用SSR标记分析1949—2005年华南地区常规籼稻主栽品种遗传多样性及变化趋势

利用均匀分布于水稻基因组的300个SSR标记对95个华南地区不同年代常规籼稻主栽品种进行分析,研究该地区常规稻品种的遗传多样性及其变化趋势。检测到236个SSR标记有多态性,共获得776个等位基因,每个位点等位基因2~12个,平均3.29个,共有206个位点的等位基因数介于2~4个,占全部多态性位点的87.3%。多态性标记位点的PIC值平均为0.42,变化范围为0.041~0.790。不同染色体的位点多态性差异显著,其中第10染色体的位点平均等位基因数最多,PIC值最高,而第5染色体的位点平均等位基因数最少,PIC值最低;6个年代中,50~70年代育成品种包含等位基因数呈显著的上升趋势,70年代达最高值2.83,随后逐渐下降。分子方差分析(AMOVA)结果显示不同年代间遗传变异仅占总体变异的3.77%,但仍达极显著水平(P0.001)。不同年代育成品种的遗传距离(GD)呈下降趋势。聚类分析结果显示,在遗传相似系数(GS)为0.685处可将品种区分为5大类,表明华南地区各时期的常规稻品种遗传改良都是围绕少数骨干亲本进行的。试验结果显示,华南地区籼稻品种的遗传多样性狭窄且随年代而变化,70年代以后呈下降趋势,在今后的育种中应扩大亲本选材范围、拓宽育种亲本的遗传基础以提高育成品种的遗传多样性。     

Development of novel microsatellite markers for effective applications in Anthurium cultivar identification

Anthurium andraeanum is one of the most economically important floral crops and potted flowers marketed worldwide. Microsatellite markers are currently the preferred molecular marker owing to the many desirable attributes, including hypervariability, codominance, and amenability to high-throughput genotyping; however, there are few polymorphic molecular markers available for Anthurium. The object of this study was to develop and characterize novel microsatellite markers using the Araceae sequences in GenBank of the National Center for Biotechnology Information (NCBI) to contribute to molecular identification for cultivar protection. Using 1,579 Araceae expressed sequence tags (ESTs) and the related nucleotide sequences, 100 candidates contained simple sequence repeat (SSR) motifs that were suitable for primer design. Furthermore, 100 pairs of SSR primers were screened against a set of 28 diverse genotypes representing 24 cultivars that included four registration cultivars which were bred from the Taiwan Agricultural Research Institute (TARI) and 20 commercial cultivars, appended with three hybrid progeny and a mutant line. From the selected six polymorphic SSR loci, 52 alleles were amplified and 27 distinct genotypes were found, except for ‘Tropical’ and its mutant, with a mean number of eight alleles per locus. The polymorphism information content (PIC) ranged from 0.86 to 0.93. Based on these results, we proposed a key identification set using four microsatellite markers that is sufficient to discriminate among 24 cultivars. Because the Anthurium microsatellite markers developed in this study are primarily from expressed sequence tags or related genomic sequences, they can be used for cultivar identification and, accordingly, contribute to genetic evaluations in breeding programs.     

Cross‐genera amplification of informative microsatellite markers from common bean and scarlet runner bean for assessment of genetic diversity in mungbean (Vigna radiata)

Mungbean (V. radiata) is an important Asiatic legume supplying inexpensive protein to a vast majority of vegetarian masses. To increase markers repertoire in mungbean, a study was conducted to analyse 384 microsatellite markers derived from common bean, scarlet runner bean and adzuki bean for their transferability and polymorphism. The results showed that 87 (24.71%) primer pairs could amplify DNA loci of 20 mungbean genotypes including one accession of V. trilobata, while 52 showed reliable banding and polymorphism. These showed different degrees of variability at each locus producing 250 alleles with the number of alleles varying from 2 to 9. The major allele frequency varied from 0.17 to 0.95, while the polymorphic information content of SSRs ranged between 0.09 and 0.86 with an average of 0.60 ± 0.16. UPGMA revealed three major clusters accommodating ~95% of the accessions while one accession of V. trilobata (‘NSB‐007’) did not group with any other genotype describing the discriminating power of informative microsatellites. This study identified a set of useful microsatellite markers to accelerate the genetic studies and breeding programme of mungbean.     

Abundance, polymorphism and genetic mapping of microsatellites in oilseed rape (Brassica napus L.)

The objective of the present study was to estimate the abundance and degree of polymorphism of simple sequence repeat (SSR) markers in rapeseed. By screening about 45000 clones of a small inserts library of rapeseed total DNA the abundances of GA/TC and CA/TG simple sequence repeats in the rapeseed genome were estimated to be approximately one repeat every 100 kb and 400 kb, respectively. After sequencing 13 positive clones, primer pairs could be designed for 11 microsatellite loci. Seven of these primer pairs produced reproducible amplification products in a set of 31 rapeseed genotypes, with one pair amplifying two independent products, giving a total of eight amplified loci. The different microsatellite loci displayed between one and three visible alleles. At four loci, additional null alleles were observed. With up to four alleles, polymorphic microsatellite markers show significantly higher allele numbers in rapeseed than restriction fragment length polymorphism (RFLP) markers. Four of the eight microsatellite markers could be mapped on four different linkage groups of an RFLP map of the rapeseed genome.     

Genetic diversity in cultivated Osteospermum as revealed by random amplified polymorphic DNA analysis

A collection of 28 Osteospermum clones and cultivated varieties of different origin were evaluated by random amplified polymorphic DNA (RAPD) analysis. All the clones were identified by 12 decamers selected from a set of 30. This is the first characterization by molecular markers of the genetic material of Osteospermum. The level of genetic diversity among genotypes was assayed and all the accessions tested were then classified into six groups by UPGMA cluster analysis; the clustering of genotypes using the RAPD data proved to be in accordance with their breeding group origin. RAPD analysis can therefore be a useful tool for evaluating genetic variability in other Osteospermum germplasm collections for breeding purposes and for protecting intellectual property rights of improved varieties.     

Development, characterization and utilization of microsatellite markers in pigeonpea

Pigeonpea is a major legume of the semi‐arid tropics that has been neglected in terms of molecular breeding. The objectives of this study were to develop microsatellite markers and evaluate their potential for use in pigeonpea genetics and breeding. Two hundred and eight microsatellite loci were isolated by screening a non‐enriched partial genomic library. Primers were designed for 39 microsatellite loci, 20 of which amplified polymerase chain reaction products of the expected size. Nineteen of the primer pairs were polymorphic amongst 15 cultivated and nine wild pigeonpea accessions providing evidence for cross‐species transferability within the genus Cajanus. A total of 98 alleles were detected at the 19 polymorphic loci with an average of 4.9 alleles per locus. The observed heterozygosity ranged from 0.17 to 0.80 with a mean of 0.42 per locus. Less allelic variation (31 alleles) was observed within the cultivated species than across the wild species (92 alleles). The diversity analysis readily distinguished all wild relatives from each other and from the cultivated germplasm. Development of more microsatellites is recommended for future genomic studies in pigeonpea.     

用于区别不同棉花品种基因组特征的微卫星位点筛选

保守性强、重复性好、多态性高的微卫星位点可被有效用于构建作物DNA条形码。选取目前生产上主要推广种植、代表不同来源系统的12个棉花品种作为微卫星位点筛选材料,参考我室构建的四倍体栽培棉种种间高密度遗传图谱信息,从376对覆盖全基因组的SSR引物中,筛选出51对引物可扩增出带型清晰且多态性高的微卫星位点。这些引物在12个供试品种中共产生155个等位位点,每对引物揭示的等位基因位点在2~7之间,平均值为3.04。参照微卫星位点的染色体定位和多态信息,在每条染色体上选择一个多态性相对高的SSR位点,其相应的26对SSR引物被推荐为构建棉花品种DNA条形码的一套首选引物,并初步应用于12个品种的DNA条形码编制。其余25对引物作为候选引物。使用该套引物扩增出的微卫星位点可用于大量棉花品种DNA条形码构建,为棉花品种真实性和纯度的分子鉴定奠定基础。     

甜叶菊微卫星富集文库的构建与多态性标记的筛选

甜叶菊是我国一种重要特种经济作物, 其分子标记相关遗传背景研究甚少。本研究基于生物素与链霉亲和素的强亲和性原理, 用链霉亲和素顺磁颗粒捕捉人工合成的标记有生物素的寡核苷酸探针(AG)₁₅, 间接筛选出含有甜叶菊基因组微卫星序列的DNA酶切片段, 将筛选得到的片段连接到pUC-T载体中, 构建甜叶菊微卫星序列的富集文库。挑取354个克隆进行菌落PCR检验, 从中筛选出158个阳性克隆进行测序。结果表明, 134个(84.81%)克隆中含有微卫星序列, 其中完美型85个(63.43%)、非完美型15个(11.19%)、复合型34个(25.38%)。根据微卫星序列共设计出71对微卫星引物, 其中62对能扩增出稳定的条带。利用24个甜叶菊品系对这62对引物的遗传多样性的分析表明, 有16个位点表现出多态性, 等位基因数为2~8个, 平均每个位点扩增得到4.5个等位基因, 多态性信息含量在0.3163~0.7595之间, 观测杂合度(H_o)与期望杂合度(H_e)的范围分别为0.2174~0.9167与0.3555~0.8076。通过聚类分析, 将甜叶菊分为大小叶两大类。本研究开发出的微卫星标记可为甜叶菊的分子遗传育种提供有效的遗传标记。     

The art of attrition: development of robust oat microsatellites

Microsatellite or simple sequence repeat (SSR) markers are important tools for genetic analyses, especially those targeting diversity. The primary objective of this study was to develop robust oat‐based microsatellite markers from newly enriched genomic libraries to expand on a relatively small existing oat SSR toolbox. Microsatellite motifs characterized by (CA/GT), (AAT/TTA), (ATG/TAC) and (CATC/GTAG) repeats were targeted for enrichment. Preliminary screening showed that 90% of clones from the (CA/GT) and 79% of the clones from the (ATG/TAC) libraries contained repeats, while < 11% of the clones from (AAT/TTA) and (CATC/GTAG) libraries contained repeats. Subsequent sequencing of 1536 clones from the (CA/GT) and (ATG/TAC) libraries resulted in 539 and 578 SSRs for which primers were designed, respectively. A total of 246 SSRs were polymorphic across 11 oat lines. One hundred and twenty‐five of the markers produced highly reproducible assays that interrogated 369 alleles at 193 loci. Of these, 79 robust assays interrogated 146 codominant alleles. These markers will be useful for a wide range of genetic analyses in oat including assessment of diversity and marker‐assisted breeding.     

DNA typing of hops (Humulus lupulus) through application of RAPD and microsatellite marker sequences converted to sequence tagged sites (STS)

Summary Both random amplified polymorphic DNA and microsatellite repeat sequences were investigated as DNA markers for distinguishing hop cultivars. Microsatellite sequences converted to STS markers proved to be most successful. The relative abundance of microsatellite repeat sequences in the hop genome varied depending on the sequence class. Of the repeat types investigated the dinucleotide repeats (GA)_n and (GT)_n are the most highly represented in the hop genome. Microsatellite repeat sequences in hops have been shown to be highly polymorphic and are very informatives as STS molecular markers. A DNA typing system using sequence-tagged microsatellite site markers has been developed which will not only be useful for hop cultivar identification but also marker assisted breeding and quality control purposes.     

New SSR markers of Phaseolus vulgaris from sequence databases

To enlarge the number of microsatellite loci of Phaseolus vulgaris (L.) (common bean), a set of primer pairs has been obtained from public databases, to amplify DNA regions including simple sequence repeats (SSR), and they have been assayed in several P. vulgaris genotypes. Publically available DNA sequence data of P. vulgaris were searched for SSR motifs. Twenty sequences containing microsatellites were identified and primer pairs were designed to amplify those microsatellites. Primers were evaluated for their ability to detect polymorphisms within a set of P. vulgaris accessions, local landraces and hybrids and in addition, two accessions of the closely related species P. coccineus. Eighteen polymorphic SSR loci were identified. Polymorphisms were found at both levels of accessions and Phaseolus species.     

裸燕麦品质资源AFLP标记遗传多样性分析

用20对AFLP引物组合对281份栽培裸燕麦(Avena nuda)进行遗传多样性分析,共得到1 137条带,其中260条为多态性带,引物的平均多态性百分率为22.96%,平均多样性信息指数(PIC)为0.0326。以地理来源分组,不同来源的组群Simpson指数在1.235~1.495之间,Shannon指数范围为0.1558~0.4437,组群内变异贡献率为83.45%,组群间变异占16.55%。组群大小与多态性位点数、组群内变异贡献率、Simpson指数及Shannon指数显著相关。内蒙古和山西资源多样性丰富,东北地区资源独特,西部地区资源遗传结构单一,东欧组群与内蒙古组群遗传关系最近。国内组群的遗传多样性水平高于国外组群。地方品种与育成品种相比,组群内变异贡献率较高。建议在遗传多样性丰富地区进一步收集裸燕麦资源,并加强对材料少、代表性较差的地区,如西北和西南地区的裸燕麦地方品种的收集,以丰富我国的裸燕麦基因源。     

Assessing the genetic diversity of Portuguese maize germplasm using microsatellite markers

A collection of Portuguese maize accessions representing a valuable source of genes for introduction into modern cultivars is stored at the Portuguese Plant Germplasm Bank (Banco Português de Germoplasma Vegetal—BPGV). To assess genetic diversity among inbreds, microsatellite analysis was carried out for 54 inbred lines representing the diversity of Portuguese dent and flint maize germplasm. Fifty American and other European elite inbreds were also analysed for comparison. Fifteen microsatellite loci distributed throughout the maize genome were chosen based on their repeat unit and base composition. A total of 80 alleles were detected with an average allele number of 5.33 per locus. Polymorphism information content (PIC) values and observed genetic distances showed the existence of large variability among inbreds. Cluster analysis indicated that almost all of the inbreds could be distinguished from each other and Portuguese inbreds were present in all clusters formed. These associations were consistent with the known pedigree records of the inbreds, confirming a mixed origin of Portuguese materials. Comparative analysis of microsatellite diversity among groups was established according to important traits for both breeding and line identification. This revealed that, although most of the genetic diversity (>95%) was attributable to differences among inbreds of different groups, the existence of phenotypic differentiation in endosperm colour, kernel type and cob colour could be suggested for grouping. These findings support the joint use of molecular and morphological traits in management of the germplasm collection. In this study, SSR markers proved to be effective to characterise and identify maize inbred lines, and demonstrate associations among them. This revised version was published online in July 2006 with corrections to the Cover Date.     

裸燕麦种质资源AFLP标记遗传多样性分析

用20对AFLP引物组合对281份栽培裸燕麦(Avena nuda)进行遗传多样性分析,共得到1137条带,其中260条为多态性带,引物的平均多态性百分率为22.96%,平均多样性信息指数(PIC)为0.0326.以地理来源分组,不同来源的组群Simpson指数在1.235～1.495之间,Shannon指数范围为0.1558～0.4437,组群内变异贡献率为83.45%,组群间变异占16.55%.组群大小与多态性位点数、组群内变异贡献率、Simpson指数及Shannon指数显著相关.内蒙古和山西资源多样性丰富,东北地区资源独特,西部地区资源遗传结构单一,东欧组群与中国内蒙古组群遗传关系最近.国内组群的遗传多样性水平高于国外组群.地方品种与育成品种相比,组群内变异贡献率较高.建议在遗传多样性丰富地区进一步收集裸燕麦资源,并加强对材料少、代表性较差的地区,如中国西北和西南地区的裸燕麦地方品种的收集,以丰富我国的裸燕麦基因源.     

Microsatellite monitoring of recombination around the Vrn-B1 locus of wheat during early backcross breeding

The length of chromosomal segments retained around the Vrn‐B1 gene controlling sensitivity to vernalization in wheat (Triticum aestivum L.) was studied in the first and third backcrosses by using microsatellite markers. Eleven polymorphic markers located on chromosome 5B were used for microsatellite analysis. It was shown in the first backcross that plants with a donor segment around the gene of interest not longer than 50% of chromosome 5B could be selected. When selection is not molecular‐marker assisted, the length of the chromosomal donor segment with the target gene may reach 94% of chromosome 5B even in plants of the third backcross generation. The considerable length differences in the 5B microsatellite loci between the winter and spring lines of wheat studied indicate that these markers are promising in marker‐assisted backcrossing or marker‐assisted selection for the Vrn‐B1 gene using different combinations of Spring and Winter genotypes.     

Estimating Genetic Diversity in Durum and Bread Wheat Cultivars from Turkey using AFLP and SAMPL Markers

Since 1925, more than 100 wheat varieties were developed and released in Turkey, and many more were introduced from abroad, but no systematic analysis of their genetic diversity has been performed yet. In this research, a total of 34 domestic and foreign cultivars (12 durum and 22 bread wheats), released in Turkey between 1936 and 2000, were fingerprinted by means of five amplified fragment length polymorphism and three selective amplification of microsatellite polymorphic loci (SAMPL) primer combinations, to evaluate their genetic variation and to determine the existence of cultivar-specific bands. Among the 344 amplicons scored, 214 were polymorphic. The primer combination E_ACG/M_AGG yielded the highest number and the primer combination SAMPL–6/M _AGA produced the lowest number of polymorphic bands. Most cultivars were molecularly very similar, although a few distinct ones (the durum wheat 'Kunduru–1149' and the bread wheat 'İkizce–96') were also identified. Seven cultivar-specific markers for different bread wheat cultivars ('Golia', 'Seri–82', 'Adana–99', 'Pandas' and 'Sertak–52') and six cultivar-specific markers for durum wheat cv 'Kunduru' were observed. Our results show that genetic diversity among old and present–day wheat cultivar commonly grown in Turkey is limited.     

Development and variability analysis of microsatellite markers in peach

A genomic DNA library enriched with AG/CT repeats has been developed from the peach cultivar ‘Merrill O'Henry’. The enrichment method was efficient, with 61% of the clones obtained carrying a microsatellite sequence and a yield of one polymorphic microsatellite every 2.17 sequenced clones. From 35 microsatellites detected, 24 were polymorphic in a set of 25 cultivars including 14 peaches and 11 nectarines. A total of 82 alleles were found with the polymorphic microsatellites, with an average of a 37% of observed heterozygosity. Microsatellites with a high number of repeats were generally those having the largest number of alleles. All cultivars except two (‘Spring Lady’ and ‘Queencrest’) could be individually distinguished with the markers used. Just three selected microsatellites were enough for the discrimination of 24 out of the 25 possible genotypes. Cluster analysis grouped all nectarines in a single cluster. Peaches, with 75 of the 82 alleles found, were more variable than nectarines, with only 64. Microsatellites appear to be powerful and suitable markers for application in peach genetics and breeding.     

大豆微卫星诱发突变的分子分析

Microsatellite or SSR marker is an efficient tool for plant genotype identification, molecular mapping and marker-assisted selection. Objective of this study is to analyze the mutagenized microsatellite variations in soybean genome and reveal nature of these mutations. In the present study, mutations at fifteen microsatellite loci were detected in genomic DNAs of soybean mutant E182 induced by EMS (ethyne metyl sulfate) using PCR amplification of 485 pairs of SSR primers. These fifteen mutagenized microsatellite loci with repeat number variation were Satt005, Sattll7, Satt185, Satt282, Satt290, Satt420, Satt452, Satt483, Satt569, Satt579, Satt600, Satt602, Sat-086, Sat-107 and Sat-135, respectively. Sequencing results of these fifteen loci indicated that microsatellite sequences at Satt282, Satt483, Satt579, Satt600 and Satt602 loci were respectively deleted 1 -, 3 -, 8-, 20 - and 1 - trinucleotide (all [ATT]1-20 except for [CAA]8 [TAA]12 at Satt600 locus) repeats, which made allele sizes at these five loci decrease 3, 9, 24, 60 and 3 bp, respectively. And while microsatellite sequences at the other ten mutated loci, Satt005, Sattll7, Satt185, Satt290, Satt420, Satt452, Satt569 and Sat-086, Sat-107, Sat-135, were respectively inserted 1-, 6-, 6-, 3-, 4-, 3-, 8- trinu-cleotide repeats (ATT)1-8 and 12-, 6-, 16- dinucleotide repeats (AT)6-16, making allele sizes at these ten loci increase 3, 18, 18, 9, 12, 9, 24, 24, 12, 32 bp, respectively. On the other hand, eleven events of base mutations were detected in flanking regions at seven (Sat- 107, Satt185, Satt282, Satt420, Satt569, Satt579 and Satt600) of fifteen mutated microsatellite loci. These base mutations consisted of 6 transitions (4T→C and 2 A→G), 2 transvertions (A→T and T→A), 1 insertion (T) and 2 deletions (A and T). The experimental results proved that EMS mutagenesis could cause different types of mutations at microsatellite multilocus in soybean genome, including repeat number variations in microsatellite regions and random base mutations in flanking regions. We found three mutational biases, which were frequent insertion mutations of repeat units, initiating positions of microsatellite sequences of repeat unit insertions/deletions and both flanking-base T ↓ A of these insertion/deletion positions. In addition, the resolution capacity of high-quality agarose gels was sufficient to distinguish differences of only three base pairs in this experiment.