Abstract
Ramie (Boehmeria nivea L. Gaud) is one of the most important natural fiber crops, and improvement of fiber yield and quality is the main goal in efforts to breed superior cultivars. However, efforts aimed at enhancing the understanding of ramie genetics and developing more effective breeding strategies have been hampered by the shortage of simple sequence repeat (SSR) markers. In our previous study, we had assembled de novo 43,990 expressed sequence tags (ESTs). In the present study, we searched these previously assembled ESTs for SSRs and identified 1,685 ESTs (3.83%) containing 1,878 SSRs. Next, we designed 1,827 primer pairs complementary to regions flanking these SSRs, and these regions were designated as SSR markers. Among these markers, dinucleotide and trinucleotide repeat motifs were the most abundant types (36.4% and 36.3%, respectively), whereas tetranucleotide, pentanucleotide, and hexanucleotide motifs represented <10% of the markers. The motif AG/CT was the most abundant, accounting for 28.74% of the markers. One hundred EST-SSR markers (97 SSRs located in genes encoding transcription factors and 3 SSRs in genes encoding cellulose synthases) were amplified using polymerase chain reaction for detecting 24 ramie varieties. Of these 100 markers, 98 markers were successfully amplified and 81 markers were polymorphic, with 2–6 alleles among the 24 varieties. Analysis of the genetic diversity of all 24 varieties revealed similarity coefficients that ranged from 0.51 to 0.80. The EST-SSRs developed in this study represent the first large-scale development of SSR markers for ramie. These SSR markers could be used for development of genetic and physical maps, quantitative trait loci mapping, genetic diversity studies, association mapping, and cultivar fingerprinting.
Highlights
Ramie (Boehmeria nivea), popularly called ‘‘China grass,’’ is a perennial diploid (2n = 28) herbaceous plant that belongs to the family Urticaceae
We identified 1,827 expressed sequence tags (ESTs)-simple sequence repeat (SSR) markers based on ramie ESTs that had been assembled in our previous transcriptome sequencing study [15]
A few instances of primer mismatch occurred, and in such cases, some EST-SSRs might have failed to amplify because the primers were designed across splice sites or because large introns were present within the target amplicon [13,22,23,24]
Summary
Ramie (Boehmeria nivea), popularly called ‘‘China grass,’’ is a perennial diploid (2n = 28) herbaceous plant that belongs to the family Urticaceae It is one of the most important natural fiber crops. Some markers that are not specific for location, such as sequence-related amplified polymorphism (SRAP), random amplified polymorphic DNA (RAPD), and inter simple sequence repeat (ISSR) markers, were used to analyze the genetic diversity in ramie [1,2,3]. These markers have the common shortcomings of poor repeatability and dominance. Fewer than 100 SSR markers have been identified for ramie, including the markers generated for SSR-enriched genomic libraries and the expressed sequence tags (ESTs) deposited in public databases [10,11]
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