Genome-wide development of lncRNA-derived-SSR markers for Dongxiang wild rice (Oryza rufipogon Griff.)

Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) is the northernmost distributed wild rice found in the world. Similar to other populations of O. rufipogon, DXWR contains a large number of agronomically valuable genes, which makes it a natural gene pool for rice breeding. Molecular markers, especially simple repeat sequence (SSR) markers, play important roles in plant breeding. Although a large number of SSR markers have been developed, most of them are derived from the genome coding sequences, rarely from non-coding sequences. Meanwhile, long non-coding RNAs (lncRNAs), which are derived from the transcription of non-coding sequences, play vital roles in plant growth, development and stress responses. In our previous study, we obtained 1655 lncRNA transcripts from DXWR using strand-specific RNA sequencing. In this study, 1878 SSR loci were detected from the lncRNA sequences of DXWR, and 1258 lncRNA-derived-SSR markers were developed on the genome-wide scale. To verify the validity and applicability of these markers, 72 pairs of primers were randomly selected to test 44 rice accessions. The results showed that 42 (58.33%) pairs of primers have abundant polymorphism among these rice materials; the polymorphism information content values ranged from 0.04 to 0.87 with an average of 0.50; the genetic diversity index of SSR loci varied from 0.04 to 0.88 with an average of 0.56; and the number of alleles per marker ranged from 2 to 11 with an average of 4.36. Thus, we concluded that these lncRNA-derived-SSR markers are a very useful source for future basic and applied research.

Genome-wide identification and characterization of functionally relevant microsatellite markers from transcription factor genes of Tea (Camellia sinensis (L.) O. Kuntze)

Tea, being one of the most popular beverages requires large set of molecular markers for genetic improvement of quality, yield and stress tolerance. Identification of functionally relevant microsatellite or simple sequence repeat (SSR) marker resources from regulatory “Transcription factor (TF) genes” can be potential targets to expedite molecular breeding efforts. In current study, 2776 transcripts encoding TFs harbouring 3687 SSR loci yielding 1843 flanking markers were identified from traits specific transcriptome resource of 20 popular tea cultivars. Of these, 689 functionally relevant SSR markers were successfully validated and assigned to 15 chromosomes (Chr) of CSS genome. Interestingly, 589 polymorphic markers including 403 core-set of TF-SSR markers amplified 2864 alleles in key TF families (bHLH, WRKY, MYB-related, C2H2, ERF, C3H, NAC, FAR1, MYB and G2-like). Their significant network interactions with key genes corresponding to aroma, quality and stress tolerance suggests their potential implications in traits dissection. Furthermore, single amino acid repeat reiteration in CDS revealed presence of favoured and hydrophobic amino acids. Successful deployment of markers for genetic diversity characterization of 135 popular tea cultivars and segregation in bi-parental population suggests their wider utility in high-throughput genotyping studies in tea.

Insights Into Comparative Analyses and Phylogenomic Implications of Acer (Sapindaceae) Inferred From Complete Chloroplast Genomes

Acer L. (Sapindaceae) is one of the most diverse and widespread plant genera in the Northern Hemisphere. It comprises 124–156 recognized species, with approximately half being native to Asia. Owing to its numerous morphological features and hybridization, this genus is taxonomically and phylogenetically ranked as one of the most challenging plant taxa. Here, we report the complete chloroplast genome sequences of five Acer species and compare them with those of 43 published Acer species. The chloroplast genomes were 149,103–158,458 bp in length. We conducted a sliding window analysis to find three relatively highly variable regions (psbN-rps14, rpl32-trnL, and ycf1) with a high potential for developing practical genetic markers. A total of 76–103 SSR loci were identified in 48 Acer species. The positive selection analysis of Acer species chloroplast genes showed that two genes (psaI and psbK) were positively selected, implying that light level is a selection pressure for Acer species. Using Bayes empirical Bayes methods, we also identified that 20 cp gene sites have undergone positive selection, which might result from adaptation to specific ecological niches. In phylogenetic analysis, we have reconfirmed that Acer pictum subsp. mono and A. truncatum as sister species. Our results strongly support the sister relationships between sections Platanoidea and Macrantha and between sections Trifoliata and Pentaphylla. Moreover, series Glabra and Arguta are proposed to promote to the section level. The chloroplast genomic resources provided in this study assist taxonomic and phylogenomic resolution within Acer and the Sapindaceae family.

Genetic Diversity of Fig (Ficus Carica L.) Germplasm From The Mediterranean Basin As Revealed by SSR Markers

Fig (Ficus carica L.) tree is cultivated worldwide and is highly appreciated for its fruit, which is consumed fresh or dried, having high nutritional and pharmaceutical value and for these reasons there is an increasing interest for its cultivation. In the present study, an ex situ collection of 60 fig accessions (41 indigenous Greek and 19 from other Mediterranean countries) was established and its diversity was analyzed using eight simple sequence repeat (SSR) loci. Greek fig genotypes showed relatively low allelic variation (average number of SSR alleles per locus was 3.3), an excess of heterozygosity (mean He = 0.449 and Ho = 0.537), and extensive outbreeding (mean F index -0.184). Cluster analysis showed that the established fig population exhibited weak genetic structure with the majority of the genetic variation (69%) being present within individual members of the clusters. Both cluster and principal coordinate analysis confirmed that there is no correlation between genetic makeup and geographical origin of the fig accessions. Polymorphism information content (PIC) with an average of 0.398 was reasonably informative. An identification key scheme for fig cultivars that will be useful in cultivar discrimination and intellectual property protection was developed. This work will contribute to a sustainable fig production regionally and worldwide, through the establishment and conservation of a reference fig collection, providing germplasm for future breeding efforts.

Search of the new SSR-loci of DNA for development of effective technology for soybean genotyping

Soybean is the major protein-oil crop of a huge economic importance. Currently, to describe the new cultivars being applied for a patent there are used the modern methods based on an analysis of microsatellite (SSR) loci of DNA. The purposes of this work were a search of the new microsatellite markers to optimize the existing technology of soybean cultivars certification and identification as well as selection of conditions for PCR analysis and to test them on cultivars from the VIR’s collection. Seven microsatellite loci demonstrated the high polymorphism level on soybean cultivars and located in the different chromosomes were chosen in the literary sources and librarian data bases. The optimal temperatures for annealing were selected empirically for all the pairs of SSR-markers. The results of DNA amplification of 20 soybean genotypes showed all seven studied SSR-loci were polyallel. In general, we revealed 22 alleles that on average are 3.1 per a locus. The effective number of alleles Ne for the studied soybean genotypes varied from 1.69 to 2.27 and on average was equal to 2.01. An average meaning of an index of the polymorphic information content (PIC) was 0.50. All the investigated soybean samples have the unique sets of alleles by the studied loci. Seven approbated loci can be used in development of an effective technology for identification and certification of the soybean genotypes.

Analyses of Lysin-Motif Receptor-like Kinase (LysM-RLK) Gene Family in Allotetraploid Brassica napus L. and Its Progenitor Species: An In Silico Study

The LysM receptor-like kinases (LysM-RLKs) play a crucial role in plant symbiosis and response to environmental stresses. Brassica napus, B. rapa, and B. oleracea are utilized as valuable vegetables. Different biotic and abiotic stressors affect these crops, resulting in yield losses. Therefore, genome-wide analysis of the LysM-RLK gene family was conducted. From the genome of the examined species, 33 LysM-RLK have been found. The conserved domains of Brassica LysM-RLKs were divided into three groups: LYK, LYP, and LysMn. In the Brassica LysM-RLK gene family, only segmental duplication has occurred. The Ka/Ks ratio for the duplicated pair of genes was less than one indicating that the genes’ function had not changed over time. The Brassica LysM-RLKs contain 70 cis-elements, indicating that they are involved in stress response. 39 miRNA molecules were responsible for the post-transcriptional regulation of 12 Brassica LysM-RLKs. A total of 22 SSR loci were discovered in 16 Brassica LysM-RLKs. According to RNA-seq data, the highest expression in response to biotic stresses was related to BnLYP6. According to the docking simulations, several residues in the active sites of BnLYP6 are in direct contact with the docked chitin and could be useful in future studies to develop pathogen-resistant B. napus. This research reveals comprehensive information that could lead to the identification of potential genes for Brassica species genetic manipulation.

Assessment of tea plant (Camellia sinensis L.) accessions for pollen sources in natural crossing by using microsatellites

Tea (Camellia sinensis L. [O.] Kuntze) is a highly cross-pollinated and self-incompatible plant. Seeds can be harvested from specific individual mother plants in polyclonal tea gardens. Whether the pollen donor plays an important role in seed formation remains unclear. This study aimed to identify the male parents of 72 natural hybridized progenies (F1) from one female parent on the basis of a putative specific allele by using simple-sequence repeat (SSR) markers and the exclusion-likelihood method with Cervus 3.0 software. The genetic material, which comprised seven accessions of C. sinensis L., was acquired from Assamica planted in the Kayulandak polyclonal seed garden of the Pagilaran tea plantation in Batang District, Central Java, Indonesia, and was studied during 2019 and 2020. The genotype PGL-15 was used as the female parent, whereas the six candidate genotypes PGL-10, GMB-9, GMB-7, TPS-93, GMB-11, and TRI-2025 were used as the male parents. In this study, 13 SSR loci were used to identify the male parents of the F1 progenies obtained through natural hybridization between one female and six male tea accessions. Results indicated that the exclusion-likelihood method, which correctly predicted 100% of the male parents, was more effective than the putative specific allele approach, which correctly predicted only 34.72% of the male parents in the 72 hybridized F1 progenies of tea plants.

Identifying Traits Associated With Terminal Drought Tolerance in Sesame (Sesamum indicum L.) Genotypes

Sesame is predominantly cultivated in rainfed and low fertile lands and is frequently exposed to terminal drought. Sesamum species inhabiting dryland ecosystems adaptively diverge from those inhabiting rainfed habitats, and drought-specific traits have a genetic basis. In sesame, traits associated with drought conditions have not been explored to date, yet studies of these traits are needed given that drought is predicted to become more frequent and severe in many parts of the world because of climate change. Here, 76 accessions from the available Indian core set were used to quantify variation in several traits under irrigated (WW) and terminal drought stress (WS) conditions as well as their association with seed yield over two consecutive years. The range of trait variation among the studied genotypes under WW and WS was significant. Furthermore, the traits associated with seed yield under WW and WS differed. The per se performance of the accessions indicated that the expression of most traits was reduced under WS. The correlation analysis revealed that the number of branches, leaf area (LA), leaves dry weight (LDW), number of capsules plant–1, and harvest index (HI) were positively correlated with seed yield under WW and WS, and total dry matter (TDM), plant stem weight, and canopy temperature (CT) were negatively correlated with seed yield under WW and WS, indicating that smaller and cooler canopy genotypes had higher yields. The genotypes IC-131936, IC-204045, IC-204861, IC-205363, IC-205311, and IC-73576 with the highest seed yields were characterized by low canopy temperature, high relative water content, and high harvest index under WS. Phenotypic and molecular diversity analysis was conducted on genotypes along with checks. Phenotypic diversity was assessed using multivariate analysis, whereas molecular diversity was estimated using simple sequence repeat (SSR) loci to facilitate the use of sesame in breeding and genetic mapping. SSRs showed low allelic variation, as indicated by a low average number of alleles (2.31) per locus, gene diversity (0.25), and polymorphism information content (0.22). Cluster analysis (CA) [neighbor-joining (NJ) tree] revealed three major genotypic groups and structure analysis showed 4 populations. The diverse genotypes identified with promising morpho-physiological traits can be used in breeding programs to develop new varieties.

Genetic (SSRs) versus morphological differentiation of date palm cultivars: Fst versus Pst estimates

Date Palm (Phoenix dactylifera L.) is one of the oldest domesticated fruit trees. For future breeding program, knowledge on genetic structure of cultivars is necessary. Therefore, the present study was performed with the following aims: 1- To provide data on genetic diversity and genetic structure of 36 date palm cultivars, 2- To provide data on the association between fruit characteristics and the genetic features of the cultivars. We used nine SSRs and EST-SSR loci for our genetic investigation. The most of SSR loci obtained have a high Gst value (0.70), and therefore have a good discrimination power for date palm cultivar differentiation task. K-Means clustering grouped date palm cultivars either in two broad clusters, or in 16 smaller genetic groups. This was supported by delta K = 2 of the STRUCTURE analysis. AMOVA produced significant genetic difference among date palm cultivars (PhiPT = 0.70, P = 0.001). New genetic differentiation parameters estimated also produced significant difference among date palm cultivars (G’st(Nei) = 0.673, P =0.001; G’st(Hed) = 0.738, P = 0.001). Test of assignment revealed that some of the cultivars have 33-66% misassignment, probably due to genetic admixture. Heatmaps of genetic versus morphological/or agronomical characters in date palm cultivars differed from each other showing the cultivars morphological changes is not merely related to their genetic content. It points toward the potential role played either by environmental conditions or local selection practice. The new findings can be utilized in future conservation and breeding of date palms in the country.