- In the past decade, significant progress has been made in pear molecular genetics based on DNA research, but the number of molecular markers is still quite limited, which hardly satisfies the increasing needs of geneticists and breeders.. - A total of 101,694 pairs of SSR primers were designed from the SSR loci, and 80,415 of the SSR loci were successfully located on 17 linkage groups (LGs). - Eighteen polymorphic SSR markers were randomly selected from the 332 polymorphic SSR markers in order to perform a further analysis of the genetic diversity among 44 pear cultivars. - Conclusions: The results of the present study showed that the use of next-generating sequencing to develop SSR markers is fast and effective, and the developed SSR markers can be utilized by researchers and breeders for future pear improvement.. - The genus Pyrus belongs to the subtribe Malinae of the tribe Maleae in the subfamily Amygdaloideae of the family Rosaceae [1], with three known secondary centers of. - With the development of molecular breeding technology, marker-assisted selec- tion, an important tool now in the improvement of many crops, permits the rapid identification of key individ- uals that harbor useful genes and offers promise for pear breeding. - 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - as possible in the whole genome is the prerequisite for the construction of a genetic linkage map, gene mapping, molecular marker assisted selection (MAS) and other genomics studies [3–5].. - developed 73 expressed sequence tag (EST)-simple sequence re- peat (SSR) markers based on ESTs derived from 11 cDNA libraries of the Japanese pear cultivar ‘Housui. - and the SSR markers showed good transferability to other species in the Rosaceae [20]. - [11] developed 1341 SSR markers based on the genome sequence of the ‘Dangshansuli’ pear [27].. - In the present study, we aimed to: (1) develop SSR markers from publicly available scaffold sequence of the pear genome;. - (2) anchor the SSR markers to the existing reference gen- etic map. - and (3) evaluate SSR polymorphism and applica- tions in the analysis of genetic diversity.. - Identification and distribution of SSRs in the genome A total of 156,396 SSR motifs were identified within the scaffold sequences. - Of the total SSRs identified, mono-nucleotide repeat motifs were pre- dominant, followed by di-nucleotide repeat motifs . - Of the tri-nucleotide repeats, AAG/CTT and AAT/ATT were the most abundant, accounting for 27.61 and 26.45%, respectively. - Further comparison of the number of SSRs with differ- ent repeat motifs revealed that the number of SSRs with shorter motifs was much higher than that with longer motifs. - Among them, the number of SSRs in the inter- genic region was . - which was the highest, followed by the number of SSRs located in the intron of the gene . - There are 5052 SSRs in total in the region of 5’ UTR, or in the region of 3’ UTR. - Only 2135 SSRs were located in the exon region of coding genes (Additional file 5: Table S4).. - These anno- tations were summarized into 48 GO-terms of the three major categories. - Of them, biological process comprised the majority of the GO annotations, followed by cellular component and molecular function . - Genes were mostly enriched in the terms of metabolic process (10257), cel- lular process (10158), cell (9421), cell part (9405) and binding (9088) (Fig. - After deletion of the SSR loci with more than one identical primer, a total of 101,694 SSR loci (Additional file 6: Table S5, 65.02%) were. - According to the data of the high-density genetic map of pear [28], we designated the SSR loci to the corre- sponding linkage group (LG) and provided the scaffolds’. - genetic position to the SSR loci derived from the scaf- folds as an estimated reference position in the linkage group. - Of the 101,694 SSR loci, a total of 61,160 SSR loci were localized to a single linkage group. - The 19,255 SSR loci may be located in one of the 2–3 linkage groups, but the specific linkage group could not be identified. - Of the 17 linkage groups, the SSR loci were the least in the seventh linkage group, 1011 in total. - and the rest of the 507 SSR primer pairs could amplify the expected product (94.94%).. - 1 Result of the KEGG pathway annotation. - They can be used not only in the genetic population analysis but also in germplasm identifica- tion and a genetic diversity analysis.. - Assessment of the genetic relationship among 44 pear cultivars by SSR markers. - To verify the applicability of the newly developed SSR primer, a total of 44 pear varieties were genotyped by 18 SSR primers. - Most of the actual size of the amplified frag- ments matched or was very close to the size of the ex- pected fragments. - The 18 pairs of SSR primers detected 5–15 al- leles in the tested cultivars, with an average allele number of 10.56. - In the UPGMA cluster analysis, the 44 pear accessions were classified into three groups (For similarity coeffi- cient among 44 pear cultivars, see Additional file 12:. - The ‘Deqinli’ of the Zangli pear is grouped into one group individually, which is in a special position in the dendrogram. - 2 GO terms of the genes. - ‘Hongsucui’) of the Yunnan sand pear cultivar ‘Huobali’.. - In the population structure analysis, there is an obvi- ous turning point for the estimated log probability of data when K = 2 (Fig. - 5A-c) and the peak value of the ΔK score (Fig. - 4 Dendrogram for 44 pear cultivars derived from the UPGMA cluster analysis of 18 polymorphic SSR markers. - In com- parison, subpopulation 2 mainly comprised 14 out of 17 accessions from the first group obtained in the UPGMA cluster analysis (Fig. - SSRs can be divided into genomic SSRs and genic SSRs, and genomic SSRs are usually derived from SSR- enriched genomic libraries or random genomic sequences, while genic SSRs are derived from coding regions of the transcriptome or EST sequences . - which was generated by joining exons from Psy1 and an unknown gene and resulted in the yellow-fruited phenotype of tomato accession PI 114490, might be caused by an SSR with 19 A/T repeats in the downstream sequence of the Psy1 gene with Psy1/Un- known . - Clearly, the genomic SSRs located in introns and in the downstream sequence of genes may have the same enormous potential as genic SSRs in MAS applications. - In the present study, a total of 49,384 SSR loci were identified from the intron and 1 KB upstream and downstream from the transcription initi- ation site, and a total of 7917 SSR loci were identified from the extrons, 5’ UTR and 3’ UTR, which demon- strates the importance of the development of SSR markers in the whole genome.. - The functions of genic SSRs can often be inferred by performing a homology comparison of gene-containing genic SSRs, which is one of the reasons researchers think genic SSRs are more useful than genomic SSRs [29, 30]. - The functions of the genomic SSRs de- veloped from the related species can also be inferred from the gene annotation files or the function of neigh- boring genes. - In the present study, the 18 SSR loci distinguished the 44 pear individuals with an average of 10.56 alleles per locus and an average PIC value of 0.7808, indicating a high level of polymorphism. - Comparison of our results with previous findings of the SSR-based studies in Pear revealed that the average number of alleles per locus and PIC values recorded in the present study were compat- ible or higher than other SSR-based studies in Pyrus . - [40] studied the genetic diversity of 47 pear cultivars and genotypes using 28 SSR markers and showed that the average number of alleles per SSR locus. - [15] in the study of genetic variabil- ity of 99 P. - [42] found PIC values were in the range of 0.42 to 0.89 with an average of 0.65 for the 19 high polymorphic SSR markers selected from 40 tested SSR markers. - Erfani-Moghadam and Zarei [44] found PIC values of the SSR markers varied from 0.44 to 0.69 with an average of 0.59. - Some of the SSR markers developed in the present study have also been applied to construction of high-resolution linkage maps and QTL mapping ana- lyses in pear by Wang et al. - The SSR markers developed in the present study are the most abundant pear SSR loci to date, and also provided the physical location and the estimated genetic positions for reference, which could significantly increase the efficiency of the related genomics studies, such as genetic diversity, constructing high-resolution linkage maps, QTL mapping, and so on.. - In the present study, we used the UPGMA method to cluster the 44 pear varieties, and the European pears and Asian pears were clustered into different groups. - A simi- lar distinct clustering pattern has also been reported, and the Asian pear and European pear were completely separated from each other in the phylogenetic tree [17, 47] and probably evolved independently [48]. - and proved that the correctness of the white pear cultivar group was assigned as a subgroup of sand pear of the P.. - In the previous studies, P.. - In the present study, we also obtained similar results, i.e., the P. - Zangli pear, a variety of landrace, distributed in the higher altitude junction region of Tibet, Yuannan and Sichuan Provinces in southwestern China, is a kind of semi-wild or semi-cultivated type, with hardy character- istics and resistance to a hypoxic environment. - for example, Zangli var- ieties ‘Deqinli’ pear, distributed in Deqin County of the Diqing Tibetan Autonomous Prefecture in Yunnan Province, China, have morphological characteristics of both Oriental and European pear, but its genetic rela- tionship is closer to European pear, with smaller and leathery leaves. - In the present study, the results of a cluster analysis of 44 pear cultivars with newly devel- oped SSR markers also showed that the phylogenic sta- tus of ‘Deqinli’ in the dendrogram was rather special, indicating that it was closer to European pear. - ussuriensis cultivars, but the European pears were not included in the study [52]. - In this study, we developed 101,694 genomic SSR markers from the reference genome sequences of pear.. - 6 base repeats, which is the largest number of SSR markers developed from a single development in pear by far. - These SSR markers were also marked with the rela- tive position information of both the physical location and the linkage groups, and the adjacent genes of the SSR loci were also annotated, which can provide better help for related research work and the developed SSR markers can be utilized by researchers and breeders for future pear improvement.. - A total of 44 accessions, planted in the Pear Germplasm Resources Garden in Zhengzhou, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, were used in the present study (Tables 1 and 2).. - Table 1 The 44 accessions used in the polymorphism analysis. - Based on SSR motifs, the Primer 3 software [57] was used to design SSR primers with the following param- eters: the length of primers was in the range of 18–. - and the melting temperature (Tm) was in the range of 55–65 °C, with 60 °C as the optimum and a maximum Tm difference of 1 °C. - Primers of the SSRs with compound motif types were discarded. - These newly devel- oped scaffold-derived SSR markers were then aligned to the high-density genetic linkage map [28] according to the scaffolds’ genetic positions, which were defined Table 2 Eighteen polymorphic SSR markers validated in the 44 pear accessions. - as the average genetic position of the SNPs on a scaffold of the map. - Forward primers of the 18 primer pairs were 5′ end labeled with a fluorescent dye (HEX or FAM). - Determination of the fragment sizes and data collection were done using the GeneMapper 4.0 software (Applied Biosystems, Foster City, CA, USA).. - The genotyping data of the accessions in the 18 SSR loci selected in the initial screening were then used to evaluate the genetic diversity of the 44 pear accessions.. - Percentages of different motifs among mono-(a), di- (b) and tri- (c) nucleotide repeats in the ‘ Dangshansuli ’ pear genome. - Information of new designed 101, 694 SSR primers, including information of the primer sequences, annealing temperature (Ta), repeat motifs, target size, linkage groups, and positions in genetic and physical maps of pear. - Information of 534 SSR primers, including information of the primer sequences, annealing temperature (Tm), repeat motifs, target size, linkage groups, and positions in genetic and physical maps of pear. - Information of 332 good SSR primers, including information of the primer sequences, annealing temperature (Tm), repeat motifs, target size. - Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. - Genetic diversity and similarity of pear (Pyrus L.) cultivars native to East Asia revealed by SSR (simple sequence repeat) markers. - Identifying genetic diversity and a preliminary core collection of Pyrus pyrifolia cultivars by a genome-wide set of SSR markers. - Development of genic SSR markers from transcriptome sequencing of pear buds. - Transferability of newly developed pear SSR markers to other Rosaceae species. - Development of novel EST-SSR markers derived from Japanese pear (Pyrus pyrifolia). - Development of microsatellite markers in the Japanese pear (Pyrus pyrifolia Nakai). - The genome of the pear (Pyrus bretschneideri Rehd. - Development of Saccharina japonica genomic SSR markers using next-generation sequencing. - A genome-wide survey of the microsatellite content of the globe artichoke genome and the development of a web-based database. - Genetic diversity in local Tunisian pears (Pyrus communis L.) studied with SSR markers. - Assessment of genetic structure among different pear species (Pyrus spp.) using apple-derived SSR and evidence of duplications in the pear genome. - Genetic diversity and population structure of pear (Pyrus spp.) collections revealed by a set of core genome-wide SSR markers. - Genetic variation and population structure of “ Zangli ” pear landraces in Tibet revealed by SSR markers
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