants sampled in North West Russia and Belarus. The technique of random amplified polymorphic DNA was applied using five random primers (ol-1, ol-9-12). Genetic variability was measured for all tufted ducks investigated and for different sub-populations from various regions. Individual genetic structure and genetic variability was higher in ducks collected from Latvia. Gene diversity of amplified DNA bands in birds of Latvian origin was 24% with 80% polymorphism. Means of gene diversity and polymorphism for tufted ducks sampled in other countries varied from 12 to 14% and from 27 to 40%, respectively. A high number of unique bands characterized ducks breeding in Latvia. The oligonucleotide primers used in this study were suitable to analyze differences among tufted ducks of different origin. Possible explanations for the variation observed among the studied ducks are discussed.     

转基因抗虫棉SSR和AFLP遗传变异研究

利用SSR和AFLP两种分子标记技术,分析了52份转基因抗虫棉品种（系）的遗传多样性。结果表明：在61对SSR引物中,有4对引物在供试材料中表现出多态性,共扩增出102个标记,其中多态性标记25个,多态性百分率为24．51％,每对引物的扩增带数变化在17～30之间;在100对AFLP引物中,有9对引物在供试材料中产生多态性,共扩增出618个标记,多态性标记33个,占总数的5．34％,每对引物组合扩增的标记数分布于47～81之间。成对品种的欧式距离变化在2．00～5．57之间,平均值为4．21,单一品种欧氏距离的平均值分布在3．73～4．75之间,表明不同品种之间遗传差异不大。基于SSRs和AFLPs多态性数据的聚类分析,可以将供试材料划分为3个类群（SAGs）,但类群划分与品种地理来源不十分吻合。     

Distribution of Simple Sequence Repeat and AFLP Markers in Molecular Linkage Map of Rice

SSR (simple sequence repeat) and AFLP (amplified fragment length polymorphism) are PCR-based molecular markers developed in recent years. In this study, the authors analyzed the polymorphisms, inheritance and distribution of SSR and AFLP markers using an F2 population from a cross between cultivar "Aijiao Nante" ( Oryza sativa L. ssp. indica) and an accession of the common wild rice ( O. rufipogon Griff). A total of 200 new markers were obtained including 28 SSR and 172 AFLP markers. Six of the 28 SSR markers were developed by National Key Laboratory of Crop Genetic Improvement (NKLCGI) using DNA sequences from GenBank and the other 22 were from data published previously. The 172 AFLP markers were from a total 228 polymorphic bands amplified using 25 selected primer combinations. Mapping of the 200 new markers using NKLCGI' S previously developed RFLP map based on the same F2 population resolved these markers to all 12 rice chromosomes. Integration of the SSR and AFLP markers into the RFLP map resulted in a high density molecular linkage map containing 612 polymorphic loci.     

High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.     

云南栽培种及野生种芋头的AFLP指纹分析

目的:分析云南芋头种质资源遗传多样性。方法:应用扩增片段长度多态性(AFLP)指纹技术,用3对AFLP引物对采集自云南省的9份芋头栽培品种及1份野生品种进行研究,分离AFLP多态性条带。结果:共分离到60个AFLP多态性条带,AFLP多态位点百分率为96.77%,云南芋头种质资源在DNA分子水平上表现出丰富的遗传多样性。聚类分析将10份芋头品种分为2组,遗传距离为0.101～0.908。结论:AFLP指纹技术是筛选品种间差异基因的有效方法,研究结果为云南省芋头品种鉴定、遗传相关性分析、特殊功能基因的分离等工作提供了一定的理论基础。     

用RAPD和AFLP的方法对中国卤虫(Artemia)种及亲缘关系的研究

利用ＲＡＰＤ（随机扩增多态ＤＮＡ）和ＡＦＬＰ（扩增片段长度多态性）技术对不同种及种群卤虫的关系进行分析。 １０１个随机引物对４种卤虫Ａｆｒａｎｃｉｓｃａｎａ、Ａ ｕｒｍｉａｎａ、Ａ ｓｉｎｉｃａ和Ａ．ｐａｒｔｈｅｎｏｇｅｎｅｌｉｃａ基因组ＤＮＡ进行扩增,平均每个种获得７５１条带,其中４５８条带为多态性标记,每个引物提供平均７４个标记信息,聚类结果表明Ａ．ｓｉｎｉｃａ是不同于其他旧大陆两性生殖卤虫的一个独立的种。对来自 １５个种及品系的卤虫的 ＡＦＬＰ分析显示了非常好的遗传多态性,采用 １２对引物检测到 ５９４条带,其中 ４８０个为多态性标记。聚类结果表明来自西藏的两性生殖卤虫为不同于中国内陆两性生殖卤虫的新种。而孤雌生殖卤虫在进化过程中可能是多源的,中国内陆和沿海的孤雌生殖卤虫是沿着不同的途径进化的,内陆和沿海的孤雌生殖卤虫可能为不同的种。     

Changes in AFLP and SSR DNA polymorphisms induced by short-term space flight of rice seeds

Differences of both amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) polymorphisms were compared between the 60-d-old rice (Oryza sativa L. cv. DH7) and F3 rice plants (SP3) derived from seed, which endured a 7-d-space flight in March 2002. Total leaf AFLP DNA bands amplified from 22 primer pairs were 537 in DH7, whereas 562 in SP3. From the total 267 SSR DNA bands generated by 267 primer pairs, 39 were polymorphic with 22 larger (56 %) or 17 smaller (44 %) fragment size bands. The greatest numbers of AFLP DNA bands were amplified by primer E1M1 in DH7 (33) and E3M1 in SP3 (35), whilst the least by E4M3 in DH7 (14) and E5M2 in SP3 (16).     

中国食用向日葵种质资源遗传变异的RAPD及AFLP分析

本研究采用RAPD和AFLP方法对23个中国不同地区的食用向日葵（Helianthus annuus L.)骨干品种进行了遗传变异分析,同时对两种标记系统进行了比较。26个RAPD引物产生了总计192条DNA条带,大小分布 于0.26kb-1.98kb之间,其中165条（86．12％）具有多态性,每条引物产生DNA条带的平均数为7．38。8对AFLP引物组合共产生了576条带,分布于100bp-500bp之间,其中的341条具有多态性,多态百分率为76．00％,每对引物组合产生DNA条带的平均数为72。RAPD方法检测的每位点有效等位基因数（1．76）大于AFLP（1．65）,AFLP标记位点的平均多态性信息量（PIC）（0．38）低于RAPD标记位点PIC（0．41）,但AFLP标记具有很高的多态性检测效率（Ai=38．52）。用RAPD标记分析23个食用向日葵材料的亲缘关系,Nei氏相似性系数分布在47．84％－82．06％,平均相似性系数为0．6495,而采用AFLP的Nei氏相似性系数分布在54．15％－83．52％,平均相似性系数为0．6884。RAPD数据的标准差为0．13,而AFLP数据的标准差为0．08。因此,采用RAPD和AFLP方法分析食用向日葵遗传变异,RAPD标记具有较低相似性系数和较高方差而AFLP则相反。源于两种不同标记的遗传相似矩阵的相关系数为0．51,说明采用RAPD和AFLP系统分析食用向日葵遗传变异得到的结果有一定的相关性,无论采用RAPD还是AFLP标记进行聚类分析,都将23个不同基因型的食用向日葵材料分成了三个类群。     

Analysis of genetic diversity through AFLP,SAMPL, ISSR and RAPD markers in <Emphasis Type="Italic">Tribulus terrestris</Emphasis>, a medicinal herb

Tribulus terrestris is well known for its medicinal importance in curing urino-genital disorders. Amplified fragment length polymorphism (AFLP), selective amplification of microsatellite polymorphic loci (SAMPL), inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers were used for the first time for the detection of genetic polymorphism in this medicinal herb from samples collected from various geographical regions of India. Six assays each of AFLP and SAMPL markers and 21 each of ISSR and RAPD markers were utilized. AFLP yielded 500 scorable amplified products, of which 82.9% were polymorphic. SAMPL primers amplified 488 bands, 462 being polymorphic (94.7%). The range of amplified bands was 66 [(TC)₈G + M-CAG] to 98 [(CA)₆AG + M-CAC] and the percentage polymorphism, 89.9 [from (CT)₄C (AC)₄A + M-CTG] to 100 [from (GACA)₄ + M-CTA]. The ISSR primers amplified 239 bands of 0.4–2.5 kb, 73.6% showed polymorphism. The amplified products ranged from 5 to 16 and the percentage polymorphism 40–100. RAPD assays produced 276 bands, of which 163 were polymorphic (59%). Mantel test employed for detection of goodness of fit established cophenetic correlation values above 0.9 for all the four marker systems. The dendrograms and PCA plots derived from the binary data matrices of the four marker systems are highly concordant. High bootstrap values were obtained at major nodes of phenograms through WINBOOT software. The relative efficiency of the four molecular marker systems calculated on the basis of multiplex ratio, marker index and average heterozygosity revealed SAMPL to be the best. Distinct DNA fingerprinting profile, unique to every geographical region could be obtained with all the four molecular marker systems. Clustering can be a good indicator for clear separation of genotypes from different regions in well-defined groups that are supported by high bootstrap values.     

羊草种质基因组DNA的AFLP多态性研究

羊草是禾本科牧草之王 ,在当前我国西部生态建设和草原畜牧业发展中发挥着重要作用。用AFLP方法对2 7份我国不同地区分布的羊草 (Leymuschinensis (Trin .)Tzvel)材料进行了基因组DNA多态性分析 ,8对AFLP引物组合在 2 7个不同羊草基因型中共扩增出 5 37条带 ,产生出的DNA片段大小分布在 75bp - 5 30bp之间。其中单态性带 89条 ,占 16 .6 % ,多态性带 32 9条 ,占 6 1.3%。平均每对引物组合扩增的DNA带数为 6 6 .13,总的多态性比率为 78.84%。AFLP多态信息含量PIC值分布于 0 .0 - 0 .5之间 ,平均PIC值为 0 .2 16 ,出现的PIC最大值 (0 .5 )约占AFLP标记的 8.5 % ,说明羊草基因组DNA的多态性比较丰富。以 5 37个AFLP标记为原始数据 ,根据Nei和Li的方法对 2 7份羊草材料进行遗传变异和聚类分析的结果表明 :羊草种内有高频率的遗传变异发生 ,且与地理分布和生态环境密切相关 ;2 7份羊草不同基因型被划分为四大类群 ,不同类群相互间的遗传距离相对较大 ,在树状图中表现为较远的亲缘关系。对羊草种内遗传变异发生的原因和品种的形成进行了初步讨论。     

Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

利用AFLP分子标记探讨蜡梅种质资源的遗传多样性

利用AFLP分子标记技术,对中国蜡梅种质资源7个野生种群的遗传多样性进行了研究。利用筛选出的3对引物,共扩增出253条谱带,其中218条多态带,多态位点占86.17% ;种群间的基因分化系数为0.2906,说明蜡梅基因多样性主要存在于种群内;种群总的Nei s基因多样性指数为0.2933,Shannon信息多态性指数为0.4487,蜡梅总的遗传多样性水平较高。蜡梅不同种群遗传多样性水平差异较大,种群多态位点百分率在65.44% ～87.16%之间,Nei s基因多样性指数为0.1653 ～0.4012,Shannon信息多态性指数为0.3132 ～0.5603。神农架种群(SN)和保康种群(BK)的遗传多样性水平较高。用NTSYS2.01版软件对样品进行UPGMA聚类分析,结果7个种群并没有按地理距离进行聚类。最后提出要对各蜡梅野生群体采取相应的迁地和就地保护措施。     

Loss of Genetic Diversity of Domesticated Panax notoginseng F H Chen as Evidenced by ITS Sequence and AFLP Polymorphism: A Comparative Study with P. stipuleanatus H T Tsai et K M Feng

In the present study, we evaluated the genetic diversity of Panax notoginseng F H Chen, a domesticated species, and P. stipuleanatus H T Tsai et K M Feng, an endangered wild species in southeastern Yunnan and adjacent areas in Vietnam, using sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA and amplified fragment length polymorphism (AFLP) markers. Twenty-four accessions from three plantations of P. notoginseng and 51 samples from eight populations of P. stipuleanatus were assayed. A total of 694 bp of partial sequences of 18S, ITS 1, 5.8S, ITS2, and partial sequences of 26S were obtained. No sequence variation was detected within P. notoginseng and nine sites (1.30%) were variable in P. stipuleanatus. Two-thirds of the variable sites were found between Langqiao and other populations. In P. notoginseng, four pairs of AFLP primer combinations generated 312 bands, of which 240 (76.9%) were polymorphic and 60.15% of the polymorphisms were harbored within plantations. Approximately 41.0% and 66.9% of bands were polymorphic in population D7 and 5589, respectively. In P.stipuleanatus, the same four primer combinations produced 346 bands, of which 334 (96.5%) were polymorphic and approximately 62.14% of polymorphisms were maintained within populations. Considerable variations were observed. The percentage of polymorphic bands ranged from 50.2% to 84.9% and the average over populations was 70.9%. Cluster analysis did not show correlation of genetic differentiation with the distinctive leaf morphology of P. stipuleanatus (i.e. one form with bipinnatifid leaflets and the other with undivided leaflets). Because over 40% of genetic variations were maintained among populations and because of the very restricted distribution of P. stipuleanatus, all natural populations of this species should be conserved in situ. Considering that there are variations in P. notoginseng within and among plantations, we suggest establishing a genetic resource conservation garden or reintroducing P. notoginseng into its native habitats in southwestern China. Such reintroduction should be carefully executed after large-scale screening of genetic variation within the species.     

Genetic diversity and biogeography of the traditional Chinese medicine, Gardenia jasminoides, based on AFLP markers

Gardenia jasminoides Ellis is used in traditional Chinese medicine (TCM) in China. Levels of genetic variation and patterns of population structure within and among eight wild or cultivated populations of G. jasminoides Ellis in China were investigated using amplified fragment length polymorphism (AFLP) markers. Of the 11 primers screened, four produced highly reproducible AFLP bands. Using these primers, 244 discernible DNA fragments were generated with 165 bands (67.6%), were polymorphic, indicating considerable genetic variation at the species level. In contrast, there were relatively low levels of polymorphism at the population level with the percentage of polymorphic bands (PPB) ranging from 36.89% to 59.43%. Genetic diversity within populations ranged from 0.2086 to 0.3108, averaging 0.2392 at the species level. A high level of genetic differentiation among populations was detected based on Nei's genetic diversity analysis (76.59%), Shannon's index analysis (64.8%) and AMOVA analysis (72.75%). No significant statistical differences (analysis of molecular variance [AMOVA], p = 0.0639) in AFLP variation were found between regions. However, the variance among populations and within populations differed significantly (p < 0.001). An indirect estimate of historical levels of gene flow (N_m = 1.7448) was consistent with the high mean genetic identity (mean I = 0.9263) found among populations. There is an association between geographic and genetic distances between populations. Presently gene change exists between populations.     

DNA cloning and sequence analysis of chicken AFLP

Amplified fragment length polymorphisms (AFLP) have been shown to be useful for linkage mapping in chickens and other domestic animals. It is often desirable to convert AFLP bands to sequence-tagged site (STS) markers, in particular, so that AFLP-based linkage information can be integrated with recombinant DNA clone-based maps. Sixteen chicken AFLP bands were excised from gels, re-amplified, cloned and analysed. All inserts proved to be EcoRI-TaqI fragments, which suggests that unlabelled TaqI-TaqI AFLP fragments do not amplify well, and therefore do not significantly contaminate AFLP bands. For eight of the AFLP, the cloned fragment was used to probe blots of AFLP reaction fingerprints, confirming that the predominant DNA clone indeed contained the polymorphic fragment. Flanking regions of selected AFLP fragments were isolated using Vectorette cloning. The results obtained suggest that the these chicken AFLP most commonly arise from sequence polymorphism at or near the TaqI site.