HRM技术在大白菜EMS突变体筛选上的应用研究

以大白菜抗霜霉病基因相关的1段DNA序列为目标片段,利用高分辨率熔解曲线技术(HRM),对EMS诱变大白菜12-2和12-7花蕾进行小孢子培养获得的92个DH单株,鉴定出7株12-2突变株,6株12-7突变株。针对其中9株进行了PCR片段测序,结果表明全部为点突变,主要包括4种突变类型,最易突变位点为C-T类型,其次为A-C和C-A类型,没有G-C突变类型。试验明确了高效筛选大白菜EMS突变体的分析流程,验证了HRM技术筛选大白菜突变体的可行性,为大量筛选大白菜突变体库奠定了基础。     

小麦粒重基因等位变异的高通量分子检测及组合分析

TaCwi-A1、TaGW2-6A及TaSus2-2B基因是调控小麦粒重的基因,目前已发现TaCwi-A1基因存在TaCwi-A1a和TaCwi-A1b两种等位变异,TaGW2-6A存在启动子区Hap-6A-A和Hap-6A-G及编码区一个T碱基插入等位变异,TaSus2-2B存在SUS2-2B-H和SUS2-2B-L两种等位变异。为了探究小麦粒重基因的综合效应,为粒重基因的分子辅助聚合育种提供方法依据与优异材料,本研究利用高分辨率熔解曲线分析(high-resolution melting curve analysis,HRM)技术分别检测了上述4个位点在262份小麦微核心种质材料中的变异类型,分析了不同变异及变异组合与粒长、粒宽和千粒重的相关性,并对变异组合在我国十个麦区的分布情况进行了分析。结果表明,TaCwi-A1a单倍型品种的千粒重显著高于TaCwi-A1b(P0.05);Hap-6A-A单倍型与Hap-6A-G单倍型品种的粒长、粒宽及千粒重均无显著差异,T碱基插入等位变异(命名为TT单倍型)材料的粒宽和千粒重显著高于无T碱基插入等位变异(tt)材料(P0.05);SUS2-2B-H单倍型品种的粒长和粒宽显著(P0.05)高于SUS2-2B-L单倍型品种,千粒重极显著(P0.01)高于SUS2-2B-L单倍型品种。对变异组合的分析表明,262份材料中仅出现了8种组合类型,变异组合与粒长及千粒重呈极显著相关(P0.01),与粒宽相关不显著,其中Ⅳ型组合(TaCwi-A1a/Hap-6A-G/tt/SUS2-2B-H)为最优组合,Ⅲ型组合(TaCwi-A1a/Hap-6A-G/TT/SUS2-2B-L)为最差组合。我国十个麦区中七个麦区Ⅲ型组合分布频率最高,仅青藏麦区Ⅳ型组合分布频率较高。     

三疣梭子蟹线粒体基因组SNP在增殖放流家系识别中的应用

为开发三疣梭子蟹(Portunus trituberculatus)增殖放流分子标志技术,本研究在前期获得三疣梭子蟹线粒体基因组24个SNP位点的基础上,采用高分辨率熔解曲线(HRM)检测技术对4个用于增殖放流的家系(A、B、C和D)(代表约80~120万个放流个体)进行了鉴别工作。结果显示,含有SNP位点的22条PCR扩增序列中,有9条SNP位点扩增产物在亲代(母本)及其子代的28个个体之间具有基本一致的熔解峰,且子代个体间Tm的均一性较好,无明显差异;进一步以序列已知的野生型及其突变体作为同一SNP引物扩增片段,在各家系间分析HRM标准曲线,这9个SNP可以成功用于三疣梭子蟹4个放流家系的鉴别。在这9个SNP位点中,可鉴定C家系的有5个,可鉴定B家系的有2个,可鉴定A、D家系的各1个。该研究结果为三疣梭子蟹种质资源的鉴定及标志放流工作的开展提供了技术支持。     

High-resolution melt curve analysis: A real-time based multipurpose approach for diagnosis and epidemiological investigations of parasitic infections

BackgroundReal-time PCR coupled with high resolution melting curve analysis is a practical technique that could be employed in multipurpose studies. During the recent decade, this technique has been practiced for different targets, worldwide.MethodsIn the current study three major database centers consisted of PubMed, Scopus and Web of Science were searched until Aug 2019 for applications of HRM real-time PCR in parasitology studies using terms: “Parasite” AND “HRM real-time PCR” OR “High Resolution Melting curve analysis” OR “Real-time PCR”, “Protozoan parasites” AND “HRM real-time PCR” OR “High Resolution Melting curve analysis” OR “Real-time PCR”, “Helminth” AND “HRM real-time PCR” OR “High Resolution Melting curve analysis” OR “Real-time PCR”.ResultsTotally, 83 papers met our criteria and were included in our study. This method was more frequently used for protozoan parasites (52/83; 62.65%), while lower (31/83; 37.35%) studies were incorporated on helminths parasites. Furthermore, Plasmodium spp., and Leishmania spp., were the most prevalent protozoan parasites, and Taenia spp., and filers were the most frequent helminths that were studied by HRM real-time PCR.ConclusionHRM real-time PCR is a sensitive, flexible and cost-effective method that could be used for multipurpose studies.     

Development of a molecular marker for self‐compatible S4′ haplotype in sweet cherry (Prunus avium L.) using high‐resolution melting

Sweet cherry (Prunus avium L.) has stylar gametophytic self‐incompatibility, which is controlled by the multi‐allelic S‐locus and encompasses the highly polymorphic genes for the S‐ribonuclease (S‐RNase) and S‐haplotype‐specific F‐box (SFB), which are female and male determinants, respectively. The self‐compatible mutant SFB4′ corresponds to an allele variant of SFB4 and presents a frameshift mutation. Even though male‐determinant molecular markers can discriminate between SFB4 and SFB4′ alleles, the methods required are laborious, time‐consuming and expensive, and not suitable for massive analysis and integration into breeding programmes. Our aim was to develop molecular markers for the evaluation of self‐compatibility alleles in sweet cherry, that could be used as a high‐throughput screening strategy to identify SFB4 and SFB4′ alleles, based on a marker for male determinacy. Our results were consistent using primers flanking the mutation responsible for the SFB4′ allele. We designed a specific molecular marker and confirmed it in sweet cherry commercial varieties. This new molecular marker is feasible for self‐compatibility alleles in the male determinant in sweet cherry‐assisted breeding programs.     

Modification of Campylobacterjejuni Broiler Colonization by a Feed Additive Composed of Encapsulated Organic Acids and Essential Oils

The objective of this study was to evaluate the effect of a novel feed additive on chicken intestinal colonization and carcass contamination by Campylobacterjejuni. The feed additive was composed of microencapsulated organic acids and essential oils （OA/EO）. The feed additive tested was provided by Jefo Nutrition Inc., St-Hyacinthe, Quebec, Canada. Day-old birds were separated into two rooms and subdivided into two groups. Chicken were fed with OA/EO or not fed with OA/EO until they reached 35 d of age. At 14 d of age, chickens received an oral suspension of two well characterized C. jejuni strains, depending on the room they were housed in. The levels of C. jejuni were periodically monitored in the caecum and on the carcasses. C. jejuni colonization was further characterized by the use of high-resolution melt analysis of the C. jejuniflaA gene （HRM-flaA）. The effect of the feed additive was strain-dependent. In room two, the feed additive had no effect on the caecal counts. In room one, at 35 d of age, caecal C. jejuni counts were higher with OA/EO, as opposed to carcasses counts which were lower in the treated group. The HRM-flaA analysis showed that an amplification profile was predominant in birds fed with OA/EO at 35 d of age in room one, suggesting the selection of a C. jejuni strain. In conclusion, the OA/EO seemed to be effective to reduce C. jejuni levels but this effect appeared strain dependent.     

应用高分辨率熔解曲线技术分析水稻分子标记基因型

 【目的】验证高分辨熔解曲线（high-resolution melting curve analysis，HRM）技术在水稻分子标记分析中应用的可行性和可靠性，应用该技术对水稻重组自交系（RIL）群体及其亲本进行SSR、InDel和SNP标记基因型分析。【方法】以粳稻品种丽江新团黑谷（LTH）和籼稻品种三黄占2号（SHZ-2）及其衍生的RIL群体为材料，以纯合亲本LTH作为参比基因型，利用HRM技术分析一个G/A转换的SNP标记以及非变性聚丙烯酰胺凝胶电泳（PAGE）难以分辨的一个SSR标记和一个InDel标记的基因型。【结果】通过加入适量的参比DNA，HRM能够分辨2个纯合亲本及其衍生的纯合体和杂合体RI系的分子标记基因型，并且以加入20%模板量的参比DNA对3种基因型的分辨最好。利用该方法对作图群体的部分RI系进行分子标记基因型分析，结果与变性PAGE的分析结果完全吻合。【结论】验证了HRM应用于水稻SSR、InDel以及SNP标记分析的可行性和可靠性。相对于凝胶电泳分析，HRM具有分辨率高、高效、快速、操作简单、安全等优点，是一种在水稻分子标记分析中很有应用前景的技术。     

苹果抗炭疽菌叶枯病基因SNP和InDel标记的HRM筛选

以207株‘金冠’与‘富士’苹果的F1杂交分离群体实生树为试材,挑选抗炭疽菌叶枯病基因R_(gls)位点区域内的,第15条染色体上,位于SSR标记S0304673和S0405127之间500 kb物理距离内,由全基因组重测序技术所获得的与抗该病相关的10个SNP及10个InDel标记,通过高分辨率熔解曲线(High Resolution Melting,HRM)技术筛选出与基因R_(gls)位点紧密连锁的2个SNP及4个InDel标记。通过对重组个体的基因型及表型分析,发现标记InDel4227和InDel4254表现出与R_(gls)基因位点共分离,将基因R_(gls)精细定位于标记InDel4199和SNP4299之间100 kb的物理距离内。对该基因位点两侧4.1~4.3Mb距离内的基因进行统计分析,表明在该区段内存在40个有功能注释的基因,涉及到细胞组成、分子功能和生物过程方面共19个GO分类。     

苹果不同HRM反应体系分析效果评价

高分辨率熔解曲线(high resolution melting,HRM)分析是一种新型的DNA多态性快速筛查技术。本研究以苹果品种"富士"和"舞姿"为试材,用来自苹果基因组的SSR标记CH03d11对不同反应体积、不同DNA浓度以及不同PCR退火程序下的HRM分型效果进行了测试与评估。结果表明:采用5μL反应体积可以获得与10μL或20μL反应体积相同的检测效果。在5μL反应体积中,DNA浓度小到0.25ng/μL时,PCR扩增及随后的HRM分析效果依然良好。另外,研究表明,采用降落PCR扩增模式也能取得良好的HRM多态性检测结果。