高粱SSR和EST-SSR标记在割手密中的通用性分析

目前, 割手密中开发的分子标记有限, 为增加其分子标记数量, 本研究对高粱分子标记在割手密中的通用性进行分析。选用高粱的 29对基因组 SSR标记和 20对 EST-SSR标记在割手密种质 GSM39中进行筛选, 以评价其通用性。进一步利用 14份割手密材料评价标记的多态性和遗传多样性。结果显示： 70%的 EST-SSR引物在割手密中得到成功扩增, 可用的 EST-SSR引物中多态性比率占 50%。SSR引物在割手密中的通用性比率只有 34.5%, 但多态性比率为 70%。14对多态性引物在 14份割手密中共检测到33个等位基因, 平均观测等位基因数为2.4286, 平均有效等位基因数为1.7, 平均观测表观杂合度为0.544, 平均期望杂合度为 0.3858, Nei’ s基因多样性指数平均值为 0.3716。结果表明, EST-SSR引物的通用性高于 SSR引物, 但 SSR引物的多态性更高。这对割手密遗传多样性研究和比较基因组学研究具有重要意义。     

Genetic diversity,structure and fruit trait associations in Greek sweet cherry cultivars using microsatellite based (SSR/ISSR) and morpho-physiological markers

It is important to couple phenotypic analysis with genetic diversity for germplasm conservation in gene bank collections. The use of molecular markers supports the study of genetic marker-trait associations of biological and agronomic interest on diverse genetic material. In this report, 19 Greek traditional sweet cherry cultivars and two international cultivars, which were used as controls, were grown in Greece and characterized for 17 morpho-physiological traits, 15 simple sequence repeat (SSR) loci and 10 inter simple sequence repeat (ISSR) markers. To our knowledge, this is the first report on molecular genetic diversity studies in sweet cherry in Greece. Principal component analysis (PCA) of nine qualitative and eight quantitative morphological parameters explain over 77.33% of total variability in the first five axes. The SSR markers yielded a combined matching probability ratio (MPR) of 9.569 × e−12. The 15 SSR loci produced a total of 92 alleles. Ten ISSR primers generated 91 bands, with an average of 9.1 bands per primer. Expected heterozygosity (gene diversity) values of 15 SSR loci and 10 ISSR markers averaged at 0.683 and 0.369, respectively. Based on stepwise multiple regression analysis (MRA), SSR alleles were found associated with harvest time and fruit polar diameter. Furthermore, three ISSR markers were correlated with fruit harvest and soluble solids and four ISSR markers were correlated with fruit skin color. Stepwise MRA identified six SSR alleles associated with harvest time with a high correlation (P < 0.001), with linear associations with high F values. Hence, data analyzed by the use of MRA could be useful in marker-assisted breeding programs when no other genetic information is available.     

西瓜核心种质遗传多样性分析及指纹图谱构建

深入了解西瓜核心种质资源的群体结构及遗传多样性,可以准确指导亲本间杂交组合的有效配置,避免具有相似遗传背景材料的组合,以便提高育种效率。据此,本研究通过采用45对具有多态性的SSR标记对不同来源的78份西瓜核心种质进行了多元统计分析。结果表明,45个SSR标记共检测出175个位点,其中具有多态性位点数为165个,多态性比率达95.41%,且每对引物平均检测到等位基因位点3.67个,PIC大小范围在0.049~0.781之间,平均值为0.415,其有效等位基因数(Ne)、期望杂合度(He)、观测杂合度(Ho)、多样性指数(I)及Nei's基因多样性指数(H)在该群体水平上分别为2.258、0.479、0.521、0.850和0.476,同时筛选出了28对多态性高、带型稳定、易于统计的核心引物,构建了78份西瓜核心种质的SSR指纹图谱。群体结构分析表明,78份西瓜核心种质资源划分为2大类群和4个亚群,其中来自不同地域的美国材料和日本材料分别独立趋于2大类群中,而中国种质材料普遍交叉分布于2大类群中,且各亚群之间存在着明显的基因交流,分子方差分析表明种质间遗传多样性主要存在于品种间和居群间。     

Mining of favorable marker alleles for flag leaf inclination in some rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) accessions by association mapping

Flag leaf angle (FLA) in rice (Oryza sativa L.) is one of the important traits affecting F₁ seed production by mechanization. To elucidate the genetic mechanism of FLA and mine favorable marker alleles for F₁ seed production in rice, we performed a genome-wide association study using phenotypic data over 2 years and genotypic data of 262 pairs of simple sequence repeat (SSR) markers collected from 441 rice accessions. We detected seven SSR marker loci associated with FLA and four loci were novel. The four newly found loci were RM6266 on chromosome 3, RM348 on chromosome 4, RM258 on chromosome 10 and RM7303 on chromosome 11. We found a total of 27 favorable alleles, of which four, i.e., RM348-130 bp, RM7303-90 bp, RM258-180 bp, and RM4835-230 bp, had phenotypic effects larger than 10°. Nine combinations, which increased FLA by 45.7°–94.7° through pyramiding the favorable alleles contained in seven typical accessions, were predicted.     

Genetic Diversity of Farmers’Preferred Sorghum Accessions and Improved Lines from ICRISAT Reveal a Disconnect Between Innovation and Technology Transfer

The genetic diversity of 65 accessions of sorghum [Sorghum bicolor (L.) Moench] collected from various farmers and germplasm lines from ICRISAT-Kenya were analyzed. Simple sequence repeats (SSR) markers were used in order to determine the extent and distribution of its genetic diversity. Twenty-nine (29) SSRs markers were polymorphic and a total of 192 alleles were detected which showed diversity. The number of alleles per primer ranged from 2 to 17, with an average of 6.62. The range of polymorphism information content (PIC) ranged from 0.03 to 0.86, with total average of 0.82. According to the results analyzed, estimates of the mean allelic pattern across the two populations was generated; expected heterozygosity (He; 0.45, 0.54), average observed alleles (Na; 3.40, 6.20), number of private allele (0.23, 3.03), and Shannon information index (I; 0.85, 1.13) for farmer and ICRISAT-Kenya germplasm, respectively. The expected heterozygosity (He) varied from 0 to 0.26 with an average of 0.05. The Neighbor-joining phenogram based on Nei’s genetic distance grouped the 65 accessions into three main groups. The analysis of molecular variance (AMOVA) revealed that 99% of the total genetic variation was within accessions in a population whereas the genetic variation among populations in accessions accounted for 1% of the total genetic variation. Genetic diversity in ICRISAT sorghum material compared to the farmer’s collection suggested little infiltration of improved germplasm to the farmers.     

Construction of an integrated genetic map based on maternal and paternal lineages of tea (Camellia sinensis)

Genetic study on important traits of tea is difficult because of its self-incompatibility in nature. Moreover, development of a new variety usually needs more than 20 years, since it takes many years from seedling to matured plants for trait investigation. Genetic map is an essential tool for genetic study and breeding. In this study, we have developed an integrated genetic map of tea (Camellia sinensis) using a segregating F1 population derived from a cross between two commercial cultivars (‘TTES 19’ and ‘TTES 8’). A total of 574 polymorphic markers (including SSR, CAPS, STS, AFLP, ISSR and RAPD), 69 markers with highly significant levels of segregation distortion (P < 0.001) (12.0 %) were excluded from further analyses. Of the 505 mapped markers, there were 265 paternal markers (52.5 %), 163 maternal markers (32.3 %), 65 doubly heterozygous dominant markers (12.9 %), and 12 co-dominant markers (2.4 %). The co-dominant markers and doubly heterozygous dominant markers were used as bridge loci for the integration of the paternal and maternal maps. The integrated map comprised 367 linked markers, including 36 SSR, 3 CAPS, 1 STS, 250 AFLP, 13 ISSR and 64 RAPD that were assigned to 18 linkage groups. The linkage groups represented a total map length of 4482.9 cM with a map density of 12.2 cM. This genetic map has the highest genetic coverage so far, which could be applied to comparative mapping, QTL mapping and marker assisted selection in the future.     

基于SSR标记的80份烟草种质指纹图谱的构建及遗传多样性分析

利用均匀分布于烟草24个连锁群上的48个SSR标记对80份烟草材料进行分析。结果显示,48个SSR标记共扩增出211个等位基因,平均每个标记4.396个,Shannon′s信息指数I为1.034,多态信息含量值为0.229~0.905,观测杂合度（H_o）、期望杂合度（H_e）和Nei′s多样性指数（H）平均值分别为0.320、0.572、0.431。聚类结果表明,在遗传距离为0.68时可以将80份烟草种质分为2个类群。5个烟草居群间的遗传一致度在0.643~0.765范围内,遗传距离分布在0.268~0.442。筛选4对核心引物构建了不同烟草种质资源的数字指纹图谱, 可将这80份烟草种质资源全部区分开。本研究在分子水平上为筛选优质烟草种质资源、挖掘重要基因以及拓宽烟草育种遗传基础等工作提供科学依据。     

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。     

Analysis of population structure and genetic diversity reveals gene flow and geographic patterns in cultivated rice (<Emphasis Type="Italic">O. sativa</Emphasis> and <Emphasis Type="Italic">O. glaberrima</Emphasis>) in West Africa

To fully exploit the diversity in African rice germplasm and to broaden the gene pool reliable information on the population genetic diversity and phenotypic characteristics is a prerequisite. In this paper, the population structure and genetic diversity of 42 cultivated African rice (Oryza spp.) accessions originating from West Africa (Benin, Mali and Nigeria, Liberia etc.) were investigated using 20 simple sequence repeats (SSR) and 77 amplified fragment length polymorphisms (AFLP). Additionally, field trials were set up to gain insight into phenotypic characteristics that differentiate the genetic populations among rice accessions. The analysis revealed considerably high polymorphisms for SSR markers (PIC mean?=?0.78) in the germplasm studied. A significant association was found between AFLP markers and geographic origin of rice accessions (R?=?0.72). Germplasm structure showed that Oryza sativa accessions were not totally isolated from Oryza glaberrima accessions. The results allowed identification of five O. glaberrima accessions which grouped together with O. sativa accessions, sharing common alleles of 18 loci out of the 20 SSR markers analyzed. Population structure analysis revealed existence of a gene flow between O. sativa and O. glaberrima rice accessions which can be used to combine several interesting traits in breeding programs. Further studies are needed to clarify the contributions of this gene flow to valuable traits such as abiotic and biotic stresses including disease resistance.     

Transferability of non-genic microsatellite and gene-based sunflower markers to safflower

Safflower (Carthamus tinctorius L.) DNA marker resources are currently very limited. The objective of this study was to determine the feasibility of transferring non-genic microsatellite (SSR) markers and gene-based markers, including intron fragment length polymorphism (IFLP) and resistance gene candidates (RGC)-based markers from sunflower (Helianthus annuus L.) to safflower, both species belonging to the Asteraceae family. Cross-species amplification of 119 non-genic SSRs, 48 IFLPs, and 19 RGC-based sunflower markers in 22 lines and germplasm accessions of safflower was evaluated. Additionally, 69 EST-SSR markers previously reported to amplify in safflower were tested. The results showed that 17.6% of the non-genic SSR, 56.2% of the IFLP, and 73.7% of the RGC-based markers were transferable to safflower. The percentage of transferable markers showing polymorphic loci was 66.6% for non-genic SSR, 70.6% for EST-SSR, 55.5% for IFLP, and 71.4% for RGC-based markers. The highest polymorphism levels were found for non-genic SSR. The average number of alleles per polymorphic locus and mean heterozygosity values were 2.9 and 0.46, respectively, for non-genic SSR, 2.2 and 0.35 for EST-SSR, 2.1 and 0.24 for IFLP, and 2.0 and 0.34 for RGC-based markers. The results of this study revealed a low rate of transferability for non-genic SSR sunflower markers and a better rate of transferability for IFLP and RGC-based markers. Transferable genic and non-genic sunflower markers can have utility for trait and comparative mapping studies in safflower.     

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.     

Expressed sequence tags from organ-specific cDNA libraries of tea (Camellia sinensis) and polymorphisms and transferability of EST-SSRs across Camellia species

Tea is one of the most popular beverages in the world and the tea plant, Camellia sinensis (L.) O. Kuntze, is an important crop in many countries. To increase the amount of genomic information available for C. sinensis, we constructed seven cDNA libraries from various organs and used these to generate expressed sequence tags (ESTs). A total of 17,458 ESTs were generated and assembled into 5,262 unigenes. About 50% of the unigenes were assigned annotations by Gene Ontology. Some were homologous to genes involved in important biological processes, such as nitrogen assimilation, aluminum response, and biosynthesis of caffeine and catechins. Digital northern analysis showed that 67 unigenes were expressed differentially among the seven organs. Simple sequence repeat (SSR) motif searches among the unigenes identified 1,835 unigenes (34.9%) harboring SSR motifs of more than six repeat units. A subset of 100 EST-SSR primer sets was tested for amplification and polymorphism in 16 tea accessions. Seventy-one primer sets successfully amplified EST-SSRs and 70 EST-SSR loci were polymorphic. Furthermore, these 70 EST-SSR markers were transferable to 14 other Camellia species. The ESTs and EST-SSR markers will enhance the study of important traits and the molecular genetics of tea plants and other Camellia species.     

21种不同类型葡萄种质资源遗传多样性的SSR分析

本研究通过SSR标记解析不同类型葡萄种质遗传多样性差异和亲缘关系,为资源分类与品种鉴定提供理论依据。利用15对SSR引物对21种不同类型葡萄种质进行PCR扩增,对扩增情况、遗传距离与亲缘关系等进行分析。15对SSR引物共扩增出等位基因位点91个,其中多态性位点89个,多态性比率高达97.8%,其中13对引物扩增多态性百分率达到100%。21份供试材料间遗传相似系数变化范围在0.45~0.91之间,并在遗传相似系数0.62处被分为2个类群。研究表明SSR标记从分子水平上解析了不同类型葡萄种质的遗传差异性,揭示了材料间亲缘关系,为育种研究提供了一定参考依据。     

基于SSR分子标记的闽楠(Phoebe bournei)核心种质的构建

为更好地发掘和利用现有闽楠种质资源,本研究利用7个SSR位点对江西和福建16个闽楠群体的237份材料进行基因型分析,采用逐步聚类(随机取样策略,位点优先取样策略)和模拟退火算法(等位基因数目最大化策略,遗传距离最大化策略)4种取样方法构建闽楠核心种质,并将各遗传多样性指标进行分析。结果表明:7个SSR位点共检测到50个等位基因,平均为7.143,平均有效等位基因为2.115,Shannon信息指数为0.778,观测杂合度为0.302,期望杂合度为0.442。基于逐步聚类方法构建的核心种质相较于基于模拟退火算法构建的核心种质各遗传参数指标都相对较高。对其进行t检验后,选择以基于逐步聚类位点优先取样策略在25%的取样比例下选取的种质为核心种质,其等位基因数、平均有效等位基因数、多态性位点百分率、Shannon多样性指数的保留率分别为初始种质的98%、104.92%、90.09%、97.94%。筛选出的59份核心种质材料能够较好地代表闽楠种质资源的遗传多样性,为闽楠的种质资源保存提供科学依据。     

Association analysis of physicochemical traits on eating quality in rice (Oryza sativa L.)

Improvement of rice eating quality is an important objective in current breeding programs. In this study, 130 rice accessions of diverse origin were genotyped using 170 SSR markers to identify marker–trait associations with physicochemical traits on eating quality. Analysis of population structure revealed four subgroups in the population. Linkage disequilibrium (LD) patterns and distributions are of fundamental importance for genome-wide mapping associations. The mean r ² value for all intrachromosomal loci pairs was 0.0940. LD between linked markers decreased with distance. Marker–trait associations were investigated using the unified mixed-model approach, considering both population structure (Q) and kinship (K). In total, 101 marker–trait associations (p < 0.05) were identified using 52 different SSR markers covering 12 chromosomes. The results suggest that association mapping in rice is a viable alternative to quantitative trait loci mapping, and detection of new marker–trait associations associated with rice eating quality will also provide important information for marker-assisted breeding and functional analysis of rice grain quality.     

用于中国小豆种质资源遗传多样性分析SSR分子标记筛选及应用

以375份小豆核心种质为试验材料, 利用从小豆及其近缘种SSR引物中筛选出的13对引物进行遗传多样性分析。检测结果显示, 小豆种质资源具有丰富的遗传多样性, 共检测到133个等位变异, 每对SSR引物检测到等位变异4~19个, 平均10.23个, 国内各省多态信息含量(PIC)平均为0.561, 多态位点比例(P)平均为93.523%。聚类结果表明, 小豆资源遗传关系与生态分区间有明显的联系, 且东北地区资源与中南部资源遗传关系较近。湖北、安徽、陕西3省资源的PIC较高, 且基本位于主坐标三维图的中心区域, 推断湖北、安徽、陕西是中国栽培小豆的起源地或多样性中心。该结果有助于更好地对小豆种质资源进行收集、保护和利用。