| Most of aquatic vegetables originat in China, and have a long history of cultivation. They are very popular with the farmers and consumers, because of their rich nutrient substances, unique taste, and good health function. However, genetics researches and genomic informations are limited in aquatic vegetable. In this study, transcriptome data were conducted by deep sequencing technique, and EST-SSR markers were developed, and used to analyse the genetic relationships in four aquatic vegetables (Nelumbo nucifera, Colocasia esculenta (L.) Schoot, Sagittaria Trifolia L. var. sinensis (Sims) Makino, Eleocharis dulcis), respectively, the fingerprinting maps were constructed and seed purity were also discussed. The main results are as follows:1. Lotus(Nelumbo nucifera) transcriptome was sequenced by using Illumina HiSeqTM 2000 platform and carried out bioinformatics analysis. A total of 10.1Gbp and 9.9Gbp short reads of wild flower lotus and cultivated rhizome lotus apical bud transcriptome were obtained, respectively. Two sets of reads data were subsequently de novo assembled into 111,925 and 100,016 contigs with a length of over 200bp with Trinity software, and then further assembled into 105,834 uni-contigs with an average length of 722bp. Using MISA tools,11,178 SSR locus were detected, and 6,568 primer pairs were designed, of which 72 primers were randomly synthesized and used for validation of the amplification in 51 individuals; 38 in-silico polymorphic primers were obtained using in-house perl scripts. The results showed that 91(82.7%) primers yielded amplification products, in which 80 were polymorphism with the number of alleles ranged from 2 to 17. The polymorphism information content was valued ranging from 0.19 to 0.87. An UPGMA dendrogram based on Jaccard’s similarity coefficients was shown that the correlation between geographical source and genotype was low, and there was a certain connection between genetic relationship and morphological characters.2. Taro(Colocasia esculenta (L.) Schoot) transcriptome was sequenced by using Illumina TruSeqTM platform and carried out bioinformatics analysis. A total of 4.5Gbp short reads of taro mature leaf transcriptome were obtained, with average length of 100bp. The reads data were subsequently de novo assembled into 52,935 contigs with average length of 588.5bp with Trinity software. The data of sequence were analyzed against four public databases (NR, GO, KEGG, KOG). Using MISA tools,5,278 SSR locus were detected, and 2,858 primer pairs were designed, of which 100 primers were randomly synthesized and used for validation of the amplification in 69 individuals (including 68 taro,1 Alocasia macrorrhiza). The results showed that 72 (72.0%) primers yielded amplification products, in which 62 were polymorphism with the number of alleles ranged from 2 to 14. The polymorphism information content was valued ranging from 0.19 to 0.87. The SSR markers developed from C. esculenta could be successfully applied to A. macrorrhiza, with a transferability rate of 36.11% (26/72). An UPGMA dendrogram based on Jaccard’s similarity coefficients was shown that there was no obvious connection between genetic relationship and morphological characters or geographic origin.3. Arrowhead (Sagittaria Trifolia L. var. sinensis (Sims) Makino) transcriptome was sequenced by using Illumina TruSeqTM platform and carried out bioinformatics analysis. A total of 4.7Gbp short reads of arrowhead mature leaf transcriptome were obtained, with average length of 100bp. The reads data were subsequently de novo assembled into 51,836 contigs with average length of 680bp with Trinity software. The data of sequence were analyzed against four public databases (NR, GO, KEGG, KOG). Using MISA tools,3,861 SSR locus were detected, and 2,476 primer pairs were designed, of which 100 primers were randomly synthesized and used for validation of the amplification in 81 individuals (including 79 arrowhead,2 Sagittaria pygmaea). The results showed that 78 (78.0%) primers yielded amplification products, in which 68 were polymorphism with the number of alleles ranged from 2 to 11. The polymorphism information content was valued ranging from 0.01-0.84. The SSR markers developed from Sagittaria Trifolia L. could be successfully applied to Sagittaria pygmaea, with a transferability rate of 91.14%(72/79). An UPGMA dendrogram based on Jaccard’s similarity coefficients was shown that there was a certain connection between genetic relationship and geographic origin.4. Water chestnut(Eleocharis dulcis) transcriptome was sequenced by using Illumina TruSeqTM platform and carried out bioinformatics analysis. A total of 4.7Gbp short read of water chestnut mature leaf transcriptome were obtained, with average length of 100bp. The reads data were subsequently de novo assembled into 40,796 contigs with average length of 616.6bp with Trinity software. The data of sequence were analyzed against four public databases (NR, GO, KEGG, KOG). Using MIS A tools,2,570 SSR locus were detected, and 1,606 primer pairs were designed, of which 100 primers were randomly synthesized and used for validation of the amplification. The results showed that 77 (77.0%) primers yielded amplification products.5. SSR fingerprinting map and seed purity were studied in lotus, taro and arrowhead. For 22 lotus cultivars, fingerprinting map were constrcted on the basis of SSR markers. Among them, the key 6 pairs of polymorphic primers which could differentiate all cultivars and the candidate 3 primers with high polymorphism information content were selected from the previous primers to analyse the seed purity. The purity of Taikong lotus 36 was 93.3%(84/90). For 46 taro cultivars, fingerprinting map were constrcted on the basis of SSR markers. Among them, the key 20 pairs of polymorphic primers which could differentiate all cultivars were selected from the previous primers. The purity of Shandong 8502 taro was checked based on the first 12 key primers. The purity was 98.9%(89/90). For 10 arrowhead cultivars, fingerprinting map were constrcted on the basis of SSR markers. Among them, the key 3 pairs of polymorphic primers which could differentiate all cultivars and the candidate 9 primers were selected from the previous primers to analyse the seed purity. The purity of Wuhan arrowhead was 100.0%(90/90).In this study, this is the first attempt to exploit a transcriptome database and develop a large set of SSR markers in four aquatic vegetables. It is important for the researches of function gene cloning, genetic diversity analysis, germplasm characterization and molecular marker-assisted breeding etc. |
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