分子标记在植物上的应用

综述分子标记技术ＲＦＬＰ、ＲＡＰＤ、ＡＦＬＰ的基本原理、优缺点及其在植物研究中的应用情况。     

家蚕基因组中的分子标记遗传图谱

遗传标记既是基因组研究的重要内容，又是构建遗传图谱、研究生物多样性、育种、基因定位与克隆等的基因。目前，已有多项分子标记技术用于家蚕基因组的研究，并构建了多种分子标记遗传图谱，包括限制性酶切片段长度多态性（简称RFLP）、DNA随机扩增多态性（简称RAPD）、扩增片段长度多态性（简称AFLP）、简单重复序列PCR（简称SSR－PCR）等。本文就分子标记技术构建的家蚕基因组分子标记遗传图谱进行了讨论。     

桑树多倍体育种材料遗传背景的AFLP分析

利用AFLP(Amplified Fragment Length Polymorphism)分子标记技术,即扩增片段长度多态性,从DNA分子水平对19份人工三倍体桑育种材料进行了遗传背景分析,构建了指纹图谱.19份育种材料可组配70个杂交组合,各材料间AFLP相似系数大于0.750 0的有54个组合,经AFLP分析认为,它们不宜作亲本间杂交组合;AFLP相似系数小于0.750 0的有16个组合,经AFLP分析认为它们适于作亲本间杂交组合.本研究从基因组DNA分子水平上为人工三倍体桑品种选育以及杂交亲本的选择和组配提供了遗传背景依据.     

东亚飞蝗蝗蝻不同体色的AFLP分析

应用AFLP技术对来自不同种群的东亚飞蝗5龄蝗蝻的9种体色类型进行分析,结果表明:在东亚飞蝗不同体色的蝗蝻中,浅绿色、黄色、红色、黑色种群与散居型相关的绿色关系较近,而赤褐色、红绿色、灰色种群与群居型相关的褐色关系较近.     

Similarities and differences between Amaranthus species and cultivars and estimation of outcrossing rate on the basis of electrophoretic separations of urea-soluble seed proteins

Wheat-rye translocation line (2BS/2RL) has been developed for resistance to biotype L of Hessian fly and agronomically useful traits. AFLP analysis using 64 primer pairs was conducted in order to identify 2RL-specific polymorphisms between “Coker 797” (non-2RL), near-isogenic line (NIL) carrying 2RL, and “Hamlet”. Nine primer combinations identified twelve reproducible polymorphic fragments in the NIL carrying 2RL. These twelve fragments were cloned and sequenced with an aim towards converting AFLP markers into sequence tagged sites (STS). A comparison of the 12 sequences with non-redundant accessions in the NCBI database using the BLAST search option indicated that one fragment of approximately 200 bp in length (amplified using primer combination E+AAC / M+CTA) was highly homologous with the rye-specific repetitive sequence R173-1 and Wis-2-lA, a retrotransposon-like element in wheat. Two STS primers (SJ07 and SJ09) out of twelve STS primer sets enabled the detection of polymorphisms between Coker 797 and NIL carrying 2RL. In order to verify whether the polymorphism detected by primers SJ07 and SJ09 was in fact the result of the presence of 2RL, additional plant material was examined. Amplified products of about 260 bp fragment with the SJ07 primer set were generated in rye cvs.“Chilbohomil” and “Jochunhomil”, triticale experimental line Suwon 15, and wheat experimental line K-14 (1AL/1RS & 2BS/2RL), as well as NIL carrying 2RL and Hamlet, but not in Coker 797 (non-2RL), “Keumgangmil” (non-translocation wheat), KS92WGRC17 (PI592729 /;/ 6BS/6BL-6RL), KS92WGRC19 (P1592731 /;/ 4BS/4BL-6RL), “TAM200” (1AL/1RS), and “Siouxland” (1BL/1RS). Our data suggest that primer set SJ07 amplifies a “2RL-specific” fragment of diagnostic value. This revised version was published online in August 2006 with corrections to the Cover Date.     

利用双杂合位点标记资料构建芒果遗传图谱

为了建立芒果 (MangiferaindicaL )的分子标记遗传图 ,用 15对AFLP (AmplifiedFragmentLengthPolymorphism)引物组合扩增了芒果品种间杂交组合 (Keitt×Tommy Atkins)的 6 0个F1单株 ,获得了 191个多态性位点。它们的分离表现为双杂合 (Aa×Aa)和测交 (Aa×aa)分离两种类型 ,但前者占了 5 9 7%。为了充分利用双杂合位点分离所提供的遗传信息 ,我们根据群体中任意两个双杂合位点隐性个体出现的数目 ,利用二项式分布概率理论推断它们是否连锁以及它们彼此间的相引或相斥关系。在该芒果群体呈 3:1分离的 81个多态性标记中 ,39个被分为 14组 ,以此为基础构建了 15个连锁群 ;这些连锁群共覆盖了 35 4 1cM的芒果基因组。其中 ,最小与最大遗传距离分别为 3 7cM和 2 8 9cM。此外 ,对 18个 1∶1分离类型的标记 ,直接利用Mapmaker作图软件构建了两个芒果连锁群。本文对所提出的利用双杂合位点构建果树遗传图谱的策略进行了讨论。     

Intraspecific olive diversity assessed with AFLP

Amplified fragment length polymorphism (AFLP) was used to study diversity within and among Spanish olive varieties. A high degree of diversity was found among the varieties present in each growing region. Olive oil production and quality relies on appropriate cultivar selection as well as good orchard management. Production based only on a few superior cultivars would result in improved yield, oil quality, and production management. Amplified fragment length polymorphism were evaluated as a tool to identify the intraspecific and intravarietal diversity of olive. Amplified fragment length polymorphism analysis of 38 accessions belonging to 10 cultivars using six primer combinations produced 106 polymorphic bands. Results were analyzed for similarity among accessions via unweighted pair‐group means cluster analysis, resulting in 10 clusters corresponding to named variety designations. Similarity among varieties ranged from 0.60 to 0.72. Diversity within varieties was identified. Similarity within named varieties (accessions with the same varietal name) ranged from 0.75 to 0.96. Differences in several markers were found among 34 accessions. Intravarietal diversity was shown to exist within the Spanish olive varieties grown in the region surrounding Valencia.     

No DNA loss in autotetraploids of Arabidopsis thaliana

To address the issue of genome evolution in autopolyploids and particularly to investigate whether rapid sequence elimination also occurs in autopolyploids as in allopolyploids, amplified fragment length polymorphism (AFLP) fingerprinting was employed to examine a large number of genomic loci in F₁ hybrids between two different autotetraploids of Arabidopsis thaliana accessions, namely Ler and Col. Using this approach, perfect additivity in the F₁ hybrids was found between the newly‐formed autopolyploids when compared with their parental lines. Using flow cytometry, the study was extended in a quantitative manner, in which the nuclear DNA contents in one autotetraploid A. thaliana accession Ler, was determined. The increase in genome size of the autotetraploid line was additive. Taken together, no evidence was found for genome size reduction due to autopolyploidization of A. thaliana. The results indicating that there was no DNA loss in autotetraploid A. thaliana suggest that a different type of genome evolution may occur in autopolyploids during the initial stages of their formation when compared with allopolyploids.     

用AFLP技术构建棉花雄性不育三系及其杂种F1的DNA指纹图谱

实验应用AFLP同位素标记法,获得了棉花细胞质雄性不育系、保持系、恢复系及其杂种F1的DNA指纹图谱.较为详尽地介绍了有关AFLP的技术核心及注意点,并探讨了其适用于棉花研究的方法.结果表明,AFLP是一种十分有效的DNA指纹技术,它具有多态性丰富、稳定性高、重复性好等优点.     

Integration of AFLP markers into a linkage map of sugar beet (Beta vulgaris L.)

The AFLP (amplified fragment length polymorphism) technique has been applied in establishing an extended linkage map of sugar beet. A total of 120 AFLPs were integrated into an existing linkage map based on RFLP markers. Four primer combinations yielded between 19 and 40 polymorphic bands in an F₂ population consisting of 94 plants. The AFLP loci were evenly distributed over the nine linkage groups, with the exception of linkage group V where the number of AFLPs was significantly low. The AFLPs were found to be reproducible even against the background of different combinations of Taq DNA polymerases and buffers. However, the quantity of higher molecular weight fragments (>400 bp) was reduced when using plant DNA of poor quality as a template. The results of these experiments are discussed, together with possible applications of AFLPs in sugar beet breeding.