Evaluation of newly developed SSR markers and identification of quantitative trait loci for bast fibre cellulose in white jute (Corchorus capsularis)

Cellulose is one of the main chemical component of bast fibre in jute. However, quantitative trait loci (QTL) for bast fibre cellulose content remains elusive. In this study, we identified 846 new SSR markers from 70,792 unigenes in the NCBI and validated them in a panel of 24 diverse jute accessions. Of 846 SSRs, 748 (88.41%) were successfully amplified, and 585 (69.14%) showed polymorphisms, implying that these are high‐quality SSRs. Furthermore, 585 SSRs along with 5,074 polymorphic SLAF (specific locus amplified fragment) and 173 InDel markers were used to reconstruct a high‐dense linkage map in a recombinant inbred population with 104 F₈ lines. Totally, 835 markers were successfully mapped to a whole length of 604.5 cM with a mean distance of 2.84 cM between adjacent markers. Furthermore, five QTLs for bast fibre cellulose were identified. One major QTL (qBFC1‐1) was stable in 2 years and explained average phenotypic variance with 14.34%. These results may be useful for developing enhanced bast fibre quality in white jute through marker‐assisted selection (MAS) breeding.     

黄麻全基因组SSR鉴定与特征分析

黄麻是世界上重要的天然韧皮部纤维作物之一。然而, SSR标记的缺乏限制了黄麻的遗传改良。本研究从圆果种黄麻测序品种CVL-1的基因组、基因、CDS和cDNA中挖掘SSR信息,利用SSR Primer软件查找SSR位点,并分析其分布特征。结果表明,基于基因组序列共开发了153,242个基因组SSR,平均密度为467.20个SSRMb~(–1);基于cDNA序列开发了10,747个SSR,平均密度为260.85 SSR Mb~(–1)。大部分重复基元为二至四核苷酸,占76.91%,其中cDNA序列SSR中三核苷酸重复基元数量较多而基因组SSR中二核苷酸重复基元数量较多。对于不同类型的SSR重复基元,随着重复单元数量的增加,其基因组和cDNA的SSR分布频率呈现逐步降低特征。黄麻全基因组SSR标记鉴定,不仅可以丰富黄麻分子标记的数量,而且为剖析黄麻重要农艺性状的遗传机制奠定基础。     

Development of EST‐SSR markers for diversity and breeding studies in opium poppy

All publicly available opium poppy expressed sequence tag (EST) sequences, totalling 20 885, were assembled into unigenes and examined for simple sequence repeats (SSRs). Nearly 19% of the 14 957 unigenes contained SSRs with 4% harbouring more than one SSR. Average density of the SSRs was 1 SSR per 3.6 kb of non‐redundant EST sequence. Trinucleotide SSRs were most frequently identified (39%), and many of the most prevalent motifs were AT‐rich. Flanking primers were designed for 86% of the SSRs and 67 primer pairs were tested on 37 opium poppy accessions and seven related species. All markers were transferable to the related species. Polymorphism information content (PIC) values for the markers were intermediate for comparisons within opium poppy (average of 0.27) and slightly higher for comparisons across species (average of 0.29). The markers were found to be useful for diversity analysis as they successfully distinguished among Turkish opium poppy accessions and land races.     

基于转录组测序的槜李EST-SSR标记开发

为开发槜李EST-SSR标记,本研究利用MISA软件筛选了槜李花转录组测序获得的35584条Unigenes,对其SSR信息进行分析后,利用Primer Premier 3.0软件设计EST-SSR引物,并随机选取40对SSR引物对12个李品种进行EST-SSR引物筛选及多态性分析。结果发现,在槜李花转录组中共搜索到个10791个SSR位点,分布于8433条Unigenes,SSR发生频率为23.70%,平均每3.71 kb含有1个SSR;SSR重复基元中二核苷酸重复出现频率最高,占总SSR数量的52.98%,其次为三核苷酸重复(占24.00%)和单核苷酸重复(占20.95%);二核苷酸重复基元以AG/CT为主(85.95%),三核苷酸重复基元以AAG/CTT为主(31.24%)。利用Primer Premier 3.0软件共设计出9870对候选引物,随机选择40对引物对12个李品种进行SSR引物筛选及多态性分析。40对引物均能扩增出预期大小的条带,有效扩增效率为100%,40对引物中有5对引物在12个李品种中表现出多态性。本研究开发的EST-SSR标记可为李属植物遗传多样性分析提供丰富的候选标记,同时可为槜李发育相关功能基因定位、遗传图谱构建、及分子标记辅助育种等研究提供帮助。     

Dissection of a major QTL for seed colour and fibre content in Brassica napus reveals colocalization with candidate genes for phenylpropanoid biosynthesis and flavonoid deposition

A major quantitative trait locus (QTL) influencing seed fibre and colour in Brassica napus was dissected by marker saturation in a doubled haploid (DH) population from the black‐seeded oilseed rape line ‘Express 617’ crossed with a yellow‐seeded B. napus line, ‘1012–98’. The marker at the peak of a sub‐QTL with a strong effect on both seed colour and acid detergent lignin content lay only 4 kb away from a Brassica (H+)‐ATPase gene orthologous to the transparent testa gene AHA10. Near the peak of a second sub‐QTL, we mapped a copy of the key phenylpropanoid biosynthesis gene cinnamyl alcohol dehydrogenase, while another key phenylpropanoid biosynthesis gene, cinnamoyl co‐a reductase 1, was found nearby. In a cross between ‘Express 617’ and another dark‐seeded parent, ‘V8’, Bna.CCR1 was localized in silico near the peak of a corresponding seed fibre QTL, whereas in this case Bna.CAD2/CAD3 lay nearby. Re‐sequencing of the two phenylpropanoid genes via next‐generation amplicon sequencing revealed intragenic rearrangements and functionally relevant allelic variation in the three parents.     

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.     

Genetic diversity in groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) genotypes and detection of marker trait associations for plant habit and seed size using genomic and genic SSRs

Groundnut (Arachis hypogaea L.) an important oilseed crop in India is known to have narrow genetic base. Therefore, the assessment of genetic diversity and detection of marker-trait association are important objectives for the genetic improvement of groundnut. The present study involved the development of 192 SSR markers from Arachis genomic survey sequences. From these, seven polymorphic SSRs along with 15 other genomic SSRs, 19 genic SSRs, and three STS markers were used to detect genetic diversity among 44 groundnut genotypes. These polymorphic SSR markers amplified 155 bands (76 genomic and 79 genic), of these 128 bands (67 genomic and 61 genic) were polymorphic. The genomic SSR exhibited 88.1% and genic SSRs displayed 77.2% allelic polymorphism. The polymorphic information content (PIC) of the markers ranged from 0.04 to 0.95. The pair-wise genetic similarity ranged from 24.2 to 90.7% for genomic SSR and 32.9 to 97.9% for genic SSR markers. Cluster analysis based on the pooled data from both genomic and genic SSRs revealed a dendrogram which could distinguish all the genotypes. Further, the AMOVA analysis detected 16.7% genetic variation due to differences in seed size and 13.0% due to plant habit. Based on locus-by-locus AMOVA and Kruskal-Wallis ANOVA and further confirmation by discriminant analysis and general linear model, six markers were found to be associated with plant habit and four markers with seed size.     

GenBank数据库中黄麻EST-SSR标记的开发及其通用性评价

本研究从GenBank公共数据库中下载黄麻表达序列标签838条,利用SSRPrimer软件对其进行SSR位点查找,利用Primer 3.0软件设计66对SSR引物,通过琼脂糖凝胶研究这些SSR引物的PCR扩增特点,以检测其多态性。结果表明,66对SSR引物在黄麻属6个不同类型材料的扩增中,42 (63.6%)对引物至少在2个材料之间存在多态性。(AT)n重复基元和(GC-)n丰富的三核苷酸重复基元多态性较高,可作为黄麻SSR标记引物设计的首选。黄麻EST-SSR标记开发效率较高,不仅可以丰富黄麻分子标记的数量,而且为剖析黄麻重要性状的遗传机制奠定基础,这对于黄麻的遗传基础研究具有重要应用价值。     

黄麻SSR标记与纤维产量性状的相关性

Simple sequence repeat (SSR) markers correlated to fiber yield traits would be a beneficial tool for molecular marker-assisted breeding in jute. In this study, 397 SSR markers were screened from 311 jute germplasms by the phenotype identification from 2016 to 2018 and 116 pairs of primers. The correlation analysis between SSR markers and fiber yield related traits by SPSS revealed that the range of the coefficient of variation related to 9 fiber traits was from 13.05% to 76.78%, indicating a comprehensive genetic variation. The correlation analysis of the agronomic traits showed that there were significantly correlations among these traits. The average correlation coefficient between branch height and nodes of main stem was the highest (r = 0.931), followed by the correlation coefficients (r = 0.781) of fresh bark weight per plant and fresh stem weight per plant and the coefficients (r = 0.779) of dry bark weight and fresh bark weight per plant. Analysis of variance (ANOVA) showed that the traits of main stem nodes, number of branches, plant height and branch height were relatively stable in different years with the higher broad heritability capacity. Furthermore, the SSR markers associated with fiber yield related traits were identified by Pearson correlation method. Stepwise regression analysis among each trait associated SSR markers indicated that there were six markers associated significantly with fiber yield related traits, and phenotypic variation explained by each SSR marker varied from 3.9% to 22.5%. These results will accelerate the development of molecular design breeding in jute.     

香蕉根系转录组SSR位点信息分析

分析香蕉根系转录组中的SSR位点信息，并设计SSR引物，为开发新的分子标记奠定基础。利用MISA工具对香蕉根系转录组unigene序列进行SSR检索。从25158条unigene中共发现4663个SSR，分布在3820条unigene序列中，出现频率为18.53%，平均每7.77 kb含有1个SSR位点，共有58种重复基元。香蕉根系转录组中，二、三核苷酸重复基元所占比例最大，分别为40.50%和40.63%；二者分别以AG/CT和AGG/CCT重复基元为主，分别占该重复基元的88.03%和30.83%。SSR重复次数以5~9次为主，基序长度主要分布于12~20 bp，平均长度为18.80 bp。香蕉根系转录组SSR位点频率、密度较高，类型多样，在香蕉遗传多样性研究及分子标记辅助育种中有较大应用潜能。     

Characterization of microsatellite markers,their transferability to orphan legumes and use in determination of genetic diversity among chickpea (Cicer arietinum L.) cultivars

The microsatellite or simple sequence repeat (SSR) marker is the most preferred marker because of its many desirable properties. It is important to increase the genic and genomic resources particularly in legumes because the SSR markers currently available in chickpea, pigeonpea, horsegram, blackgram, and cowpea are very limited. In the present study, 201 pairs of SSR markers comprising of 172 genic and 29 genomic SSRs were screened against 11 chickpea genotypes, among which 153 produced monomorphic and 48 produced polymorphic bands. The polymorphic information content ranged from 0.152 to 0.373 for both genic and genomic SSRs. Among the polymorphic markers, two-three alleles were detected for genic and two-four alleles for genomic SSRs. A unique banding pattern could be found for all the genotypes within 48 polymorphic SSR markers and cultivar specific markers could be identified for seed purity test. We have also studied the ability of chickpea genic and genomic SSRs to amplify distantly related but important legumes viz., horsegram, blackgram, cowpea, pigeonpea, and soybean. Out of 201 chickpea SSR primer pairs, 66.7% in blackgram, 62.2% in horsegram, 61.7% in redgram, 54.7% in cowpea, and 62.7% in soybean produced amplification. The transferability of about 60.0% of the chickpea SSRs to distantly related legumes could be considered successful. In the present study, 134, 133, 126, 124, and 110 new SSR primers for blackgram, horsegram, soybean, redgram, and cowpea pulse crops, respectively, were identified. It is an important addition to the already available genomic resources in these crops. In addition, among genic primer pairs, 12 in horsegram, three in soybean, 13 in redgram, and eight in cowpea, and among genomic primer pairs, two in horsegram and four in redgram were polymorphic even in the two-three genotypes tested indicating their potentially for application in genetic studies and mapping.     

Development of genomic simple sequence repeat markers from an enriched genomic library of grass pea (Lathyrus sativus L.)

Simple sequence repeat (SSR) or microsatellite markers are a valuable tool for several purposes such as evaluation of genetic diversity, fingerprinting, marker‐assisted selection and breeding. In this study, a SSR genomic enriched library was developed in Lathyrus sativus (grass pea) by affinity capture of restriction fragments to biotinylated microsatellite oligonucleotides. About 400 randomly selected clones were sequenced, and SSRs were present in approximately 30% of them. Clones contained 75%, 9% and 16% of simple, interrupted and compound SSRs, respectively. Of the 10 SSRs tested, 7 primer pairs produced clearly distinguishable DNA banding patterns. Successively, SSR primer pairs were successfully tested to reveal polymorphism in a set of four different grass pea germplasm accessions. The transferability of SSR markers was high among three related species of Lathyrus, namely Lathyrus cicera, Lathyrus ochrus and Lathyrus tingitanus, and the legume crop, Pisum sativum. These results indicate that the novel SSR markers are informative and will be useful and convenient for genetic analysis in grass pea and related species.     

Linkage map construction and mapping QTL for cotton fibre quality using SRAP, SSR and RAPD

Tetraploid cotton is one of the most extensively cultivated species. Two tetraploid species, Gossypium hirsutum L. and G. barbadense L., dominate the world's cotton production. To better understand the genetic basis of cotton fibre traits for the improvement of fibre quality, a genetic linkage map of tetraploid cotton was constructed using sequence‐related amplified polymorphisms (SRAPs), simple sequence repeats (SSRs) and random amplified polymorphic DNAs (RAPDs). A total of 238 SRAP primer combinations, 368 SSR primer pairs and 600 RAPD primers were used to screen polymorphisms between G. hirsutum cv. Handan208 and G. barbadense cv. Pima90 which revealed 749 polymorphic loci in total (205 SSRs, 107 RAPDs and 437 SRAPs). Sixty‐nine F₂ progeny from the interspecific cross of ‘Handan208’בPima90’ were genotyped with the 749 polymorphic markers. A total of 566 loci were assembled into 41 linkage groups with at least three loci in each group. Twenty‐eight linkage groups were assigned to corresponding chromosomes by SSR markers with known chromosome locations. The map covered 5141.8 cM with a mean interlocus space of 9.08 cM. A × test for significance of deviations from the expected ratio (1: 2: 1 or 3: 1) identified 135 loci (18.0%) with skewed segregation, most of which had an excess of maternal parental alleles. In total, 13 QTL associated with fibre traits were detected, among which two QTL were for fibre strength, four for fibre length and seven for micronaire value. These QTL were on nine linkage groups explaining 16.18‐28.92% of the trait variation. Six QTL were located in the A subgenome, six QTL in the D subgenome and one QTL in an unassigned linkage group. There were three QTL for micronaire value clustered on LG1, which would be very useful for improving this trait by molecular marker‐assisted selection.     

Development and utilization of genomic and genic microsatellite markers in Assam tea (Camellia assamica ssp. assamica) and related Camellia species

Microsatellite or simple sequence repeat (SSR) markers are valuable tools for many purposes, such as phylogenetic, fingerprinting and molecular breeding studies. However, such marker resources are unavailable in Assam tea (Camellia assamica ssp. assamica; Masters). With an objective to enrich the repertoire of microsatellite markers in traditional tea, 185 novel microsatellite (150 genomic and 35 genic) markers were identified from (GA)n‐enriched genomic libraries and public expressed sequence data in Assam tea. High‐quality 0.412‐Mb non‐redundant (NR) genomic data set derived from nucleotide sequencing of 1297 (GA)n‐enriched genomic positive clones and 2723 unigenes (1.33 Mb) predicted from 10 803 random public expressed sequence tags (ESTs) in C. assamica ssp. assamica were utilized for identification of genomic and genic microsatellite markers, respectively. The average number of alleles and polymorphic information content (PIC) recorded for the newly developed SSR markers were 6.17 and 0.398, respectively. The average observed (H_o) and expected (H_e) heterozygosity varied from 0.626 to 0.697, respectively. These markers were found to be highly transferable (74.5–100%) to cultivated (C. sinensis, C. assamica ssp. lasiocalyx) and five wild Camellia species. Genetic diversity coefficient detected a high level of divergence in 24 cultivated tea accessions (69.3%). Phylogenetic analysis revealed that major groupings were broadly in accordance with taxonomic classification of tea, and all the wild Camellia species remained as an out‐group. The high polymorphic content coupled with high rate of cross‐transferability demonstrates wider applicability of novel microsatellite markers in genotyping, genetic diversity, genome mapping and evolutionary studies in various Camellia species.     

Development and characterization of tomato SSR markers from genomic sequences of anchored BAC clones on chromosome 6

Simple sequence repeat motifs are abundant in plant genomes and are commonly used molecular markers in plant breeding. In tomato, currently available genetic maps possess a limited number of simple sequence repeat (SSR) markers that are not evenly distributed in the genome. This situation warrants the need for more SSRs in genomic regions lacking adequate markers. The objective of the study was to develop SSR markers pertaining to chromosome 6 from bacterial artificial chromosome (BAC) sequences available at Solanaceae Genomics Network. A total of 54 SSR primer pairs from 17 BAC clones on chromosome 6 were designed and validated. Polymorphism of these loci was evaluated in a panel of 16 genotypes comprising of Solanum lycopersicum and its wild relatives. Genetic diversity analysis based on these markers could distinguish genotypes at species level. Twenty-one SSR markers derived from 13 BAC clones were polymorphic between two closely related tomato accessions, West Virginia 700 and Hawaii 7996 and were mapped using a recombinant inbred line population derived from a cross between these two accessions. The markers were distributed throughout the chromosome spanning a total length of 117.6 cM following the order of the original BAC clones. A major QTL associated with resistance to bacterial wilt was mapped on chromosome 6 at similar location of the reported Bwr-6 locus. These chromosome 6-specific SSR markers developed in this study are useful tools for cultivar identification, genetic diversity analysis and genetic mapping in tomato.     

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。     

A preliminary genetic analysis of fibre traits and the use of new genomic SSRs for genetic diversity in jute

Jute is one of the most important fibre crops, which is second only to cotton in providing environment-friendly (biodegradable and renewable) ligno-cellulose fibre. In order to improve this largely neglected crop, we conducted a preliminary study involving the following: (i) analysis of nature and extent of the genetic variability for fibre yield and four other related traits in a set of 81 genotypes belonging to two commercially cultivated Corchorus species (45 genotypes of C. olitorius + 36 genotypes of C. capsularis), (ii) development and analysis of a set of simple sequence repeat (SSR) markers from C. olitorius, and (iii) use of a sub-set of SSRs for assessment of genetic diversity in the above set of 81 genotypes. The results suggested quantitative nature of fibre yield and other related traits, with a preponderance of dominance component in genetic variance. A sub-set of 45 SSRs derived from C. olitorius, when used for a study of DNA polymorphism and genetic diversity, showed high transferability of these C. olitorius SSRs to C. capsularis. The average number of alleles for individual SSRs was surprisingly low (3.04 for both species, 2.02 for C. capsularis and 2.51 for C. olitorius), and so was the average polymorphic information content (PIC; 0.23 and 0.24 in two species). In the dendrogram obtained using a similarity matrix, the 81 genotypes were grouped into three clusters, which largely corresponded to the two species, Cluster I belonging mainly to C. capsularis and the other two closely related clusters (clusters II and III) belonging to C. olitorius. It was also shown that a minimum of 15 SSRs could give the same information as 41 SSRs, thus making many SSRs redundant. The SSR markers developed during the present study and to be developed in future will prove useful not only for evaluation of genetic diversity, but also for molecular mapping/QTL analysis, and for comparative genome analysis of the two Corchorus species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.