Ecotilling is a new approach based on enzyme-mediated heteroduplex cleavage to discover DNA polymorphisms in natural population. We used mung bean nuclease（MBN） instead of routinely used CELI to cleave single base p...Ecotilling is a new approach based on enzyme-mediated heteroduplex cleavage to discover DNA polymorphisms in natural population. We used mung bean nuclease（MBN） instead of routinely used CELI to cleave single base pair mismatches in heteroduplex DNA templates. Nested set of primers were designed to amplify targeted region to avoid the influence of the variation in quality and quantity of the genomic DNA. To reduce the costs in fluorescently labeled primers, we added M13 adapter to 5＇end of gene specific primers to make IRD dye labeled M13 forward and reverse primers possibly universal for different genes. A Brassica rapa ZIP gene homologue was subjected to the analysis to practise the feasibility of the method in polymorphisms detection. Our experiment showed this method is efficient in discovering DNA polymorphisms in Brassica rapa natural population.展开更多
叶绿体rps16基因作为叶绿体重要基因,广泛应用于植物系统发育研究,而甘蓝型油菜rps16基因多态性研究尚未见报道。本研究利用二维Eco TILLING(ecotype targeting induced local lesions in genomes)技术及Sanger测序技术对全球256份甘蓝...叶绿体rps16基因作为叶绿体重要基因,广泛应用于植物系统发育研究,而甘蓝型油菜rps16基因多态性研究尚未见报道。本研究利用二维Eco TILLING(ecotype targeting induced local lesions in genomes)技术及Sanger测序技术对全球256份甘蓝型油菜种质资源的rps16基因进行多态性分析。结果发现,256份甘蓝型油菜rps16基因有5个多态性位点,经测序检测其存在4种单倍型;5个SNP位点都位于非编码序列上。研究结果表明,rps16基因在不同材料中具有丰富的遗传变异,可为其它基因遗传多样性研究提供借鉴,有助于对油菜种质资源进行评估和多样性分类。展开更多
TILLING(Targeting induced local lesions in genomes,定向诱导基因组局部突变技术)是一种高通量的等位变异创制和突变体快速鉴定技术,其实质是将传统的化学诱变方法和突变的高效筛选有效结合的反向遗传学研究方法。其技术原理是将传...TILLING(Targeting induced local lesions in genomes,定向诱导基因组局部突变技术)是一种高通量的等位变异创制和突变体快速鉴定技术,其实质是将传统的化学诱变方法和突变的高效筛选有效结合的反向遗传学研究方法。其技术原理是将传统的酶切技术与PCR技术相结合后采用红外双色荧光系统进行结果鉴定,从而筛选出相应的突变体。传统的TILLING技术主要用于筛选由人工诱导产生的突变体。Ecotilling技术由TILL-ING技术延伸而来,主要用于鉴定自然界中已经存在的突变体,其与传统的TILLING技术的区别主要为构建DNA池时略有差异。随着该项技术在拟南芥等模式植物中的成功应用,越来越多的人开始将其用于基因组较大的植物之中。本文对近年来TILLING技术在麦类作物中的应用进行了分析,并通过比较不同植物突变体库中的突变频率发现,经EMS处理的小麦等麦类作物突变体库中的突变频率显著高于其他植物,因此相信,TILLING技术将会作为一种常规手段在麦类作物尤其是普通小麦改良中得到越来越广泛的应用。展开更多
The value of crop wild relatives has long been acknowledged and this wild resource has been used to improve crop performance with clear economic benefits. Sugar beet (Beta vulgaris subsp. vulgaris) is the most economi...The value of crop wild relatives has long been acknowledged and this wild resource has been used to improve crop performance with clear economic benefits. Sugar beet (Beta vulgaris subsp. vulgaris) is the most economically valuable crop species in the order Caryophyllales, B. vulgaris subsp. maritima being the ancestor of the cultivated beets. The wild species of the genus Beta s.l. are commonly found in coastal areas of Europe and Mediterranean Region, where a rich genetic heritage still exists. Broadening the genetic base of sugar beet by introgression with wild relatives is a growing need regarding the maintenance of ecologically important traits. Since wild relatives have adapted to specific habitats, they constitute an important source of novel traits for the beet breeding pool. So, we conducted a broader research project aiming to delimit taxa and identify priority locations to establish genetic reserves of the wild Beta species occurring in Portugal (Western Iberian Peninsula). The aim of this study was: 1) to identify and characterize the main habitats of these wild Beta species;and 2) to present a review of some genetic tools available for future application in sugar beet breeding. In this review, we have focused on EcoTILLING as a molecular tool to assess DNA polymerphisms in wild populations of Beta and identify candidate genes related to drought and salt tolerance, as well as addressed some issues related to next-generation sequencing (NGS) technologies as a new molecular tool to assess adaptive genetic variation on the wild relatives of sugar beet.展开更多
Plant height is an important agronomic trait. Dramatic increase in wheat yield during the ＂green revolution＂ is mainly due to the widespread utilization of the Reduced height （Rht）-1gene. We analyzed the natural a...Plant height is an important agronomic trait. Dramatic increase in wheat yield during the ＂green revolution＂ is mainly due to the widespread utilization of the Reduced height （Rht）-1gene. We analyzed the natural allelic variations of three homoeologous loci Rht-A1, Rht-B1, and Rht-D1 in Chinese wheat （Triticum aestivum L.） micro-core collections and the Rht-B1/D1 genotypes in over 1,500 bred cultivars and germplasms using a modified EcoTILLING. We identified six new Rht-A1 allelic variations （Rht-Alb-g）, eight new Rht-B1 allelic variations （Rht-Blh-o）, and six new Rht-D1 allelic variations （Rht-Dle-j）. These allelic variations contain single nucleotide polymorphisms （SNPs） or small insertions and deletions in the coding or uncoding regions, involving two frame-shift mutations and 15 missenses. Of which, Rht-Dle and Rht-Dlh resulted in the loss of interactions of GID1-DELLA-GID2, Rht-Blicould increase plant height. We found that the Rht-Blh contains the same SNPs and 197 bp fragment insertion as reported in Rht-Blc. Further detection of Rht-Blh in Tibet wheat germplasms and wheat relatives indicated that Rht-Blc may originate from Rht-Blh. These results suggest rich genetic diversity at the Rht-1 loci and provide new resources for wheat breeding.展开更多
文摘Ecotilling is a new approach based on enzyme-mediated heteroduplex cleavage to discover DNA polymorphisms in natural population. We used mung bean nuclease（MBN） instead of routinely used CELI to cleave single base pair mismatches in heteroduplex DNA templates. Nested set of primers were designed to amplify targeted region to avoid the influence of the variation in quality and quantity of the genomic DNA. To reduce the costs in fluorescently labeled primers, we added M13 adapter to 5＇end of gene specific primers to make IRD dye labeled M13 forward and reverse primers possibly universal for different genes. A Brassica rapa ZIP gene homologue was subjected to the analysis to practise the feasibility of the method in polymorphisms detection. Our experiment showed this method is efficient in discovering DNA polymorphisms in Brassica rapa natural population.
文摘叶绿体rps16基因作为叶绿体重要基因,广泛应用于植物系统发育研究,而甘蓝型油菜rps16基因多态性研究尚未见报道。本研究利用二维Eco TILLING(ecotype targeting induced local lesions in genomes)技术及Sanger测序技术对全球256份甘蓝型油菜种质资源的rps16基因进行多态性分析。结果发现,256份甘蓝型油菜rps16基因有5个多态性位点,经测序检测其存在4种单倍型;5个SNP位点都位于非编码序列上。研究结果表明,rps16基因在不同材料中具有丰富的遗传变异,可为其它基因遗传多样性研究提供借鉴,有助于对油菜种质资源进行评估和多样性分类。
文摘TILLING(Targeting induced local lesions in genomes,定向诱导基因组局部突变技术)是一种高通量的等位变异创制和突变体快速鉴定技术,其实质是将传统的化学诱变方法和突变的高效筛选有效结合的反向遗传学研究方法。其技术原理是将传统的酶切技术与PCR技术相结合后采用红外双色荧光系统进行结果鉴定,从而筛选出相应的突变体。传统的TILLING技术主要用于筛选由人工诱导产生的突变体。Ecotilling技术由TILL-ING技术延伸而来,主要用于鉴定自然界中已经存在的突变体,其与传统的TILLING技术的区别主要为构建DNA池时略有差异。随着该项技术在拟南芥等模式植物中的成功应用,越来越多的人开始将其用于基因组较大的植物之中。本文对近年来TILLING技术在麦类作物中的应用进行了分析,并通过比较不同植物突变体库中的突变频率发现,经EMS处理的小麦等麦类作物突变体库中的突变频率显著高于其他植物,因此相信,TILLING技术将会作为一种常规手段在麦类作物尤其是普通小麦改良中得到越来越广泛的应用。
基金supported by the Portuguese Foundation for Science and Technology with the FCT/Ciência 2008 to DB and MMREcologi-cal data presented is an output from the project PTDC/AGR-AAM/73144/2006 funded by the Portuguese Foundation for Science and Technology.
文摘The value of crop wild relatives has long been acknowledged and this wild resource has been used to improve crop performance with clear economic benefits. Sugar beet (Beta vulgaris subsp. vulgaris) is the most economically valuable crop species in the order Caryophyllales, B. vulgaris subsp. maritima being the ancestor of the cultivated beets. The wild species of the genus Beta s.l. are commonly found in coastal areas of Europe and Mediterranean Region, where a rich genetic heritage still exists. Broadening the genetic base of sugar beet by introgression with wild relatives is a growing need regarding the maintenance of ecologically important traits. Since wild relatives have adapted to specific habitats, they constitute an important source of novel traits for the beet breeding pool. So, we conducted a broader research project aiming to delimit taxa and identify priority locations to establish genetic reserves of the wild Beta species occurring in Portugal (Western Iberian Peninsula). The aim of this study was: 1) to identify and characterize the main habitats of these wild Beta species;and 2) to present a review of some genetic tools available for future application in sugar beet breeding. In this review, we have focused on EcoTILLING as a molecular tool to assess DNA polymerphisms in wild populations of Beta and identify candidate genes related to drought and salt tolerance, as well as addressed some issues related to next-generation sequencing (NGS) technologies as a new molecular tool to assess adaptive genetic variation on the wild relatives of sugar beet.
基金supported by grants from the Ministry of Science and Technology of China (2011CB100304, 2009CB118300)
文摘Plant height is an important agronomic trait. Dramatic increase in wheat yield during the ＂green revolution＂ is mainly due to the widespread utilization of the Reduced height （Rht）-1gene. We analyzed the natural allelic variations of three homoeologous loci Rht-A1, Rht-B1, and Rht-D1 in Chinese wheat （Triticum aestivum L.） micro-core collections and the Rht-B1/D1 genotypes in over 1,500 bred cultivars and germplasms using a modified EcoTILLING. We identified six new Rht-A1 allelic variations （Rht-Alb-g）, eight new Rht-B1 allelic variations （Rht-Blh-o）, and six new Rht-D1 allelic variations （Rht-Dle-j）. These allelic variations contain single nucleotide polymorphisms （SNPs） or small insertions and deletions in the coding or uncoding regions, involving two frame-shift mutations and 15 missenses. Of which, Rht-Dle and Rht-Dlh resulted in the loss of interactions of GID1-DELLA-GID2, Rht-Blicould increase plant height. We found that the Rht-Blh contains the same SNPs and 197 bp fragment insertion as reported in Rht-Blc. Further detection of Rht-Blh in Tibet wheat germplasms and wheat relatives indicated that Rht-Blc may originate from Rht-Blh. These results suggest rich genetic diversity at the Rht-1 loci and provide new resources for wheat breeding.
