The development of specific SNP markers for chromosome 14 in cotton using next‐generation sequencing

Lacking of reference genome sequence for the development of stable molecular markers for specific chromosomes (intervals) remains to be a challenge in cotton, which was a necessary step in fine mapping of gene (QTL). In this study, the feasibility of development of single‐nucleotide polymorphism (SNP) markers between CS‐B14Sh (a substitution line for short arm of Chromosome 14) and TM‐1 (the recurrent parent) was explored using next‐generation sequencing (NGS) based on reduced representation libraries (RRLs). High‐quality genome sequences, representing about 7.1%, 8.8% and 10.4% of the tetraploid cotton genome, were generated for TM‐1, 3‐79 (the donor parent) and CS‐B14Sh, respectively. A total of 397 putative SNP markers were detected between CS‐B14Sh and TM‐1, and most (358) of them were also detected between TM‐1 and 3‐79. Allele‐specific PCR method was used for validation of 40 SNP markers, and 27 of them showed polymorphism between TM‐1 and CS‐B14Sh, and a linkage group comprising of 25 SNP markers and five SSR markers was constructed. The order of SNP markers agreed well with the position of them on Chr05 of D genome, which further approved the truth of SNPs detected. The results suggested that the development of SNP markers in specific genome region using NGS was efficient in substitution or near‐isogenic lines.     

Parentage analysis of tea cultivars in Japan based on simple sequence repeat markers

Tea cultivars have been bred by individual selection of landraces and by crossbreeding, but the validation of the parentage is limited. In this study, we performed parentage analysis of 79 tea cultivars in Japan based on SSR markers to confirm or identify the parent-offspring relationships among them. The effectiveness of nine SSR markers for parentage analysis was validated by comparing them to the existing cleaved amplified polymorphic sequence markers. The former markers were detectable more alleles than the latter. Simulation of parentage analysis of the tea cultivars predicted biparental origins for 12 cultivars (‘Houshun’, ‘Mie ryokuhou no. 1’, ‘Surugawase’, ‘Tenmyo’, ‘Yamanoibuki’, ‘Harumidori’, ‘Koushun’, ‘Minekaori’, ‘Okumusashi’, ‘Saemidori’, ‘Sofu’, and ‘Toyoka’), in the first five of which candidate parents of yet-to-be-defined pedigree were newly identified. Comparisons of a total of 41 SSR genotypes confirmed the newly-identified parentages of ‘Asahi’ for ‘Tenmyo’, ‘Rokurou’ for ‘Houshun’, ‘Surugawase’, and ‘Yamanoibuki’, and ‘Yamatomidori’ for ‘Mie ryokuhou no. 1’. The maternity of seven cultivars out of the 12 was also confirmed with chloroplast DNA sequences. Uniparental origins were confirmed for 25 cultivars. This parentage analysis has improved our knowledge of tea pedigrees and will aid in the development of new cultivars.     

Exploring genetic diversity for heat tolerance among lentil (Lens culinaris Medik.) genotypes of variant habitats by simple sequence repeat markers

The study was carried out to assess genetic diversity among 119 lentil genotypes grown in different habitats for heat tolerance using morpho‐physiological and reproductive traits and SSR markers. High‐temperature stress was applied at seedling (35/33°C) and anthesis stages (35/20°C) to study the effects on morpho‐physiological and reproductive traits under hydroponic condition, which was compared with non‐stressed and stressed field conditions. A set of 209 alleles were identified by 35 SSR markers among the genotypes. Genetic diversity and polymorphism information content values varied between 0.0494–0.859 and 0.0488–0.844, with mean values of 0.606 and 0.563, respectively. Genotypes were clustered into nine groups based on SSR markers. Morpho‐physiological and reproductive traits under heat stress were found to be significantly different among SSR clusters. These findings suggest that heat adaptation is variable among the genotypes and the tolerant materials can be evolved through hybridization using parents from different clusters with diverse mechanisms of heat tolerance.     

Classification of Southeast Asian mints (Mentha spp.) based on simple sequence repeat markers

Mentha is a complex genus encompassing many species as a consequence of their interspecific hybridization and polyploidy. Southeast Asian mints have been poorly distinguished though they are widely used for culinary and medical purposes. In this study, we have analyzed Southeast Asian mints and known varieties as well as a related Lamiaceae species (Nepeta sp.) using simple sequence repeat (SSR) markers and leaf morphology. Two types of mints were clearly distinguished based on their venation pattern and leaf shape index. We developed 12 SSR markers that allowed good amplification in the Mentha and another Lamiaceae species. In the SSR-based phylogram, the Mentha lines could be delimited into groups I–VI. The Southeast Asian mints divided into groups I and II, and the phylogram separated most of the available species, with groups I and II containing the known species M. × cordifolia and M. arvensis, respectively. The separation of the two groups was supported by a population structure analysis. The SSR markers developed in this study enabled the simultaneous classification of mints and will help improve our understanding of the genetic composition of known mint varieties and as yet unclassified Southeast Asian mints.     

Development of pepper SSR markers from sequence databases

Simple sequence repeat (SSR) markers are potentiallyvaluable tools for plant breeding. The objectives ofthe work reported here were to search the EMBL andGenbank databases for the presence of SSR-containingsequences from the genus Capsicum, to assess thefrequency of different motifs, and to examine thepolymorphism of selected markers in a panel ofgenotypes, including 10 Capsicum spp. and 1tomato and 1 potato genotype. Fifty-eightmicrosatellites with different motifs were found inCapsicum sequences. A subset of twelve of thesewere selected for the polymorphism survey using PCRprimers flanking the SSR. Polymorphisms between Capsicum lines can be detected with 5 of these primerpairs. PCR products of the predicted size were alsoamplified with three primer pairs in potato and oneprimer pair in tomato. The study also showed thatshorter microsatellites could be valuable markers inCapsicum.     

Genetic linkage map of Chinese native variety faba bean (Vicia faba L.) based on simple sequence repeat markers

Simple sequence repeat (SSR) marker is a powerful tool for construction of genetic linkage map which can be applied for quantitative trait loci (QTL) and marker‐assisted selection (MAS). In this study, a genetic map of faba bean was constructed with SSR markers using a 129 F₂ individuals population derived from the cross of Chinese native variety 91825 (large seed) and K1563 (small seed). By screening 11 551 SSR primers between two parents, 149 primer pairs were detected polymorphic and used for F₂ population analysis. This SSR‐based genetic linkage map consisted of 15 linkage groups with 128 SSR. The map encompassed 1587 cM with an average genetic distance of 12.4 cM. The genetic map generated in this study will be beneficial for genetic studies of faba bean for identification of marker‐locus‐trait associations as well as comparative mapping among faba bean, pea and grasspea.     

Occurrence of microsatellites in spinach sequences from computer databases and development of polymorphic SSR markers

Microsatellites are valuable tools as molecular markers in plant breeding. To establish genetic linkage maps or for population studies, information about the occurrence and usability of microsatellite markers in different species is necessary. Sequences of spinach Spinacia oleracea from computer databases were therefore searched for the presence of microsatellites. Sixty simple sequence repeats were found in 237 spinach sequences with a total of 349.4 kb DNA. After removing duplicated sequences, 50 different microsatellites with various motifs remained. Differences between nuclear and chloroplast DNA were not in the number of microsatellites but in their type and length. Chloroplast sequences from spinach contain only short strings of A and AT repeats, whereas nuclear sequences show a wider variety of motifs. Flanking primers for polymerase chain reaction (PCR) analysis were designed for 13 of these microsatellites and tested with two different varieties of spinach. Twelve primer pairs gave amplification products and seven of these showed polymorphisms in the variety ‘Wiremona’ but only one in the variety ‘Monatol’. These markers may be used for linkage analysis or population studies in spinach.     

Development of simple sequence repeat (SSR) markers in Eucalyptus from amplified inter-simple sequence repeats (ISSR)

Eucalyptus spp. are widely used in exotic plantations. Since many of these trees are derived from vegetative propagation, the routine identification of clones has become increasingly important. The most widely used molecular based method for fingerprinting these clones is by random amplified polymorphic DNAs (RAPDs). Although this technique is useful, its results are not very repeatable, especially between laboratories. The aim of this study was to develop microsatellite markers that are highly repeatable, and to investigate their value in Eucalyptus fingerprinting. Typically, this process involves the expensive procedure of constructing an enriched genomic library. However, we used an intersimple sequence repeat (ISSR) polymerase chain reaction (PCR)‐based enrichment technique for microsatellite‐rich regions. With this relatively inexpensive method, microsatellite‐rich regions were amplified directly from genomic DNA, after which PCR products were cloned and sequenced. From these microsatellite‐rich sequences, primer sets were constructed to amplify mono‐, di‐, tri‐, hexa‐and nona‐nucleotide repeats. These markers were all inherited in a Mendelian fashion in the progeny of a test cross between two Eucalyptus grandis trees. The primer sets developed were also able to amplify the corresponding microsatellite loci from five different Eucalyptus spp., namely E. grandis, E. nitens, E. globulus, E. camaldulensis and E. urophylla.     

Highly variable ‘Arhar’ simple sequence repeat markers for molecular diversity and phylogenetic studies in pigeonpea [Cajanus cajan (L.) Millisp.]

Pigeonpea is an important tropical grain legume widely cultivated in South and South‐East Asia for versatile end usage as food, feed, fodder and fuel. Recent publication of draft genome sequence of pigeonpea (‘Arhar’) has allowed mining of large numbers of genomic simple sequence repeat (SSR) markers most of which are either not validated or show very little polymorphism in simple agarose gel–based assays. Here, we describe a special category of 370 validated highly variable ‘Arhar’ genomic SSR (HASSR) markers, which provide much higher level of polymorphism than a random set of SSR markers. These markers were validated for consistent amplification in a set of eight pigeonpea varieties and 152 of these (41%) showed allelic polymorphism on agarose gel electrophoresis. Twenty‐four highest polymorphic HASSR markers were used on a larger set of forty pigeonpea varieties and eight wild Cajanus species for the analysis of genetic diversity and phylogenetic relationship. Genomic HASSR markers described here are highly suitable for genetic diversity and phylogenic studies in pigeonpea.     

Rapid and convenient gel‐free screening of SCAR markers in wheat using SYBR green‐based melt‐profiling

Sequence characterized amplified region (SCAR) markers that are highly desirable in crop breeding for marker‐assisted selection (MAS) are routinely analysed by gel‐based methods that are low‐throughput, time‐consuming and laborious. In this study, we showed a rapid and convenient method for analysis of SCAR markers in a gel‐free manner. Seven SCAR markers, linked to rust resistance genes (Sr24, Sr26 and Sr31) and seed quality traits (Pina, Pinb and Glu‐D1) in wheat (Triticum aestivum), were amplified on a real‐time PCR machine using custom reaction mixture. Subsequently, melting curve analysis was performed, to assess the specificity of amplicons. Using the amplicon‐specific melt‐profiles, the presence/absence of SCAR markers was analysed in fifteen genotypes and five F₂ populations. Unlike the fluorescence‐based in‐tube detection methods, the present method used the amplicon‐specific melt‐profiles to evaluate the status of the SCAR markers, thus eliminating the need for gel‐based analysis. Results also showed feasibility of multiplex analysis of two markers with well‐separated melting profiles. Overall, the approach is a rapid, convenient and cost‐effective method for high‐throughput screening of SCAR markers.     

Development of a cytoplasmic male‐sterile line NJCMS4A for hybrid soybean production

Cytoplasmic male‐sterile (CMS) lines are being used to produce hybrid seeds. Thus far, four CMS sources in soybean [Glycine max (L.) Merr.] have been reported in China. However, they are not sufficient or efficient in meeting the requirements of commercial soybean hybrid seed production. In this study, 33 varieties were tested for CMS using 45 crosses among 37 landraces and 17 annual wild soybean accessions (Glycine soja Sieb. et Zucc.). The cross of N23661 × N23658 showed partial to complete male sterility in backcross generations, while the corresponding reciprocal cross showed normal male fertility. Thus, the cytoplasm of N23661 is male‐sterile, the continuously backcrossed line is a male‐sterile line (designated NJCMS4A), and N23658 is its maintainer (designated NJCM4B). The male fertility of NJCMS4A was restored by another accession, Nansheng9403. Accordingly, NJCMS4A along with its maintainer and restorer composes a complete set of three lines for producing hybrid soybean. Using mitochondrial markers and sequence analyses, NJCMS4A is a CMS line with its cytoplasm not identical to the four previously reported CMS sources in soybean.     

Genetic diversity analysis of Moroccan lentil (Lens culinaris Medik.) landraces using Simple Sequence Repeat and Amplified Fragment Length Polymorphisms reveals functional adaptation towards agro‐environmental origins

In the absence of previous molecular characterization, we assessed genetic diversity of 53 Moroccan lentil landraces including two local cultivars using simple sequence repeat (SSR) and amplified fragment length polymorphisms (AFLP). Nineteen SSRs yielded 213 alleles, and seven AFLP primer combinations gave 766 fragments of which 422 were polymorphic. Moderate to high genetic variation was observed. Several small groups of landraces were differentiated. Interestingly, one of the smallest groups contained short‐cycle landraces with high early vegetative growth. Landraces in that group were from the dry land location of Abda, where they were likely selected for adaptation to drought and heat stress over centuries. Another group contained two landraces from highland areas that may have been selected for specific adaptation to cold stress. A third group contained one landrace from the Zear region known for its seed quality and has been proposed for the protected designation of origin (PDO) quality mark. Both techniques gave evidence of differentiation of the latter landrace supporting the idea of PDO attribution. Functional grouping according to agro‐environmental origins, cycle duration and early vegetative vigour was observed.