陕西设施蔬菜根结线虫特异性分子检测

为明确陕西设施蔬菜根结线虫的主要种类,利用已报道的花生根结线虫[Meloidogyne arenaria (Neal.) Chitwood]、南方根结线虫[Meloidogyne incognita (Kofold & White) Chitwood]、北方根结线虫(Meloidogyne hapla Chitwood)和爪哇根结线虫[Meloidogyne javanica (Treub)]的特异性引物,对陕西省20个县市区的24个样区的根结线虫进行PCR检测。样品中检测到了南方根结线虫、北方根结线虫、花生根结线虫和爪哇根结线虫,其中南方根结线虫为优势种群,大部分样区为单一种群为害,但个别样区存在2种种群。     

Genetic instability of bermudagrass (Cynodon) cultivars 'Tifgreen' and 'Tifdwarf' detected by DAF and ASAP analysis of accessions and off-types

DNA amplification fingerprinting (DAF) and arbitrary signatures from amplification profiles (ASAP) were used to evaluate the genetic stability of two important bermudagrass (Cynodon) cultivars, the interspecific cross Tifgreen and its somatic mutant Tifdwarf, and study genetic diversity and origin of derived bermudagrass off-types that exhibit patches of contrasting morphology and performance. Mini-hairpin primers produced complex and reproducible DAF and ASAP profiles with high levels of polymorphic DNA, and established genetic relationships between 11 Tifgreen and 8 Tifdwarf turf plot accessions and 16 off-types. DAF analysis revealed an average 14.1 ± 5.6 (SE) polymorphic bands/primer within cultivar accessions. In contrast, the higher resolving power of ASAP detected 24.5 ± 2.1 polymorphic bands/primer. Similarly, DAF and ASAP produced 13.0 ± 5.5 (SE) and 20 ± 2.8 polymorphic bands/primer within off-types, respectively. Phenetic analysis using cluster (UPGMA) and ordination (PCO) techniques showed that both Tifdwarf and Tifgreen were genetically unstable. The analysis showed that almost all cultivar accessions and one-half of the off-types studied were genetically distinct, but very close to each other. In this case, genetic variation was probably the result of somatic mutations. The other off-types and some Tifgreen accessions represented a genetically distant and diverse bermudagrass group of interspecific hybrid (n=27) origin. Off-types were probably the result of sod contamination. Results complement a previous study that established that the interspecific Tifway bermudagrass was genetically stable whereas derived off-types were contaminants rather than somatic mutants. Tifgreen and Tifdwarf showed genetic instabilities that were readily detected by DNA amplification with mini-hairpin primers. The present study offers a direct molecular alternative capable of evaluating the genetic stability of selected cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

New self-incompatibility alleles in apricot (Prunus armeniaca L.) revealed by stylar ribonuclease assay and S-PCR analysis

Apricot (Prunus armeniaca L.) shows gametophytic self-incompatibility controlled by a single locus with several allelic variants. An allele for self-compatibility (S_C) and seven alleles for self-incompatibility (S₁–S₇) were described previously. Our experiments were carried out to ascertain whether the number of allelic variants of apricot S-locus was indeed so small. Twenty-seven apricot accessions were analysed for stylar ribonucleases by non-equilibrium pH gradient electrofocusing (NEpHGE) to determine their S-genotype. To validate the results of electrofocusing, the applicability of the S-gene-specific consensus PCR primers designed from sweet cherry sequences was tested. NEpHGE revealed 12 bands associated with distinct S-alleles in newly genotyped cultivars. Cherry consensus primers amplified 11 alleles out from 16 ones, which indicated that these primers could also recognize most of the S-RNase sequences in apricot, and provided an efficient tool to confirm or reject NEpHGE results. By combining the protein and DNA-based methods, complete or partial S-genotyping was achieved for 23 apricot accessions and nine putatively new alleles (provisionally labelled S₈–S₁₆) were found. Their identity needs to be confirmed by pollination tests or S-allele sequencing. This study provides evidence that similarly to other Prunus species, the S-locus of apricot is more variable than previously believed.     

芒草ISSR分子标记体系的确定及引物的筛选

为建立并优化适用于芒草的ISSR-PCR扩增反应体系,进一步研究野生芒草群体的遗传多样性水平。以吉林省采集的芒草(Miscanthus sinensis)为材料,采用单因子试验的方法研究模板DNA、TaqDNA聚合酶的用量及引物浓度和退火温度对PCR扩增的影响。结果显示：在20 μL反应体系中,含有模板DNA 40 ng,dNTPs 0.4 mmol/L,引物0.6 μmol/L,Taq DNA聚合酶1.5 U。此外,筛选到10 条扩增稳定、条带丰富的候选引物,并确定了各自的最佳退火温度。     

甜樱桃设施栽培温度测控系统及其应用

选择适宜的通用型电子测控仪表,经电路改进后研制成甜樱桃设施栽培温度测控系统,替代传统的水银柱、红汞柱、煤油柱玻璃棒温度计和机械感应式温度计等测温装置,用于甜樱桃栽培大棚和温室的棚温遥测,取得预期效果。与传统的测温方式相比,避免了频繁进出大棚观看温度,降低了劳动强度,提高了棚温测控精度,特别是在夜间或雨雪天气,也能及时准确地观测棚温,适于甜樱桃设施栽培应用。     

利用差异显示技术克隆小麦抗白粉病相关基因的研究

对小麦—簇毛麦抗病易位系进行差异显示分析,以期获得小麦抗白粉病基因的分子克隆。根据已克隆植物抗病基因的保守域,设计了简并引物,与锚定引物组合,对抗病诱导的小麦抗白粉病易位系进行了差异显示分析,共获得10个差异片段,其中两个片段R3-1和8C1.3-6Northern杂交为阳性。将这两个片段克隆后进行了测序,序列分析结果为两个新序列,GenBank登录号分别为AF498271和AF498272。R3-1没有发现同源性较高的植物基因序列,发现8C1.3-6与Sphenostylisstenocarpa 类几丁质酶基因有同源性。     

甘蓝型油菜SSR核心引物研究

为了确定一组适用于品种纯度鉴定和遗传多样性分析的SSR核心引物,以100份具有代表性的甘蓝型油菜品种(系)为研究材料对746对SSR引物进行筛选,综合考虑PIC(平均多态信息量)值大小、引物重复性、扩增带型清晰度、连锁群分布等因素,确定44对引物为甘蓝型油菜核心引物,其中20对引物为首选核心引物,24对引物为备选核心引物。20对首选核心引物每对可检测到3~9个多态性位点,PIC值介于0.56~0.80,共检测到102个位点,分布于11个连锁群上。随机抽取1对核心引物对一个杂交组合大田制种F1纯度进行鉴定,其鉴定结果与田间自然鉴定结果一致,并可同时鉴别出来源于父本及外来的混杂单株。将44对核心引物应用于品种的系谱分析和100份品种遗传多样性聚类分析,同样得到了很好的验证结果。该套SSR核心引物适用于甘蓝型油菜品种鉴定及遗传多样性研究。     

松墨天牛携带的松材线虫PCR检测技术

利用本研究筛选出的一对松材线虫特异性PCR引物(上游引物:5'-CTACGTGCTGTTGTTGAGTTGGC-3',下游引物:5'-TGGTGCCTAACATTGCGCGA-3'),从松材线虫DNA中,扩增出一条长度403 bp(基因库登陆号:DQ855275)的特异性DNA片段.该引物可以将松材线虫DNA与松墨天牛组织以及拟松材线虫、畸刺伞滑刃线虫、变异伞滑刃线虫、小角伞滑刃线虫、利昂伞滑刃线虫、湖南伞滑刃线虫、红松滑刃线虫、滑刃属线虫、斯坦纳长尾线虫、小茎线虫、剑尾齿杆双胃线虫和寄生小杆属线虫等12种线虫的DNA区分开来.在此基础上,建立一套利用PCR技术直接从受感染的松墨天牛组织中检测出松材线虫的技术,并在实际运用中得到检验.     

不同引物RT-PCR方法检测猪繁殖与呼吸综合征病毒的比较研究

用3对分别针对猪繁殖与呼吸综合征病毒(PRRSV)的ORF7、ORF5和ORF5的PCR引物N1/N2、AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2进行RT-PCR,检测PRRSV,从其敏感性、特异性和临床样品检出率等方面进行比较,在此基础上进一步建立一步法RT-PCR检测方法。结果显示:3对引物对PRRSV均有很高的特异性;应用N1/N2引物病毒最低检测量为7.9 TC ID50,而AdGP5.1/AdGP5.2引物和RFLP5.1/RFLP5.2引物PCR最低检测量为79 TC ID50;运用N1/N2、AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2引物分别进行RT-PCR扩增检测临床样品,PRRSV检出率分别为28/48、27/48和25/48,且用AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2引物检测的阳性样品,用N1/N2引物检测也都呈阳性。运用N1/N2引物,通过一步法RT-PCR成功地从PRRSV S1株中扩增出374 bp的目的基因片段。结果表明,用N1/N2引物扩增PRRSV目的基因,其敏感性和临床样品检出率更高,更适合临床样品PRRSV的检测。     

斑茅NBS-LRR类抗病基因同源序列的克隆与分析

根据已知NBS-LRR抗病基因[含有核苷酸结合位点(NBS)和富亮氨酸重复(LRR)的胞内受体蛋白基因1NBS结构域蛋白质的保守序列,设计简并引物,对斑茅基因组进行体外扩增,获得了对应区段的DNA片段,回收、克隆这些特异片段,测序分析,共获得8个片段序列.序列分析发现其中7个编号分别为RGA-Q1、RGA-Q2、RGA-Q3、RGA-Q4、RGA-Q5、RGA-Q6和RGA-Q7的片段推导的氨基酸序列均具有典型的NBS结构域,即Ploop(GGVGKTr)、Kinase-2a(VLDDVW)、Kinase-3a(GSR/KILVTTR)及疏水结构域HD(hydrophobic domain).它们在NCBI上的登录号为EU685828、EU685829、EU685830、EU685831、EU685832、EU685833和EU685834.这些抗病基因同源片段(RGA)与已经克隆的N、L6、RPS2和胧等11个抗病基因在氨基酸水平上的同源性为2.3%～39.8%.可进一步用作斑茅抗病候选基因的分子筛选及遗传图谱的构建.