一个与稻瘟病菌无毒基因AVR-Pik~m连锁的SCAR标记的分离

 本研究将以前在稻瘟病菌菌株S1522获得的与决定对水稻品种梅雨明无毒性的基因(AVR-Pik^m)相连锁的1个RAPD标记OPO12₁₀₀₀进行了克隆和鉴定。核苷酸序列测定与分析结果表明:OPO12₁₀₀₀的大小为946个碱基,不含有与已报道的稻瘟病菌Mg-SINE、Fosburry、Magyy、Grasshopper、Pot2以及Pot3等同源的重复序列。根据OPO12₁₀₀₀的核苷酸序列,设计了1对24个核苷酸的特异引物,对无毒表型亲本S1522和毒性表型亲本S159、无毒表型群体基因池、毒性表型基因池以及有性杂交后代108个菌株进行了PCR扩增,所有无毒表型的菌株均能特异性地扩增出1条与OPO12₁₀₀₀大小相同的DNA条带,而毒性表型的菌株除5个重组个体外,均不能扩增出这条特异带。此结果表明,与稻瘟病菌无毒基因AVR-Pik^m连锁的RAPD标记OPO12₁₀₀₀被成功地转化为SCAR标记,为进一步通过染色体步移克隆该无毒基因奠定了基础。     

Development of IP and SCAR markers linked to the yellow seed color gene in Brassica juncea L.

Previous studies showed that the yellow seed color gene of a yellow mustard was located on the A09 chromosome. In this study, the sequences of the molecular markers linked to the yellow seed color gene were analyzed, the gene was primarily mapped to an interval of 23.304 to 29.402M. Twenty genes and eight markers’ sequences in this region were selected to design the IP and SCAR primers. These primers were used to screen a BC₈S₁ population consisting of 1256 individuals. As a result, five IP and five SCAR markers were successfully developed. IP4 and Y1 were located on either side of the yellow seed color gene at a distance of 0.1 and 0.3 cM, respectively. IP1, IP2 and IP3 derived from Bra036827, Bra036828, Bra036829 separately, co-segregated with the target gene. BLAST analysis indicated that the sequences of newly developed markers showed good collinearity with those of the A09 chromosome, and that the target gene might exist between 27.079 and 27.616M. In light of annotations of the genes in this region, only Bra036828 is associated with flavonoid biosynthesis. This gene has high similarity with the TRANSPARENT TESTA6 gene, Bra036828 was hence identified as being the gene possibly responsible for yellow seed color, in our research.     

A SCAR marker linked to the N gene for resistance to root knot nematodes (Meloidogyne spp.) in pepper (Capsicum annuum L.)

The root-knot nematodes (Meloidogyne spp.) are important nematode pests and cause serious diseases in pepper in the world. No molecular markers linked to the nematodes resistance N gene have been reported. In this paper, ‘Carolina Wonder’ (Capsicum annuum L.), a sweet pepper line resistant to root-knot nematode with N gene, ‘20080-5-29’ (C. annuum L.), an inbred line susceptible to root-knot nematode with good horticultural characteristics, and their F₂ progeny with 320 individuals were used as materials. Evaluation of resistance and susceptibility of parental lines, F₁ and F₂ progeny inoculated with root-knot nematodes (Meloidogyne incognita) were carried out. ‘Bulked segregant analysis’ method was used to search for polymorphic markers from 512 pairs of AFLP primers. Based on the assessment of resistance and susceptibility and polymorphism of the AFLP marker in F₂ population, the genetic linkage distance between the AFLP marker and the N gene was estimated. One AFLP marker E39/M41-339 was obtained and transferred to a SCAR marker amplifying a 315 bp DNA fragment linked to the N resistant allele and a 331 bp fragment linked to the N⁺ susceptible allele. The distance between the molecular marker and the nematodes resistance N gene is 6.3 cM. This research delivered a valuable tool for the marker assisted selection of nematodes resistance in pepper.     

分子标记检测棉田烟粉虱捕食性天敌种类的研究

[目的]为合理利用和保护棉田烟粉虱捕食性天敌提供依据.[方法]利用烟粉虱特异性引物TB-F/TB-R94585,通过检测单头捕食性天敌肠道内烟粉虱残体,查明新疆吐鲁番地区棉田烟粉虱捕食天敌的种类.[结果]在新疆吐鲁番棉田,烟粉虱的捕食天敌涵盖7个目,13个科,有19种.其中12种,菱斑巧斑瓢虫Oenopis conglobata linnaeus、绒螨Allothrornbium sp、全黑飘虫.ccineUa sp、小毛瓢虫Scymnus sp,短刺刺腿食蚜蝇Ischiodon scuteuaris Fabricius、叶盲蝽Phylinae sp、食中齿爪盲蝽Deraeocoris pwtctulatm Fallen、异须微刺盲蝽Campylomma divrsieornis Reuter,蜘蛛类有斜纹猫蛛Oxyopes sertalus L.Koch、八斑球蛛Theridion octomaculasum、跳蛛Salticidae sp,灌木新园蛛Neoscona adianta Walckenaer是国内首次报道的烟粉虱捕食天敌种类.[结论]通过分子标记的方法检测天敌,反应灵敏,操作简便,适合对害虫捕食性天敌的田间普查.     

甘蓝抗枯萎病SCAR标记的开发

 【目的】开发与甘蓝枯萎病抗性基因紧密连锁的SCAR标记,利用该标记对甘蓝抗枯萎病基因跟踪、鉴定,为甘蓝抗枯萎病分子标记辅助选择奠定基础。【方法】以高抗枯萎病的甘蓝自交系8024与感病自交系6A为亲本构建F2代分离群体和相应F3代家系,通过F3代家系抗病性分离表现确认F2代单株的基因型,选择10株纯合基因型显性抗病单株和10株纯合基因型隐性感病单株,利用BSA法构建甘蓝抗感基因池,筛选出与甘蓝枯萎病抗性基因紧密连锁的AFLP标记,克隆测序后根据序列差异将其转化为SCAR标记,通过142株F2代分离群体连锁验证该标记与甘蓝枯萎病抗性基因的连锁关系,并利用两个不同的抗感分离F2群体共100株对SCAR标记的通用性进行验证。【结果】获得了1个以相斥相连锁的SCAR标记S46M48199,该标记在感病亲本中扩增出199 bp的单一条带,而在抗病亲本中无扩增条带,142株F2代分离群体连锁分析表明,其遗传距离为2.78 cM,在F66和C1两个F2群体中的通用性验证结果,与抗性鉴定结果的吻合率分别为81%和83%。【结论】开发的SCAR标记可用于甘蓝抗枯萎病的分子标记辅助育种。     

基于SCAR标记的小麦禾谷孢囊线虫快速分子检测技术

【目的】建立从禾谷孢囊线虫及其近缘种的混合群体中检测禾谷孢囊线虫的快速分子检测方法。【方法】使用随机扩增多态性DNA（RAPD）和特征序列扩增区域（SCAR）标记的方法,运用PCR技术进行特异性检测。【结果】用本研究建立的SCAR标记快速分子检测体系对9种32个线虫种群进行检测,能够直接从混合线虫样品中检测出禾谷孢囊线虫。该检测方法对禾谷孢囊线虫的孢囊、2龄幼虫具有扩增能力,最低检出阈值为1/2000个孢囊,1/80头2龄幼虫。【结论】本研究设计的SCAR标记快速分子检测体系能够从禾谷孢囊线虫及其近缘种的混合种群中快速检测出禾谷孢囊线虫,且检测准确、灵敏度高。     

分子标记鉴别灰树花种质资源的研究

长期以来,食用菌菌种同名异种、同种异名问题给科研带来许多不必要的重复,也阻碍了菌种调剂供给.利用RAPD、ISSR、SRAP三种DNA分子标记分别对来源不同的30个灰树花(Grifola frondosa)菌株进行鉴别,并将得到的部分多态性条带转化为SCAR标记.3种分子标记对30个灰树花菌株的分析结果表明,共产生77...     

小麦 黑麦大穗型衍生后代的分子细胞学鉴定

为创制大穗型小麦种质材料,利用具有大穗多小穗性状的小麦-黑麦双二倍体材料"兰小黑"和普通小麦杂交得到一批大穗型衍生后代.综合采用基因组原位杂交(GISH)、SCAR标记、微卫星(SSR)和醇溶蛋白(A-PAGE)技术对这些大穗型后代中的8个单株进行分子细胞学鉴定.结果表明,GISH检测后代含2个外源信号;1RS特异SCAR标记检测后代均含有黑麦1.5 kb的1RS特征条带;醇溶蛋白检测后代都出现了黑麦碱基因Sec-1特征条带.筛选小麦21条染色体长短臂上各6对引物,结果发现只有1BS上的3对引物未扩增出1BS的条带,其余引物均扩增出了各自的相应条带.由此确定这8株小麦-黑麦大穗型衍生后代为1BL/1RS易位材料.     

鲤抗寒性状的RAPD标记转化为SCAR标记的研究

以黑龙江鲤Cyprinus carpio haematopterus、荷包红鲤抗寒品系、荷包红鲤Cyprinus carpio var.、柏氏鲤Cyprinus pellegnini pellegnini以及荷包红鲤抗寒品系(父本)与柏氏鲤(母本)杂交F2的DNA样品为材料,用300个随机引物对其进行PCR扩增,获得2个与鲤抗寒性状相关的分子标记AL04986和AR02788。回收、纯化这两个RAPD标记,连接到pMD18-T载体中,转化DH5α大肠杆菌感受态细胞,并对插入片段进行测序。根据测序结果,设计了3对SCAR引物SCAL04L/SCAL04R1、SCAL04L/SCAL04R2和SCAR02L/SCAR02R,其中SCAR02L/SCAR02R这对引物可以成功的反映出杂交F2两个群体在抗寒能力上的差异,适宜的退火温度为57℃,将此标记命名为SCAR02788。根据AL04986的测序结果设计的两对SCAR引物并没有反映出这种差异。SCAR02L/SCAR02R标记可以用作鲤抗寒基因分子辅助选择的依据。     

新扬州鸡早期增重的OPAY02-SCAR分子标记研究

OPAY02型2条多态性条带经克隆、测序和引物设计后,转换成SCAR标记,并对86个新扬州鸡随机交配后代基因组DNA进行了PCR扩增.2条带DNA序列与红色原鸡基因组序列比对结果表明,大分子量条带与位于红色原鸡第3号染色体上序列有98%的同源性,共检测到8个SNPS,其中195位的碱基T→G,316位的A→T,538位的G→A,731位的T→A,1 147位的G→A,1 329位的T→C,1 927位的C→T,2 081位的C→T,小分子量条带与红色原鸡没有同源序列,推测新扬州鸡野祖除红色原鸡外,还有其它来源.SCAR标记分析表明,经条件优化随机扩增的OPAY02型标记稳定、可靠,可用于遗传分析.2条带所在座位群体基因型平衡性测验结果表明,所测新扬州鸡群体处于平衡状态,选择可以打破平衡,有利于动物育种.