Pollen dispersal in a hinoki (Chamaecyparis obtusa) seed orchard detected using a chloroplast DNA marker

Pollen dispersal was estimated in two test plots in a hinoki (Chamaecyparis obtusa) seed orchard using a chloroplast DNA marker, the spacer region between thetrnD andtrnY genes, and SSCP (single strand conformation polymorphism). In Plot 1, 2,020 seeds from 40 trees within 30 m of the marker tree were analyzed using the PCR-SSCP method. In Plot 2, 1,850 seeds from 37 trees were analyzed in the same manner. The results revealed that the maximum pollen dispersal distance in the two plots exceeded 25 m. Pollen dispersal appeared to be inversely proportional to the distance from the marker tree. The effective pollen dispersal was suggested to be less than about 20 m in a mature hinoki seed orchard. Adjacent trees had an excessive influence when the pollen density was increased by artificial flower stimulation. Therefore, it was suggested that seed production better resembles ideal random mating when carried out as naturally as possible. In conclusion, the SSCP chloroplast DNA marker was a useful tool for amassing basic information on pollen management in seed orchards of coniferous species.     

SSCP Marker Development and Analysis of Candidate Restorer Gene Rf1 for Cotton Cytoplasmic Male Sterility

[Objective] Locating the cotton cytoplasmic male sterility (CMS) restorer gene Rf₁ is important for investigating restorer gene mechanisms and improving restorer lines. In our previous study, a gene cluster, with nine Pentatricopeptide repeat(PPR) genes and nine other genes, was found within the 160-kb Rf₁ target region in Scaffold 333. The objective here was to improve the density of Rf1-linked markers in the target region and determine the expression profiles of candidate genes. [Method] Using the sequences of the 18 genes, we designed 155 single-strand conformation polymorphism (SSCP) primers covering all of the gene sequences to identify the polymorphic SSCP markers between the fertile and sterile pools. Additionally, real-time polymerase chain reaction(PCR) was performed to analyze the expression profiles of eight candidate genes in the four developmental stages of buds of sterile, maintainer and restoring lines, respectively. [Result] In total, 15 polymorphic primers were identified. A genotype analysis of the F₂ population was conducted using the 15 primers and 3 other polymorphic simple sequence repeats (SSR) markers. The markers were distributed in a 4.8 cM range. In addition, owing to the influence of sterile cytoplasm or restorer genes, most of the genes showed different expression patterns in the four developmental stages of the three lines' buds. [Conclusion] SSCP markers tightly linked to Rf₁ were identified and the expression profiles of candidate genes were determined. This study provides a basis for the further fine mapping of restorer genes and for candidate gene screening.     

棉花油分合成相关基因的SSCP标记开发

本研究根据棉花油份合成相关基因(lysophosphatidic acid acyltransferase,LPAAT; diacylglycerol acyltransferase,DGAT; phosphoenolpyruvate carboxylase,PEPC)设计了75对引物,在8个棉花品种中运用SSCP技术...     

采用通用引物PCR配合SSCP和RFLP技术检测鱼病病原菌

采用通用引物PCR（UPPCR）、PCR－RFLP、PCR－SSCP技术，研究快速鉴别鱼病病原菌的分子生物学诊断技术。结果发现，采用细菌16S rRNA基因保守区特异性引物，以嗜水气单胞菌、鲁克氏耶尔森菌、鳗弧菌、柱状曲挠杆菌、乙型链球菌、荧光假单胞菌等部分常见鱼病病原菌为对象，可以建立一种UPPCR技术。该技术能在保证实验条件不变的基础上，检出上述所有细菌，并还可检出大肠杆菌和双歧杆菌等非鱼病病原菌。并且认为，该法与SSCP配合即采用UPPCR－SSCP技术能较好地鉴别被检菌而用于鱼病病原菌的快速诊断。     

贵州白山羊GDF9基因外显子2的多态性研究

生长分化因子9(GDF9)是卵母细胞分泌的生长因子,对早期卵泡的生长和分化起重要的调节作用。采用RFLP和SSCP方法检测贵州白山羊GDF9基因外显子2的单核苷酸多态性,结果表明:在所有检测样本中均未发现GDF9基因的FecGH突变;在高繁殖率(3羔以上/胎)母羊和公羊中检测到8个杂合的AB基因型,其中5个为高产的母羊,3个为公羊。碱基序列分析证实GDF9基因编码区第1189bp位点的碱基由G突变为A,该位点位于外显子2中,导致活性肽第79位缬氨酸突变为异亮氨酸(V79I),但在低繁殖率(1~2羔/胎)母羊中没有发现该突变。虽然贵州白山羊GDF9基因中G1189A突变的发生率仅为5%,但该位点编码的氨基酸与FecGH突变(S77F)仅相隔1个氨基酸,因此推测GDF9基因中的G1189A突变可能与贵州白山羊的高繁殖力有关。     

凝胶浓度对SSCP技术的影响

SSCP技术易受凝胶浓度、丙烯酰胺与甲叉双丙烯酰胺的比例和添加物等诸多因素的影响。本研究探讨了凝胶中丙烯酰胺与甲叉双丙烯酰胺的比例、凝胶浓度和添加物等对海胆SSCP技术的影响。结果显示:凝胶中丙烯酰胺与甲叉双丙烯酰胺的比例为29∶1、凝胶浓度为10%、凝胶中甘油为最佳的凝胶组成,单链DNA条带清晰,杂带少,容易观察。     

The Sequence Variations of Intron-3 of the α-Amylase Gene in Adzuki Bean

This study describes variation of intron-3 of a-amylase gene from 156 breeds of adzuki beansusing SSCP(single-strand conformation polymorphism)analysis. Based on a-amylase gene structure and se-quence, A pair of PCR primers, F (CCTACATTCTAACACACCCT) and R (GCATATTGTGCCAGTACAAT)were designed to amplify intron-3 fragments of a-amylase gene. 14 variant types were detected, including 13,9, 10, 4 variant types in the wild, weed, locally cultivated and modern brought-up adzuki beans respectively,9, 8, 7 variant types of the wild adzuki beans from Japan, China and Korea respectively, and some other va-riant types in the local adzuki beans from China and Bhutan. 60 % of subjects of cultivated races were found tobe EE type in the experiment. In addition, sequence analysis of intron-3 of α-amylase gene from 8 varianttypes reveals the evolution process of various variant types in adzuki beans.     

五指山猪近交系群体中DRA和DRB基因的SSCP检测

为确定五指山猪(WZSP)近交系群体SLA!ⅡDRA、DRB基因的等位基因数及其特性,利用PCR扩增SLA!ⅡDRA、DRB基因的第2外显子序列,经单链构象多态性即SSCP检测其等位基因数,选择纯合子个体的PCR产物直接测序。对所获得的序列与Genbank中所有相应的序列进行比对和聚类分析,特别是SLA!DRB!C、SLA!DRB!D、HLA!DRB*09012、HLA!DRB*1201、HLA!DRA*0101等位基因。结果表明:在WZSP近交系群体中,DRA、DRB各有2个等位基因且发现1个DRB新等位基因。DRA第2外显子有很强的保守性,而DRB基因呈现出高度的多态性,在核苷酸水平同源性为85%以上,氨基酸水平同源性为70%以上。该试验成功地检测了WZSP近交系SLA!DRA、DRB基因的等位基因及其特性,为建立WZSPSLA!DR基因抗原的分子分型及特异单倍型猪的培育研究奠定基础。     

重庆地区柑桔衰退病毒组群分析

柑桔衰退病毒(CTV)存在着许多生物学特性不同的株系。通过铲除感染强毒株植株或利用弱毒株交叉保护的方式来防治柑桔衰退病都需要对CTV株系进行准确、可靠的鉴定。根据对CTV衣壳蛋白基因(CPGs)的限制性片段长度多态性(RFLP)和单链构象多态性(SSCP)分析,发现在重庆地区CTV主要以CP/HinfⅠRFLP第1,3和6组群为主,并且在田间以多株系混合感染为主?     

Genetic variation in the ovine uncoupling protein 1 gene: association with carcass traits in New Zealand (NZ) Romney sheep,but no association with growth traits in either NZ Romney or NZ Suffolk sheep

The uncoupling protein 1 (UCP1) plays an important role in the regulation of lipolysis and thermogenesis in adipose tissues. Genetic variation within three regions (the promoter, intron 2 and exon 5) of the ovine UCP1 gene (UCP1) was investigated using polymerase chain reaction‐single‐strand conformational polymorphism (PCR‐SSCP) analyses. These revealed three promoter variants (designated A, B and C) and two intron 2 variants (a and b). The association of this genetic variation with variation in lamb carcass traits and postweaning growth was investigated in New Zealand (NZ) Romney and Suffolk sheep. The presence of B in a lamb's genotype was associated with decreased subcutaneous carcass fat depth (V‐GR) (p = 0.004) and proportion of total lean meat yield of loin meat (p = 0.005), and an increased proportion of total lean meat yield of hind‐leg meat (p = 0.018). In contrast, having two copies of C was associated with increased V‐GR (p < 0.001) and proportion of total lean meat yield of shoulder meat (p = 0.009), and a decreased hind‐leg yield (p = 0.032). No associations were found with postweaning growth. These results suggest that ovine UCP1 is a potential gene marker for carcass traits.