Genetic dissection of root architectural traits by QTL and genome‐wide association mapping in rapeseed (Brassica napus)

Variation in root architecture is essential for the adaptation of plants to target environments. A non‐destructive gel‐based mini‐rhizotron system was used for root architecture trait phenotyping. This system has facilitated the visualization of root architectural traits in large genotype collection of rapeseed including 94 double haploid (DH) lines from “Express617” x “V8” and 439 inbred lines (ASSYST diversity set). A high‐density Express617‐V8 linkage map was used for quantitative trait loci (QTLs) identification in DH population based on standard composite interval mapping. 6K SNPs were analysed for association mapping of root traits in ASSYST diversity set. A large variation, broad segregation and medium–low heritability of root architectural traits, primary root length and growth rate, lateral root number, lateral root length and lateral root density, were observed. In the double haploid population, 11 QTL regions, and in the diversity set, 38 significant marker–trait associations were detected. Significant marker–trait associations proved that these are quantitatively inherited traits controlled by multiple genes which revealed to proceed for genetic improvement and selection of rapeseed lines with improved root system.     

QTLs conferring FOV 7 resistance detected by linkage and association mapping in Upland cotton

Fusarium wilt is a worldwide disease that affects cotton production. Molecular markers tightly linked to resistance genes can be used for marker-assisted and/or genomic selection. We performed both family-based linkage mapping and population-based association mapping (AM) to detect quantitative trait loci (QTLs) conferring resistance against Fusarium oxysporum f. sp. vasinfectum race 7 (FOV 7) in Upland cotton. To identify QTLs underlying FOV 7 resistance by linkage mapping, three Upland cotton cultivars/lines, Xuzhou 142, Yumian 21 and Shang 9901, were used to obtain the composite cross population, designated as Xuzhou 142/Yumian 21//Xuzhou 142/Shang 9901. A linkage map containing 185 simple sequence repeat loci and 40 linkage groups was constructed with an average distance of 7.5 cM between adjacent markers. Seven QTLs were detected by linkage mapping, explaining 2.9–6.6 % of the total phenotypic variance. We also performed marker–trait AM with the MLM model (Q + K) in a panel composed of 356 Upland cotton cultivars. In total, 27 loci were significantly associated with FOV 7 resistance at the α = 0.01 level (?log₁₀ P ≥ 2), which were distributed on 16 chromosomes and explained 1.48–12.99 % of phenotypic variation. Three of the 7 QTLs identified by linkage mapping could be detected in AM. We identified the favorable allele for each of the 27 associated loci and investigated the number of favorable alleles in each accession. The results should increase our understanding of the genetic basis of FOV resistance and facilitate future resistance breeding in Upland cotton.     

Population structure and linkage disequilibrium of ICRISAT foxtail millet (Setaria italica (L.) P. Beauv.) core collection

Use of diverse germplasm is a key factor which allows high level of resolution due to extensive recombination in the history. Therefore, population used in association mapping should posses as many phenotypes as possible. One of the methods to obtain most of the phenotypes is to construct the core collection. The ICRISAT foxtail millet core collection consisting of 155 accessions was genotyped using 72 simple sequence repeat (SSR) markers to investigate the genetic diversity, population structure and linkage disequilibrium (LD). A high degree of molecular diversity among the accessions was found, with an average of 16.69 alleles per locus. STRUCTURE analyses classify the accessions into four subpopulations (SP) based on SSR allelic diversity. The Neighbor joining clustering and the principal coordinate analysis were in accordance with the racial classification. The distribution of molecular genetic variation among and within the four SP and three races showed high degree of variability within each group, and low level of genetic distance (GD) among the groups. LD decay of <40 cM of GD in foxtail millet core collection was observed, which suggests that it could be possible to achieve resolution down to the 40 cM level. From this investigation, it is evident that the foxtail millet core collection developed at ICRISAT is very diverse and could be a valuable resource for trait association mapping, crop breeding and germplasm management.     

ICRISAT花生微核心种质农艺性状和黄曲霉抗性关联分析

以国际半干旱热带地区作物研究所(ICRISAT)花生微核心种质146份资源为品种,鉴定农艺性状和黄曲霉抗性,用26对SSR引物检测多态性位点,在分析连锁不平衡、群体结构和Kinship的基础上进行关联分析。连锁不平衡的分布显示R²平均值为0.185,表明26对SSR引物扩增的120个位点之间具有较低的连锁不平衡程度。群体结构分析结果将146份花生品种分为2个亚群,分别对应疏枝亚种和密枝亚种,与植物学分类和遗传分化分析的结果基本一致。关联分析表明,共有39个位点与10个农艺性状(株高、总分枝数、第一分枝数、小叶宽、结果分枝数、百果重、出仁率、单株生产力、种子长、种子宽)相关联,表型变异解释率为1.50%~20.34%,16个SSR位点与黄曲霉侵染病情指数、黄曲霉产毒量相关联,表型变异解释率为5.23%~17.19%,与农艺性状、黄曲霉抗性同时相关联的SSR位点有13个。关联位点的等位变异效应分析表明,10个农艺性状和2个黄曲霉抗性性状共有63个增效等位变异和47个减效等位变异,并发掘了ICG6022等携有优良等位变异的载体品种。     

Detection of candidate R genes and single nucleotide polymorphisms for downy mildew resistance in maize inbred lines by association analysis

Downy mildew (DM) is a plant disease that strongly limits maize production. The resistance (R) genes for this disease have been identified and characterized and serve as important tools for plant disease evaluation. In this study, partial genes for PIC15, PO145579, and zmcf5 were identified and analyzed in a panel of 60 public and private maize inbred lines using bioinformatics and statistics tools. These candidate R genes exhibited an average nucleotide diversity value of 0.015. The negative Tajima’s D values for these genes suggested that purifying selection has played a role in the evolution of maize DM resistance genes. In addition, linkage disequilibrium among these genes occurred across an extent of ~ 200 bp. Using association analysis methods, a general linear model, and a mixed linear model, five polymorphisms within the partial PIC15 and PO145579 genes were detected and associated with DM disease. These are new polymorphisms that have not previously been reported in association with DM disease traits. Therefore, correlations between these new polymorphisms and DM disease in these germplasm collections could advance the development of a functional marker for marker-assisted selection of DM disease resistance in maize breeding programs.     

Population structure and genetic diversity in elite sugar beet germplasm investigated with SSR markers

The assessment of genetic diversity and structuration of germplasm is essential for the efficient organization of breeding material. The objectives of the study were to (i) examine the population structure of elite sugar beet germplasm, (ii) investigate genetic diversity within and among subgroups of elite sugar beet germplasm, and (iii) assess the extent of linkage disequilibrium (LD) within elite sugar beet germplasm. A total of 111 and 178 inbred lines from the seed and pollen parent (SP and PP) heterotic gene pools, respectively, which had been genotyped with 23 SSR markers, were used in this study. Two distinct subgroups were detected within the entire germplasm set by STRUCTURE and principal coordinate analysis (PCoA). This observation was not expected because the SP heterotic pool of sugar beet was developed out of the PP heterotic pool in the late 1970s. Our observation of high LD in elite sugar beet germplasm suggests that association mapping will be possible in the examined germplasm set using a relatively low numbers of markers. However, to reduce the problem of false-positive marker-phenotype association, it might be necessary to examine the subgroups separately or apply association mapping methods which take into account this structure.     

Identification and validation of a core set of microsatellite markers for genetic diversity analysis in watermelon, <Emphasis Type="Italic">Citrullus lanatus</Emphasis> Thunb. Matsum. &; Nakai

Watermelon, Citrullus lanatus Thunb. Matsum. & Nakai is an important vegetable crop worldwide. Due to its narrow genetic base, detection and utilization of the genetic variations, cultivar identification and increasing genetic diversity are some important tasks for watermelon breeders. Molecular markers, especially microsatellites or simple sequence repeats (SSRs) are playing increasingly important roles for these purposes. In the present study, a core set of 23 highly informative SSR markers was developed for watermelon genetic diversity analysis. Based on whole genome sequencing of 17 watermelon inbred lines, we identified 3.9 million single nucleotide polymorphisms (SNPs) which were used to construct a SNP-based dendrogram for the 17 lines. Meanwhile, from the sequenced genome, 13,744 SSRs were developed, of which 704 were placed on a high-resolution watermelon linkage map. To develop the core set SSR markers, 78 of the 704 mapped SSRs were selected as the candidate markers. Using the SNP-based dendrogram as calibration, 23 SSR markers evenly distributed across the genome were identified as the core marker set for watermelon genetic diversity analysis. Each marker was able to detect 2–7 alleles with polymorphism information content values ranging from 0.45 to 0.82. The dendrograms of 17 watermelon lines based on SNPs, the base set of 78 SSRs and the core set of 23 SSRs were highly consistent. The utility of this core set SSRs was demonstrated in 100 commercial watermelon cultivars and elite lines, which could be placed into six clusters that were largely consistent with previous classification based on morphology and parentage data. This core set of SSR markers should be very useful for genotyping and genetic variation analysis in watermelon.     

玉米自交系遗传关系的SSR标记分析

选用系谱明确的和系谱来源复杂的38个玉米自交系为材料,在玉米基因组上均匀选取62个SSR引物进行遗传关系分析：(ⅰ)分析SSR引物在这些自交系中的差异程度;(ⅱ)进行自交系的类群划分;(ⅲ) 明确SSR标记在不同来源类型玉米自交系的类群划分和遗传关系分析上的应用价值。62对SSR引物共检测到238个等位基因变异,平均每个位点的等位基因数4.08个,平均多态性信息量(PIC)0.612,平均标记索引系数(MI)2.58,三个指标对标记多态性的分析不完全一致。UPGMA聚类分析将38个玉米自交系分为瑞德、旅大红骨、塘四平头、兰卡斯特、P1、P2和热带素湾7个类群,划群结果与系谱基本吻合,同时对系谱来源复杂的自交系进行分析,明确了它们的归属。     

栽培种花生荚果大小相关性状QTL定位

以远杂9102为母本,徐州68-4为父本杂交衍生的F5和F6共188个家系,构建了一张包含365个标记,总长度713.07 c M,标记间平均距离1.96 c M的栽培种花生遗传图谱。图谱包含22个连锁群,各连锁群平均长度12.37~81.39 c M,连锁群上标记数量3~46个。结合2013和2014年采集的荚果表型数据,采用Win QTLcart 2.5软件的复合区间作图法(composite interval mapping,CIM)进行QTL定位和效应估计。2个环境下共检测到41个QTL,其中与荚果长、宽、厚和百果重相关的QTL分别为13、7、13和8个,表型变异解释率为3.14%~18.27%。有6个QTL在2种环境下被重复检测到,其中百果重相关的2个(q HPWLG13.1、q HPWLG14.1),分布在LG13和LG14连锁群,遗传贡献率为6.95%~14.60%;与荚果长相关的3个(q LPLG2.2、q LPLG13.1、q LPLG14.1),分布在LG2、LG13和LG14连锁群,遗传贡献率为3.14%~18.27%;与荚果厚相关的1个(q TPLG3.4),分布在LG3连锁群,遗传贡献率为8.24%~9.24%。本研究涉及性状存在9个QTL热点区,每个热点区涉及2~3个性状,表型贡献率为3.57%~18.27%。     

玉米丝裂病的抗性QTL定位

选用感丝裂病的玉米自交系R08与抗丝裂病的自交系Es40组配F₂群体共348个单株,构建了包含115个SSR标记的分子遗传连锁图谱,覆盖玉米基因组2 178.6 cM,平均图距为18.9 cM。采用复合区间作图法,对F_2:4家系丝裂病数据进行抗性QTL分析,共检测到12个QTL,分别位于第1、2、4、5和7染色体,贡献率为4.22%~37.95%。其中在第1、3染色体上检测到主效QTL,贡献率均大于30%,基因作用方式均为显性,其余10个QTL的作用方式多为加性或部分显性。     

糯玉米自交系SSR标记遗传多样性及群体遗传结构分析

为了解糯玉米种质的遗传基础,利用29对SSR标记对87份糯玉米自交系进行遗传多样性分析,共检测出180个等位变异,平均每个位点6个等位变异,多态性信息含量变幅为0.308～0.915,平均为0.572。材料间遗传相似系数为0.49～0.93,平均为0.66。通过聚类分析UPGMA (unweighted pair-group method with arithmetic means)方法在遗传相似系数0.64处将87份糯玉米自交系划分为4个类群,分别包含9、66、10和2份材料。此外,利用Structure群体遗传结构分析也将87份糯玉米自交系分为4个类群,分别包含24、25、19和19份材料;进一步分析表明,供试群体中大部分糯玉米自交系的遗传变异较单一。本研究为糯玉米新品种选育和遗传进化分析提供了种质基础和理论依据。     

Molecular diversity and genome-wide linkage disequilibrium patterns in a worldwide collection of <Emphasis Type="Italic">Oryza sativa</Emphasis> and its wild relatives

Genetic diversity analysis within a species is vital for understanding evolutionary processes at the population and genomic levels. We report a detailed study of molecular diversity, polymorphism and linkage disequilibrium in three groups of rice (Oryza) germplasm accessions based on 176 SSR markers. The first group included 65 rice (O. sativa L.) accessions introduced from seven countries, including five regions of China. The second group included 58 US rice varieties released in the past 25 years. The third group consisted of 54 accessions of rice wild relatives represented by ten different species. The number of alleles per SSR marker ranged from 4 to 32 with a mean of 16 alleles and the polymorphism information content values ranged from 0.43 to 0.91 with a mean of 0.70. The variation in SSR alleles was a significant contribution to the genetic discrimination of the 177 accessions within the three Oryza groups. Analysis of molecular variance identified deviation from Hardy–Weinberg equilibrium. Principal coordinates analysis clearly separated the accessions into their respective three groups. Neighbor-joining phylogenetic cluster reflects the ordination of each accession. Linkage disequilibrium (D′) averaged 0.75 in wild Oryza spp., and about 0.5 in both US and international O. sativa accessions. Our results showed that LD among adjacent loci in both O. sativa and Oryza spp. accessions is strong enough to be detecting marker-trait association via genome-wide scans.     

Efficiency of SNP and SSR-based analysis of genetic diversity,population structure,and relationships among cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp.) germplasm from East Africa and IITA inbred lines

The extent of genetic diversity and relatedness of cowpea germplasm from East Africa are poorly understood. A set of 13 microsatellites (SSR) and 151 single nucleotide polymorphisms (SNPs) markers were applied to assess the levels of genetic diversity in a sample of 95 accessions of local cowpea germplasm and inbred lines of Vigna unguiculata. The average genetic diversity (D), as quantified by the expected heterozygosity, was higher for SSR loci (0.52) than for SNPs (0.34). The polymorphic information content was 0.48 for SSR and 0.28 for SNP while the fixation index was 0.095 for SSR and 0.15 for SNPs showing moderate differentiation and high gene flow among cowpea accessions from East African countries. The results of data analysis of both SSR and SNP markers showed similar clustering patterns suggesting a substantial degree of association between origin and genotype. Principal coordinate analysis (PCoA) with SSR and SNP markers showed that accessions were grouped into two and three broad groups across the first two axes, respectively. Our study found that SNP markers were more effective than SSR in determining the genetic relationship among East African local cowpea accessions and IITA inbred lines. Based on this analysis, five local cowpea accessions Tvu-13490, Tvu-6378, Tvu-13448, Tvu-16073, and 2305675 were identified to be tightly clustered sharing several common alleles with the drought tolerant variety Danila when analyzed with SSR and SNP markers. The findings will assist and contribute to future genetic diversity studies aimed at the genetic improvement of local Eastern Africa cowpea accessions for improved overall agronomic performance in general and breeding for drought tolerant in particular.     

Genetic diversity of maize inbred lines in relation to downy mildew

A major emphasis in maize breeding in Asian countries has been the improvement for resistance to downy mildew, a serious disease that causes significant yield losses. A total of 102 inbred lines, including lines from Asian breeding programs, Mexico, USA and Germany, were analyzed with 76 SSR markers to measure diversity and investigate the effect of selection for downy mildew resistance. A mean polymorphism information content of 0.59, with a range of 0.14 to 0.83, was observed. Diversity at the gene level showed an average of 5.4 alleles per locus and a range of two to 16 alleles per locus, with a total of 409 alleles. About half of the alleles in the Asian lines had frequencies of 0.10 or less, and only 2% had frequencies > 0.80, indicating the presence of many alleles, and thus a high level of diversity. Some of the high-frequency alleles were in chromosomal regions associated with disease resistance. However, the frequencies of alleles in three SSR loci that are linked to a QTL for resistance to downy mildews in Asia were not significantly different in the subtropical/tropical Asian lines as compared to all the lines in the study. Lines from the US, Germany, and China, comprised three clusters of temperate maize(GS = 0.31), while those from India, Indonesia, Philippines, Thailand, Vietnam and CIMMYT comprised seven indistinct clusters of subtropical and subtropical maize (GS = 0.29). We conclude that maize breeding activity in Asia has not caused a decline in the overall amount of diversity in the region. This revised version was published online in July 2006 with corrections to the Cover Date.     

黄瓜种子长度的遗传分析及分子标记

为了今后正确制定籽用黄瓜育种方案和有效地进行品种选育,有必要对黄瓜种子大小进行详细研究。选用种子长短不同的5个黄瓜自交系作为杂交亲本,按照Griffing双列杂交试验方法Ⅱ配制杂交组合。以长种子自交系D06157和短种子自交系D0603为亲本,构建F2分离群体,采用SSR分子标记构建遗传连锁图谱,利用复合区间定位方法进行QTL定位。结果表明：黄瓜种子长度属于数量性状,以显性效应为主,同时存在加性效应,广义遗传力及狭义遗传力较低,分别为28.6777%、10.8384%。构建的4个连锁群含有16个SSR位点,连锁群总长度为287.1 cM,标记间平均距离为17.94 cM。检测出6个与黄瓜种子长度相关的QTL,其中QTL5和QTL6有着较高的对种子长度性状变异的解释率(>5%),距离最近标记的图距分别为2.1 cM、4.3 cM,LOD值分别为7.84、9.69,可解释遗传变异分别为6.07%、6.08%,加性效应值分别为39.12%、37.21%。     

甘肃部分玉米自交系SSR遗传多样性分析

分析甘肃省部分骨干玉米自交系的遗传多样性并划分群体结构。利用全自动DNA分析仪荧光SSR-PCR技术和66 对核心SSR引物研究了148 份玉米(Zea mays L.)自交系的遗传多样性,并分别使用模型结构聚类和遗传距离聚类法进行群体结构分析。在148 份自交系中,66 对SSR标记共检出279 个等位变异,每对引物检测到等位基因2~8 个,平均4.2273 个。多态性信息量(PIC)和标记指数(MI)的平均值分别为0.4879 和2.1808。模型结构聚类将148 份自交系分为SS 群和NSS群;遗传距离聚类分为5 个类群,根据常规测验种自交系可将5 个类群归并为SS 群和NSS群。2 种聚类方法所得结果与材料的系谱信息基本一致。甘肃省部分玉米自交系在育种实践中正在向2 个相对独立的优势类群转化,2 个类群之间的遗传多样性水平存在一定的差异。     

Identification of QTLs Related to Verticillium Wilt Resistance Based on RIL Population

[Objective] The objective of this study was to identify the stable quantitative trait loci (QTLs) related to Verticillium wilt resistance in cotton. [Method] In this study, a population of 111 recombinant inbred lines (RILs) strains was developed by crossing a highly resistant parental line "Changkangmian" to Verticillium wilt and the susceptible parent TM-1. The complete composite interval mapping method was adopted to detect QTLs by Verticillium wilt disease index in multiple environmental conditions and periods in Anyang and Verticillium wilt affected areas of Xinjiang. Simple sequence repeat (SSR) markers of polymorphism were screened for genetic mapping. [Result] The genetic map was constructed by 40 simple sequence repeat (SSR) markers, consisted of 12 linkage groups with total length of 212.5 centimorgan (cM). A total of six QTLs related to the resistance to Verticillium wilt were obtained. The likelihood of odd (LOD) values ranged from 2.51 to 5.55. The maximum phenotypic variation explained (PVE) 20.34%, and the minimum PVE 6.93% were recorded. Among detected QTLs, qVR-D05-1 was detected in both Verticillium wilt affected fields in Anyang and Xinjiang in July 2015 and July 2016 with PVE of 12.96% and 20.34%, respectively. [Conclusion] This study can provide a potential reference for mapping stable QTLs related to resistance to Verticillium wilt.     

Developing new markers and QTL mapping for greenbug resistance in sorghum [Sorghum bicolor (L.) Moench]

Greenbug is a major damaging insect to sorghum production in the United States. Among various virulent greenbug biotypes, biotype I is the most predominant and severe for sorghum. To combat with the damaging pest, greenbug resistant sources were obtained from screening sorghum germplasm collection. This experiment was conducted to identify the genomic regions contributing resistance to greenbug biotype I in a sorghum accession, PI 607900. An F₂ mapping population consisting of 371 individuals developed from a cross of the resistant line with an elite cultivar, BTx623 (susceptible) were tested and scored for their response to greenbug feeding in the greenhouse. Significant differences in resistance were observed between the two parental lines and among their F₂ progeny in response to greenbug feeding at 7, 10, 14 and 21 days after infestation. A linkage map spanning a total length of 729.5 cM across the genome was constructed with 102 polymorphic SSR markers (69 genomic and 33 EST SSRs). Of those microsatellite markers, 48 were newly developed during this study, which are a useful addition for sorghum genotyping and genome mapping. Single marker analysis revealed 29 markers to be significantly associated with the plant response to greenbug feeding damage. The results from interval mapping, composite interval mapping and multiple interval mapping analyses identified four major QTLs for greenbug resistance on chromosome 9. These QTLs collectively accounted for 34–82 % of the phenotypic variance in greenbug resistance. Minor QTLs located on chromosome 3 explained 1 % of the phenotypic variance in greenbug resistance. The major allele for greenbug resistance was on chromosome 9 close to receptor-like kinase Xa21-binding protein 3. These markers are useful to screen more resistant genotypes. Furthermore, the markers tagged to QTL regions can be used to enhance the sorghum breeding program for greenbug resistance through marker-assisted selection and map-based cloning.     

Evidence suggests that Mycosphaerella pinodes infection of Pisum sativum is inherited as a quantitative trait

Before likely sources of resistance in pea to Mycosphaerella pinodes can be exploited in a breeding program, the inheritance of genetic mechanisms governing the variation in resistance need to be understood. Disease responses were first examined in a diallel cross of nine Pisum sativum genotypes. An analysis of variance of parental and F₁ data detected significant additive and dominance variation but no reciprocal effects. Gene interactions were not significant in leaves with the exception of the line JI 252 and there was no association detected between the frequencies of dominant or recessive alleles and parental disease response. In stems, the slope of the regression line of the covariance if array members with their non recurrent parent on the variance of an array was significantly different from one and the additive-dominance model was therefore rejected. The non-significance of interactions among genes determining disease response in leaves was supported by the triple test cross analyses. These analyses showed that the narrow-sense heritability of disease response was higher in crosses involving SA 1160 than in the cross Wirrega × Austrian Winter. These crosses are likely to generate inbred lines more resistant to M. pinodes infection than either parent. This revised version was published online in July 2006 with corrections to the Cover Date.     

玉米分子遗传框架图谱构建

以48-2×5003的166个F2单株为作图群体,利用135个RFLP探针和131对SSR引物对亲本48-2、5003之间的多态性进行了检测,筛选出109个RFLP多态性探针和81对SSR多态性引物用于F2群体分析,利用上述109个RFLP标记和81个SSR标记,构建了具190个RFLP、SSR标记199个标记位点的玉米分子遗传图谱,覆盖整个基因组2984.1 cM,标记间平均间