Transferability of rice SSR markers to Miscanthus sinensis, a potential biofuel crop

Miscanthus sinensis (M. sinensis) is a perennial C₄ photosynthesis grass, with high yield, high efficiency of water usage, low fertilizer requirement, tolerance to extreme environments, and is one of the plant species with good biofuel potential. Simple sequence repeat (SSR) markers are highly informative and widely used in plant genetic studies. In this study, 88 SSR primer pairs derived from the rice genome, including 47 EST-SSRs (eSSRs) and 41 genomic SSRs (gSSRs), were evaluated for cross-species transferability to M. sinensis. Forty-one SSR primer pairs in total could successfully amplify DNA fragments in M. sinensis, showing an overall transferability rate of 46.6 % between rice and M. sinensis. The transferability rate of eSSR (51.1 %) was higher than that of gSSR (41.5 %). A total of 140 SSR loci and 340 alleles in the set of rice and M. sinensis germplasm collections were detected. Nei’s gene diversity varied from 0 to 0.72 and averaged 0.35. Shannon’s information index varied from 0 to 1.49 and averaged 0.56. The observed heterozygosity ranged from 0 to 0.95 and averaged 0.08. Thirty-nine loci (27.86 %) were shown heterozygosity out of 140 SSR loci. A dendrogram based on genetic distance showed a significant geographic differentiation. Our results indicated that 46.6% of the rice SSR markers are transferable to M. sinensis, and are useful for germplasm evaluation and genetic analysis in M. sinensis.     

杨树SSR标记在柳树中的通用性分析

简单重复序列（simple sequence repeat,SSR）分子标记因其具有稳定性好、多等位基因、共显性遗传、数量丰富、基因组覆盖性好等优点,现己广泛应用于多种植物的遗传育种研究中。本研究利用杨树的48对基因组SSR引物及48对EST—SSR引物对6个苏柳品种进行了通用性分析。结果表明,杨树EST-SSR引物在柳树中的通用性达54．2％,而基因组SSR的通用性仅10．4％;但EST-SSR引物在有效引物中的多态性比例为80％,基因组SSR引物在有效引物中的多态性比例为100％。同时研究结果也表明,杨树SSR标记完全可以用于柳树群体或品种间的群传多样性分析。     

绿豆基因组SSR引物在豇豆属作物中的通用性

分子标记的种间通用性可降低其开发成本,提高利用效率,也有助于促进遗传研究较薄弱物种的分子遗传学研究。本文选取绿豆、小豆、豇豆及饭豆材料各3份, 分析1 205对新开发的绿豆基因组SSR引物在这些材料中的扩增效果,结果显示绿豆基因组SSR引物在豇豆、小豆和饭豆中的通用性比率分别为50.0%、73.3%和81.6%;多态性比率分别为4.1%、1.7%和1.5%;在4个种间均通用的引物469对。这些通用性SSR引物将有助于这4种食用豆类在多样性评价、连锁图谱的构建、基因定位及比较基因组学等方面的研究。     

小麦EST-SSR标记的开发和染色体定位及其在追踪黑麦染色体中的应用

根据小麦盐胁迫诱导和茎秆组织相关EST序列开发了81对EST-SSR引物, 其中67、46、18和61对分别在小麦、黑麦、簇毛麦和大麦基因组中稳定扩增, 在不同小麦和大麦品种间具有多态性的引物分别有22和23对。利用小麦缺体-四体系共定位了43对引物的81个位点, 其中A、B和D染色体组上分别有29、30和22个位点, 涉及除4B、3D和6D外的18条染色体。此外30对引物在黑麦基因组中具有特异扩增, 其中8对分别在黑麦1R、4R、5R和R7染色体上具有特异扩增, 7对在多条黑麦染色体具有相同扩增。这些新标记可有效用于小麦及其近缘物种的遗传作图与比较遗传研究。     

普通菜豆基因组SSR标记开发及在豇豆和小豆中的通用性分析

分子标记具种属间通用性可提高其利用效率,并降低标记开发成本。本研究基于Roche 454超高通量测序技术获得普通菜豆基因组测序结果,共开发了560个普通菜豆基因组SSR标记。利用2份普通菜豆品种对标记进行初步筛选,有421个标记能够有效扩增。用新开发的标记分析16份豇豆和16份小豆的通用性。结果显示,185个普通菜豆基因组SSR标记在豇豆中能有效扩增,通用性比率为43.9%;161个SSR标记在小豆中能有效扩增,通用性比率为38.2%;在豇豆和小豆中都能获得有效扩增条带的标记共138个;并且普通菜豆基因序列SSR标记在豇豆和小豆中的通用性比率高于基因间序列SSR标记。通用性标记的多态性分析表明,豇豆和小豆的多态性比率分别为34.0%和24.8%;且豇豆和小豆中基因间标记的多态性都比基因内标记的多态性高。上述通用性标记为豇豆属作物的多样性评价、连锁图谱的构建及基因定位等方面的研究提供了便利。     

芝麻EST-SSR标记的开发和初步研究

为了加速分子标记在芝麻研究中的应用,利用网上现有的芝麻EST(expressed sequence tags)数据信息,开展了芝麻EST-SSR功能性标记的开发和利用研究。 在所有的3 328条芝麻EST序列中共确认得到1 785条非冗余EST序列。其中,在含有微卫星重复的148条序列中共检测有155个EST-SSR。非冗余EST序列总长为774.266 kb,平均每4.99 kb含有一个EST-SSR。EST-SSR的分布频率和特征分析表明,以AG/TC为重复基元(motif)的SSR出现最多,占总SSR的37.42%。利用这些序列,设计开发了50对EST-SSR引物,并分别选用36个芝麻、2个棉花、2个大豆和2个油葵进行多态性和通用性研究。其中44对引物在供试芝麻材料中扩增出条带,共产生108个位点,平均每对引物产生2.45个位点,多态信息含量(polymorphism information content, PIC)平均值为0.390。根据遗传相似性系数进行聚类,有26个芝麻材料聚类在两个大的亚类(III和IV)中,聚类结果表明芝麻的基因型与地理来源之间没有必然的联系。此外,分别有2对、3对和4对引物可以在棉花、大豆和油葵中进行通用性扩增。本研究证实这种全新开发的芝麻EST-SSR标记在芝麻遗传多样性分析、遗传图谱构建以及比较基因组等研究方面有广阔的利用前景。     

High‐throughput development of SSR marker candidates and their chromosomal assignment in rye (Secale cereale L.)

Shotgun survey sequences of flow‐sorted individual rye chromosomes were data mined for the presence of simple sequence repeats (SSRs). For 787,850 putative SSR loci, a total of 358,660 PCR primer pairs could be designed and 51,138 nonredundant SSR marker candidates were evaluated by in silico PCR. Of the 51,138 SSR primer candidates, 1,277 were associated with 1,125 rye gene models. A total of 2,112 of the potential SSR markers were randomly selected to represent about equal numbers for each of the rye chromosomes, and 856 SSRs were assigned to individual rye chromosomes experimentally. Potential transferability of rye SSRs to wheat and barley was of low efficiency with 4.3% (2,189) and 0.4% (223) of rye SSRs predicted to be amplified in wheat and barley, respectively. This data set of rye chromosome‐specific SSR markers will be useful for the specific detection of rye chromatin introgressed into wheat as well as for low‐cost genetic and physical mapping in rye without the need for high‐tech equipment.     

Identification of informative SSR markers capable of distinguishing hybrid rice parental lines and their utilization in seed purity assessment

With the objective of identifying SSR markers that can distinguish parental lines of rice hybrids, we characterized 10 each of cytoplasmic male sterile (CMS) and restorer (R) lines along with 10 popular Indian rice varieties using a set of 48 hyperpolymorphic SSRs distributed uniformly across the rice genome. All the SSR markers were polymorphic, amplifying a total of 163 alleles, with an average of 3.36 ± 1.3 allelic variants per locus. Twenty-seven SSR markers showed amplification of an allele, which was very specific and unique to a particular parental line and not amplified in any other rice genotype tested. Through multiplex PCR, SSR marker combinations that were unique to a particular parental line or hybrid were also identified. With a set of 10 SSR markers, all the public bred Indian rice hybrids along with their parental lines could be clearly distinguished. To utilize these SSR markers effectively for detection of impurities in parental lines, a two dimensional bulked DNA sampling strategy involving a 20 × 20 grow-out matrix has been designed and used for detection of contaminants in a seed-lot of the popular CMS line IR58025A. We have also designed a multiplex PCR strategy involving single tube analysis using 2–3 markers for hybrid seed purity assessments and demonstrate its superiority over single marker analysis in accurate detection of impurities in hybrids. Implications of parental and hybrid specific SSR markers and strategies to utilize the informative SSR markers for detection of contaminants in a cost effective manner are discussed.     

微卫星分子标记鉴定黑龙江主要水稻品种及遗传多样性的研究

选用分布于水稻12条染色体上的75对SSR引物,对25个水稻品种进行DNA扩增,筛选到特异扩增条带的SSR引物25对。共检测出71条多态性条带,用UPGMA聚类分析结果分析,遗传相似性系数为0.54处可分成2类,在第一类中遗传相似系数在0.73~0.94之间,除了松粳3号和龙粳14不能区分之外,其它品种均能区别,表明近缘品种之间的检出率较低。     

东乡野生稻与日本晴多态性标记的开发

东乡野生稻是分布于全球最北端的一种普通野生稻,与亚洲栽培稻基因组差异较大,目前缺少覆盖其全基因组的分子标记。本文以东乡野生稻和日本晴为材料,通过筛选已有的1017个标记,并利用东乡野生稻基因组重测序信息设计的217个InDel标记,共检测出203个标记在东乡野生稻与日本晴间呈现多态性。这些标记均匀分布于12条染色体,平均间隔1.9 Mb,基本覆盖东乡野生稻全基因组区域。通过对籼粳亚种的检验分析,发现该套多态性分子标记在东乡野生稻与粳稻杂交后代群体基因型分析上具有较高的应用价值。本研究结果为发掘东乡野生稻的有利基因以及分子标记辅助选择育种提供了有力的工具。     

玉米和高粱SSR标记在薏苡中的通用性研究

旨在分析玉米和高粱SSR标记在其近缘种薏苡中的通用性和多态性,以期筛选可用于薏苡种质资源和遗传学研究的分子标记。以11份薏苡资源为供试材料,利用SSR-PCR扩增和毛细管电泳检测方法,对364对SSR引物进行了测试和筛选。结果表明,364对玉米和高粱来源的标记中有163对能在薏苡中扩增并得到清晰的条带,其中44对标记在薏苡中表现出良好的多态性。高粱SSR的通用性和多态性比例分别为58.02%和30.85%,玉米SSR标记的通用性和多态性比例分别为23.45%和26.47%,高粱SSR标记在薏苡中通用性和多态性更好。44对引物在11份薏苡材料中扩增出110条多态性条带,每对引物扩增出的条带数在1～5条之间,平均为2.5条,PIC为0.15～0.79,其中15对引物的PIC值大于0.5,占全部多态性引物的34.09%。筛选出的通用SSR引物可为薏苡种质资源多样性和分子遗传学研究提供可用的遗传标记,也是高粱、玉米和薏苡3种作物之间比较基因组学研究的有力工具。     

茄子SSR标记在番茄上的通用性分析

微卫星标记(SSR)是分子育种中常用的遗传标记之一,但目前报道的番茄SSR标记并不能满足番茄分子育种的需要,有必要发展出更多的SSR标记,近缘物种转移法是一种发展SSR标记简便快速的方法。本研究分析了茄子SSR标记在番茄上的通用性情况,结果表明:300对茄子SSR引物中有111对能在番茄基因组DNA上扩增出产物,97对引物扩增出的带型在茄子、番茄间相似程度高,标记通用率为32.3%;EST-SSR比基因组SSR的通用性更好,前者通用率为36.6%,后者为30.0%。将这些标记转移到番茄上,能节省开发番茄SSR标记的成本。     

应用RSAP、SRAP和SSR分析苎麻种质亲缘关系

分别采用RSAP、SRAP和SSR 3种分子标记和田间性状对16份苎麻种质进行亲缘关系聚类,结果表明, 每对引物扩增出的多态性位点,SRAP标记最多,RSAP次之,SSR较少;根据SRAP标记和RSAP标记分类的结果都与RSAP+SRAP+SSR联合分类的结果基本一致,相关达到了极显著水平,而与SSR标记分类结果差异较大;分子标记聚类结果与田间性状的聚类结果接近程度, 依次为SRAP+RSAP+SSR>SRAP>RSAP>>SSR。在检测苎麻种质亲缘关系中,SRAP标记效果最优,RSAP标记稍逊,SSR标记最差。RSAP标记能较好地显示苎麻种属间的多态性和亲缘关系。以RSAP、SRAP、SSR标记联合分析,能更好地揭示种质之间的亲缘关系。     

绿豆遗传连锁图谱的整合

利用绿豆及其近缘种的701对SSR引物,对现有绿豆遗传连锁图谱进行补充,结果在高感豆象绿豆栽培种Berken和高抗豆象绿豆野生种ACC41两亲本间筛选到多态性SSR引物104对。群体分析后,结合其他分子数据,使用作图软件Mapmaker/Exp 3.0b,获得一张含有179个遗传标记和12个连锁群,总长1831.8cM、平均图距10.2cM的新遗传连锁图谱,包括97个SSR标记,91个来自绿豆近缘种;RFLP标记76个;RAPD标记4个;STS标记2个。对32个绿豆、小豆共用SSR标记在遗传连锁图谱的分布分析发现,二个基因组间有一定程度的同源性,共用标记在连锁群上的排列顺序基本上一致,只有部分标记显示绿豆和小豆基因组在进化过程中发生了染色体重排;利用新图谱对ACC41的抗绿豆象主效基因重新定位,仍定位于I(9)连锁群,与其相邻分子标记的距离均小于8cM,其中与右翼SSR标记C220的距离约2.7cM。与原图谱比较,新定位的抗性基因与其相邻标记的连锁更加紧密。     

水稻功能性插入缺失标记（InDel）的筛选与应用

开展水稻品种纯度和真实性鉴定对水稻遗传育种和种子生产有着重要的意义。以245个长江中下游主推水稻品种为研究材料,从643个插入缺失（InDel）位点中筛选出124个功能性多态性位点,建立了InDel高效检测体系。进一步利用InDel标记vf0121641804和SSR标记RM7120分析水稻样品的纯度,同时选用覆盖12条染色体的24对InDel引物和48对SSR标准引物分析水稻品种真实性,结果表明,采用InDel标记检测水稻品种纯度和真实性的结果与采用SSR标记检测结果一致,说明水稻功能性InDel标记也可用于水稻品种纯度和品种真实性的鉴定。在此基础上,鉴定了245个水稻品种124个InDel标记的基因型遗传多样性并进行了聚类分析,结果表明,水稻材料间存在明显的群体结构,基本反映了不同品种间的亲缘关系。水稻功能性InDel标记的筛选与应用,不仅为品种纯度和真实性鉴定、遗传多样性分析提供了新方法;而且可用于分子辅助育种,提高强优势组合的预见性。     

Isolation and characterization of microsatellites in Bambusa arundinacea and cross species amplification in other bamboos

Isolation and characterization of microsatellites was analysed in Bambusa arundinacea and cross species amplification studied in other bamboos. Microsatellites, tandem repeats of short nucleotide (1–6 bp) sequences, are the DNA marker of choice because of their highly polymorphic, ubiquitous distribution within the genome, ease of genotyping through Polymerase chain reaction, selectively neutral, co‐dominant and multiallelic nature. Six microsatellites, three polymorphic and three monomorphic have been characterized for the first time in a bamboo species, Bambusa arundinacea belonging to the family Poaceae. The numbers of alleles per locus ranged from 2 to 13. Cross species amplification was tested in 18 other bamboo species. Monomorphic simple sequence repeats (SSRs) were found to be cross amplified in most of the species tested and polymorphic ones in only three to four species. The utility of SSR loci in a genetic diversity study of B. arundinacea and other cross‐amplified bamboo species has been discussed. This study will help in population genetic studies in bamboo species.     

水稻分蘖相关性状的QTL定位与分析

应用147个SSR标记,对水稻测序品种日本晴(Nipponbare以下简称Nipp) ×广陆矮4号(部分测序,以下简称GLA)的F2群体进行基因检测,构建了全长为1736.6cM、覆盖水稻基因组12条染色体的连锁图,采用QTLCart1.7 统计软件对水稻分蘖相关性状如分蘖数、穗数、有效分蘖数、分蘖率等4个性状的基因座位进行定位分析,检测到5个主效果基因和17个微效基因,同时结合基因组研究的最新成果,对7个与标记间遗传距离为0(QTL POS=0)的QTLs进行生物信息学的分析,尝试用QTL定位预测基因的功能。     

Molecular breeding in the genus Musa: a strong case for STMS marker technology

Musa species are among the tallest monocotyledons and include major food-producing species. The principal cultivars, derived from two major species Musa acuminata (‘A’ genome) and Musa balbisiana (‘B’ genome), are polyploid hybrids (mainly AAA, AAB and ABB triploids), medium to highly sterile, parthenocarpic and clonally propagated. Bananas and plantains are crops to which molecular breeding is expected to have a positive impact. In order to better understand banana genetics, more knowledge has to be accumulated about the complex genome structure of hybrids and cultivars. Therefore, the aim of our work is to develop molecular markers that are codominant, reliable, universal, highly polymorphic and that are applicable to collaborative Musa germplasm genotyping and mapping. Two size-selected genomic libraries have been screened for the presence of simple sequence repeats (SSR). Our data demonstrate that SSR are readily applicable to the study of Musa genetics. Our comprehensive analyses of a significant number of banana sequence tagged microsatellite sites (STMS) will add to our knowledge on the structure and phylogeny of genomes of the Musa species, and suggest that microsatellites be used as anchor markers for a banana genetic core map. Additional markers, such as e.g. CAPS have also been tested in order to increase the detection of polymorphisms exceeding that revealed by STMS technology. The utility of PCR-derived markers for collaborative genetic analyses of the banana genome, and the transferability of 'streamlined’ laboratory techniques and data analysis to Developing Countries are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

水稻ACGM遗传标记在谷子中的可转移性研究与应用

为了检验水稻基因组分子标记在谷子基因组中的可转移性及多态性,选取59份不同的谷子材料,利用基于水稻基因组数据设计的38个扩增共有序列遗传标记(ACGM)进行分析,并采用DARwin 5.0.156软件对供试材料进行了分子聚类分析。结果表明：33对引物可以在至少5份以上谷子材料中获得扩增产物,占引物总数的87%。其中,在供试材料之间有多态性的引物共7对,占引物总数的18.4%。供试材料的分子聚类结果表明,采用水稻ACGM标记进行的聚类并没有按谷子的表型数据或生态类型聚在一起,其原因尚需进一步研究。这些结果表明基于水稻基因组序列开发的ACGM标记在谷子中具有较高的可转移性,但与其他作物相比,这些ACGM标记在谷子中的多态性偏低。