棉属多倍化研究进展

多倍化在许多植物的物种形成及其随后的进化过程中发挥了关键作用。棉花是一个大属,一些棉种的起源发生过多倍体化,部分二倍体种是染色体二倍化的多倍体,比克氏棉最典型,拟似棉最复杂,还有D基因组的旱地棉和B基因组的3个种。陆地棉等5个异源四倍体种是多倍体化典型,是由A与D基因组近似的祖先种经过杂交和染色体加倍的双二倍体。它们的D亚组供体亲本种,前人认同的是雷蒙德氏棉,然而近期荧光原位杂交和叶绿体基因组学研究,提出了雷蒙德氏棉可能不是陆地棉亲本种的观点。四倍体棉种形成的多倍体化过程有多种推论,较为共识的时期是中更新世,结合近期包括叶绿体在内的基因组学的研究支持中更新世或更后些时候。荧光原位杂交、包括叶绿体在内的基因组学将为棉花多倍化分析提供更多的证据。     

Sexing of Dog Sperm by Fluorescence In Situ Hybridization

Effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing. The standard validation method is flow cytometric remeasurement of the DNA content of the sexed sperm. Since this method relies on the same instrument that produced the original sperm separation, it is not truly independent. Therefore, to be able to specifically produce either male or female offspring in the dog, we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X- and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that the FISH protocol worked reliably for both unsorted and sexed sperm samples.     

RNAi-mediated Knockdown of Xist Does Not Rescue the Impaired Development of Female Cloned Mouse Embryos

In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.     

新疆红花随体染色体及25S rDNA位点分析

利用荧光原位杂交技术研究我国新疆红花25S rDNA在染色体上的位点数目,同时分析了其随体染色体数目。研究结果显示：新疆红花有2对随体染色体;4对染色体存在25S rDNA位点,其中3对染色体上的杂交信号较强,可稳定检出,有时还可检测到1对染色体上较弱的杂交信号。与国外红花品种相比,新疆红花rDNA位点数目多,表明新疆红花与国外红花品种间核仁组织区结构存在较大差异。这些结果将为研究红花染色体进化及其准确的核型分析提供一定的参考。     

390份小麦-黑麦种质材料主要农艺性状分析及优异材料的GISH与FISH鉴定

将小麦近缘属植物黑麦中的优良基因导入小麦可以拓宽小麦的遗传基础,丰富小麦的遗传变异。本研究调查并分析了390份小麦-黑麦种质材料。在这390份种质材料中,6个主要农艺性状值均有较大的极差,说明其遗传多样性丰富。与10份小麦主栽品种相比,90%以上的材料具有穗长和分蘖数的显著优势,60%以上的材料具有小穗数优势,约30%的材料穗粒数和千粒重显著高于主栽品种。利用基因组原位杂交(genomic in situ hybridization,GISH)和多色荧光原位杂交(multicolor fluorescent in situ hybridization,mc-FISH)技术,对8份农艺性状优良的代表性材料进行染色体组成分析,发现3份为六倍体小黑麦(AABBRR),2份为八倍体小黑麦(AABBDDRR),1份为1RS·1BL易位系,其余2份不具有可见的黑麦染色体或染色体片段。值得指出的是,3份六倍体小黑麦与2份八倍体小黑麦所含的黑麦染色体不完全相同。八倍体小黑麦中有1对来源于黑麦的小染色体,而六倍体小黑麦中没有类似小染色体;并且,不同材料中黑麦4R染色体端部的GISH杂交带有明显差异。本研究结果为这些小麦-黑麦种质材料进一步应用于小麦育种提供了依据。     

45S rDNA在7种竹子植物染色体上的定位

编码18-5.8-25S核糖体RNA的45S rDNA基因,是1个简单的多基因家族基因,一般以串联方式相连,对应于核仁组织区(NOR).首次利用荧光原位杂交技术(FISH),研究45S rDNA在散生竹类的毛竹和斑竹,混生竹类的茶秆竹、日本矮竹、菲白竹和铺地竹,以及丛生竹类的白绿竹染色体上的分布.结果表明:本研究所涉及的散生竹和混生竹的几个竹种,只观察到1对随体染色体的次缢痕区域有45S rDNA位点,而丛生竹类的白绿竹除随体染色体具有45S rDNA位点外,某些非随体染色体上也有不同拷贝数的45S rDNA位点存在.     

Analysis of radiation-induced W chromosome aberrations in the codling moth, <Emphasis Type="Italic">Cydia pomonella</Emphasis> (L.), by fluorescence in situ hybridization techniques

The structure of the female sex-determining W chromosome was examined in wild-type females and in three mutant female lines of the codling moth, Cydia pomonella. The mutant lines were isolated from progeny of irradiated females using the abnormal appearance of sex chromatin body in female somatic polyploid nuclei as a cytogenetic marker. A detailed cytogenetic analysis was carried out using three different fluorescence in situ hybridization techniques: genomic in situ hybridization (GISH), comparative genomic hybridization (CGH), and fluorescence in situ hybridization (FISH) with the codling moth W-chromosome painting probe. The FISH techniques enabled us to identify the W chromosome and its fragments in mitotic and meiotic chromosome complements and thus helped us to resolve complicated W chromosome aberrations induced by irradiation.     

First case of sterility associated with sex chromosomal abnormalities in a jenny

Chromosomal abnormalities are one of the main causes of genetic infertility in horses. Currently, their detection rate is rising due to the use of new diagnostic tools employing molecular markers linked to the sex chromosome pair. Despite genetic similarities, there are no previous reports of sterility associated with chromosomal abnormalities in the domestic donkey (Equus asinus). Hereby, we determined the presence of a chromosomal mosaicism in a female donkey with reproductive problems using molecular methodologies developed for horses. A two‐and‐a‐half‐year‐old jenny characterized by morphological abnormalities of the reproductive tract was cytogenetically analysed using conventional and fluorescent techniques and a group of microsatellite markers (short tandem repeat, STR). At the same time, five ultrasound measures of the reproductive tract were taken and compared with eight contemporary jennies of the same breed. After slaughter, morphological examinations showed that the case study had a blind vaginal vestibule defining an empty pouch that covered the entrance of the cervical os. Histopathological studies demonstrated that this abnormal structure was compatible with a remnant hymen. Molecular markers, STR and fluorescent in situ hybridization determinations revealed that the animal was a 62, XX/61,X mosaic and, therefore, the first case of chromosomal abnormalities in the sex pair reported in donkeys.     

蒙古冰草Afa家族串联重复序列克隆及染色体定位

Afa家族串联重复序列因只出现在小麦及小麦族近缘属物种而得名,本研究从蒙古冰草中克隆得到一个Afa家族序列,长度为233 bp,命名为pAmAfa1,该序列在GENBANK中进行同源序列比对,结果表明该序列与大多数小麦族其他物种的Afa家族串联重复序列存在较高的相似性;系统进化分析表明,蒙古冰草pAmAfa1序列与大赖草pLrAfa3,pLrAfa5序列聚在一起,表明蒙古冰草P染色体组与大赖草的N、X染色体亲源关系较近。为了明确Afa家族串联重复序列在蒙古冰草染色体上的位置,双色荧光原位杂交技术被采用,以pAmAfa1为探针检测到杂交信号出现在染色体的末端或近端部的区域,每条染色体上都有杂交信号,表明Afa家族串联重复序列普遍存在于P染色体组中。     

小麦品种绵麦37和绵麦367抗白粉病基因的FISH分析及分子检测

为了进一步研究和利用小麦品种绵麦37和绵麦367所携带的白粉病抗性基因,首先以OligopSc119.2-1(可检测小麦染色体结构变异)、Oligo-pTa535-1(可检测小麦染色体结构变异)和pDb12H(可检测簇毛麦染色体)为探针,对这两个小麦品种进行了荧光原位杂交(FISH)分析,然后利用与Pm21基因连锁的分子标记NAU/xibao15对这两个小麦品种进行分子检测。结果表明,绵麦37和绵麦367都含有1对6VS/6AL易位染色体,且均携带Pm21基因。