| Dioscorea polystachya Turczaninow is belong to genus Dioscorea,Dioscoreaceae.Nowadays in Chinese market, there are three main confusions: the confusion between D.polystachya and its 5 related species, the confusion among cultivars of D. polystachya, and confusion between genuine place and non-genuine place. Unfortunately, these productions are difficult to identify on traditional morphological characteristics and microscopic identification needs more professional knowledge and complicated processes. Many molecular fingerprints cost a lot and require more time. So, it's urgent to develop a more simple, fast and reliable method to identify the productions above. In this research, we have collected D. polystachya and its 5 related species, 14 main cultivars in Chinese market and 8 accessions of D. polystachya 'tiegun' for genuine authentication of species, cultivar and accession. The bulbil is an important vegetative and reproductive organ of yam species and has more nutrition and medicine values. To our knowledge, there is no molecular research about the formation of the bulbil of yam species. In this study, we present transcript data for the bulbil transcriptome of D. polystachya for the first time. Our further goal was to find the candidate genes which control the bulbil formation. The main contents and results of this study are as follows:(1) In this study, D. polystachya and its 5 relatives showed a high level of genetic diversity based on SRAP (Sequence-Related Amplified Polymorphism) marker. There was a high level of genetic diversity among species: the survey of 21 highly polymorphic primers amplify 355 fragments, 338 of them were polymorphic bands(95.27%), with an average of 16.90 bands and 16.10 polymorphic bands per primer pair. the number of alleles(Na) was from 1.8963, the effective number of alleles (Ne) was 1.7506, the Nei's genetic diversity index (H) was 0.4840; the total heterozygosity (Ht) was 0.4541 and the Shannon's information index (1) was 0.6662. The mean heterozygosity in the species (Hs) was 0.1661.The Gst (0.5711) means 57.11% of total genetic diversity existed among the species. The gene flow between these species was high (Nm=0.3754) for the reason that the wild species can exchange their genepool by sexual reproduction. The genetic structure among these species showed the lowest Fis value observed in D. japonica which means there was high genetic differentiation in this species. At the same time, the lowest genetic differentiation was observed in D.persimilis for the highest Fis value. The highest Fst has been observed in D. fordii indicating this species has the most genetic differentiation with other species while D. polystachya showed lest genetic differentiation with other species. The genetic relationship among these species indicated that D. polystachya and D. japonica had the closest relationship. In conjunction with SCAR (sequence characterized amplified region)marker, the SRAP-SCAR marker developed in this study has economic value for the chaos market of D. polystachya because it could be used to discriminate D. polystachya and its relatives.(2) ISAP (Intron Sequence Amplified Polymorphism) was carried out to study the genetic relationships among 14 D. polystachya. Our results revealed that there is a high level of polymorphism among these cultivars: 11 highly polymorphic primers amplified a total of 95 fragments with 91 polymorphic band (95.79%), the number of alleles (Na) was from 1.8999 to 2.0000 with an average of 1.9651; the effective number of alleles (Ne)ranged from 1.1529 to 1.4271 with an average of 1.3018; the difference value between Na and Ne was from 0.5729 to 0.8471 with an average of 0.6983; the Nei's genetic diversity index (H) was from 0.1327 to 0.2673 with an average of 0.2002; and the Shannon's information index (1) was from 0.2011 to 0.5832 with an average of 0.3102. Base on genetic identity and genetic distance, the genetic relationship with 'Tiegun' cultivar from close to distant was arranged as: 'Taigu', 'Caoshanyao','Mashanayo','Anshun','Wujiashuangbao', 'Xichangmao', 'Mishanyao', 'Huaying', 'Huazi', 'Xiaobaizui','Cuniutui','Jiujinhuang' and'Baiyu'. Furthermore,in this study,ISAP markers were consistent with the morphological characters of D. polystachya cultivars and these cultivars could be group into 2 clusters. An ISAP fragment (435bp) specific for 'Tiegun' cultivar was converted into SCAR maker. All the 140 individuals of all cultivars were tested with this maker. This ISAP-SCAR marker could be used to sufficiently discriminate 'Tiegun' cultivar from the other 13 cultivars.(3) The ISSR (Inter-Simple Sequence Repeat) markers were used to estimate the genetic diversity among 8 accessions of D. polystachya 'Tiegun'. The result showed; the number of alleles (Na) was from 1.7789 to 2.0000; the effective number of alleles (Ne) ranged from 1.0417 to 1.5503; the Nei's genetic diversity index (H) was from 0.0794 to 0.3751 and the Shannon's information index (I) was from 0.1864 to 0.5388. All these data showed there was a high level of genetic diversity among accessions. The genetic distance with Henan Wenxian from close to distant was arranged as: Jiangsu Xuzhou, Shanxi Taiyuan, Shandong Jining, Hunan Liuyang, Hebei Cangzhou, Sichuan Nanchong and Guangdong Dongguan.The genetic differentiation was consistent with the introduction history. A SCAR (Sequence Characterized Amplified Region) maker, developed from ISSR4(ACACACACACACACACC) maker, was found useful for identification of all the 10 individuals from Henan Wenxian compared with the other accessions missing the SCAR band except Jiangsu Xuzhou. The ISSR-SCAR maker can be useful for the identification of the non-genuine place with the longer introduced history. Combining SRAP, ISAP and ISSR markers, all the samples in different accessions, different cultivars, and different species were analysed. The result showed that the positions in the phylogenetic tree between D. polystachya and its 5 relatives were similar with the result using SRAP marker separately. The genetic relationship with D. polystachya from close to far arranged as D.japonica, D. alata, D. exalata, D.persimilis and D. fordii. The result combining three markers also supported the independent systematic position of all the species. Through coalition analysis, all cultivars were belonged to D. polystachya. All the cultivars of D.polystachya were originated from 'Tiegun' cultivar.(4) In this study, the tissues of 'Tiegun' cultivar with bulbil, no bulbil and the cultivar'Huazi' which never generate bulbil were selected to study. The result of de novo yielded 70480 high quality unigenes. There were 6263 unigenes up-regulated and 8929 unigenes down-regulated when contrast the tissues of 'Tiegun' cultivar which generate bulbil to the tissues which don't generate bulbil. There were 13026 unigenes that were up-regulated and 11352 unigenes down-regulated when contrast the tissues of 'Tiegun' cultivar which generate bulbil to 'Huazi' cultivar. There were 2698 unigenes up-regulated and 4949 unigenes down-regulated in the tissues which generates bulbil compare with the tissues don't generate bulbils. Theses unigenes were blast in many data base. In conjunction with the genes of bulbil formation of Agave tequilana Linn. and Titanotrichum oldhamii(Hemsley) Solereder, the genes of lateral branch formation, at last, we found 23 candidate genes which might be related to the bulbil formation. Furthermore, a total of 11102 pairs of SSR (simple sequence repeats) primers were developed based on RNA-seq, of which will provide more markers for yam breeding and genetic map structure. |
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