Emerging Horizons in Plant Genetics and Breeding.

Plant genetics and breeding have made significant progress in recent years, especially with the emergence of genomics [...].

Maintenance and expansion of genetic and trait variation following domestication in a clonal crop.

Clonal propagation enables favourable crop genotypes to be rapidly selected and multiplied. However, the absence of sexual propagation can lead to low genetic diversity and accumulation of deleterious mutations, which may eventually render crops less resilient to pathogens or environmental change. To better understand this trade-off, we characterize the domestication and contemporary genetic diversity of Enset (Ensete ventricosum), an indigenous African relative of bananas (Musa) and a principal starch staple for 20 million Ethiopians. Wild enset reproduction occurs strictly by sexual outcrossing, but for cultivation, it is propagated clonally and associated with diversification and specialization into hundreds of named landraces. We applied tGBS sequencing to generate genome-wide genotypes for 192 accessions from across enset's cultivated distribution, and surveyed 1340 farmers on enset agronomic traits. Overall, reduced heterozygosity in the domesticated lineage was consistent with a domestication bottleneck that retained 37% of wild diversity. However, an excess of putatively deleterious missense mutations at low frequency present as heterozygotes suggested an accumulation of mutational load in clonal domesticated lineages. Our evidence indicates that the major domesticated lineages initially arose through historic sexual recombination associated with a domestication bottleneck, followed by the amplification of favourable genotypes through an extended period of clonal propagation. Among domesticated lineages, we found a significant phylogenetic signal for multiple farmer-identified food, nutrition and disease resistance traits and little evidence of contemporary recombination. The development of future-climate adapted genotypes may require crop breeding, but outcrossing risks exposing deleterious alleles as homozygotes. This trade-off may partly explain the ubiquity and persistence of clonal propagation over recent centuries of comparative climate stability.

Genome Assembly and Analysis of the Flavonoid and Phenylpropanoid Biosynthetic Pathways in Fingerroot Ginger (Boesenbergia rotunda).

Boesenbergia rotunda (Zingiberaceae), is a high-value culinary and ethno-medicinal plant of Southeast Asia. The rhizomes of this herb have a high flavanone and chalcone content. Here we report the genome analysis of B. rotunda together with a complete genome sequence as a hybrid assembly. B. rotunda has an estimated genome size of 2.4 Gb which is assembled as 27,491 contigs with an N50 size of 12.386 Mb. The highly heterozygous genome encodes 71,072 protein-coding genes and has a 72% repeat content, with class I TEs occupying ~67% of the assembled genome. Fluorescence in situ hybridization of the 18 chromosome pairs at the metaphase showed six sites of 45S rDNA and two sites of 5S rDNA. An SSR analysis identified 238,441 gSSRs and 4604 EST-SSRs with 49 SSR markers common among related species. Genome-wide methylation percentages ranged from 73% CpG, 36% CHG and 34% CHH in the leaf to 53% CpG, 18% CHG and 25% CHH in the embryogenic callus. Panduratin A biosynthetic unigenes were most highly expressed in the watery callus. B rotunda has a relatively large genome with a high heterozygosity and TE content. This assembly and data (PRJNA71294) comprise a source for further research on the functional genomics of B. rotunda, the evolution of the ginger plant family and the potential genetic selection or improvement of gingers.

A chromosome-level reference genome of Ensete glaucum gives insight into diversity and chromosomal and repetitive sequence evolution in the Musaceae.

BACKGROUND: Ensete glaucum (2n = 2x = 18) is a giant herbaceous monocotyledonous plant in the small Musaceae family along with banana (Musa). A high-quality reference genome sequence assembly of E. glaucum is a resource for functional and evolutionary studies of Ensete, Musaceae, and the Zingiberales. FINDINGS: Using Oxford Nanopore Technologies, chromosome conformation capture (Hi-C), Illumina and RNA survey sequence, supported by molecular cytogenetics, we report a high-quality 481.5 Mb genome assembly with 9 pseudo-chromosomes and 36,836 genes. A total of 55% of the genome is composed of repetitive sequences with predominantly LTR-retroelements (37%) and DNA transposons (7%). The single 5S ribosomal DNA locus had an exceptionally long monomer length of 1,056 bp, more than twice that of the monomers at multiple loci in Musa. A tandemly repeated satellite (1.1% of the genome, with no similar sequence in Musa) was present around all centromeres, together with a few copies of a long interspersed nuclear element (LINE) retroelement. The assembly enabled us to characterize in detail the chromosomal rearrangements occurring between E. glaucum and the x = 11 species of Musa. One E. glaucum chromosome has the same gene content as Musa acuminata, while others show multiple, complex, but clearly defined evolutionary rearrangements in the change between x= 9 and 11. CONCLUSIONS: The advance towards a Musaceae pangenome including E. glaucum, tolerant of extreme environments, makes a complete set of gene alleles, copy number variation, and a reference for structural variation available for crop breeding and understanding environmental responses. The chromosome-scale genome assembly shows the nature of chromosomal fusion and translocation events during speciation, and features of rapid repetitive DNA change in terms of copy number, sequence, and genomic location, critical to understanding its role in diversity and evolution.

The Genetic Diversity of Enset (Ensete ventricosum) Landraces Used in Traditional Medicine Is Similar to the Diversity Found in Non-medicinal Landraces.

Enset (Ensete ventricosum) is a multipurpose crop extensively cultivated in southern and southwestern Ethiopia for human food, animal feed, and fiber. It has immense contributions to the food security and rural livelihoods of 20 million people. Several distinct enset landraces are cultivated for their uses in traditional medicine. These landraces are vulnerable to various human-related activities and environmental constraints. The genetic diversity among the landraces is not verified to plan conservation strategy. Moreover, it is currently unknown whether medicinal landraces are genetically differentiated from other landraces. Here, we characterize the genetic diversity of medicinal enset landraces to support effective conservation and utilization of their diversity. We evaluated the genetic diversity of 51 enset landraces, of which 38 have reported medicinal value. A total of 38 alleles across the 15 simple sequence repeat (SSR) loci and a moderate level of genetic diversity (He = 0.47) were detected. Analysis of molecular variation (AMOVA) revealed that only 2.4% of the total genetic variation was contributed by variation among the medicinal and non-medicinal groups of landraces, with an FST of 0.024. A neighbor-joining tree showed four separate clusters with no correlation to the use-values of the landraces. Except for two, all "medicinal" landraces with distinct vernacular names were found to be genetically different, showing that vernacular names are a good indicator of genetic distinctiveness in these specific groups of landraces. The discriminant analysis of the principal components also confirmed the absence of distinct clustering between the two groups. We found that enset landraces were clustered irrespective of their use-value, showing no evidence for genetic differentiation between the enset grown for 'medicinal' uses and non-medicinal landraces. This suggests that enset medicinal properties may be restricted to a more limited number of genotypes, might have resulted from the interaction of genotype with the environment or management practice, or partly misreported. The study provides baseline information that promotes further investigations in exploiting the medicinal value of these specific landraces.

Genome-Wide Novel Genic Microsatellite Marker Resource Development and Validation for Genetic Diversity and Population Structure Analysis of Banana.

Trait tagging through molecular markers is an important molecular breeding tool for crop improvement. SSR markers encoded by functionally relevant parts of a genome are well suited for this task because they may be directly related to traits. However, a limited number of these markers are known for Musa spp. Here, we report 35136 novel functionally relevant SSR markers (FRSMs). Among these, 17,561, 15,373 and 16,286 FRSMs were mapped in-silico to the genomes of Musa acuminata, M. balbisiana and M. schizocarpa, respectively. A set of 273 markers was validated using eight accessions of Musa spp., from which 259 markers (95%) produced a PCR product of the expected size and 203 (74%) were polymorphic. In-silico comparative mapping of FRSMs onto Musa and related species indicated sequence-based orthology and synteny relationships among the chromosomes of Musa and other plant species. Fifteen FRSMs were used to estimate the phylogenetic relationships among 50 banana accessions, and the results revealed that all banana accessions group into two major clusters according to their genomic background. Here, we report the first large-scale development and characterization of functionally relevant Musa SSR markers. We demonstrate their utility for germplasm characterization, genetic diversity studies, and comparative mapping in Musa spp. and other monocot species. The sequences for these novel markers are freely available via a searchable web interface called Musa Marker Database.

Transcriptome wide SSR discovery cross-taxa transferability and development of marker database for studying genetic diversity population structure of Lilium species.

Lily belongs to family liliaceae, which mainly propagates vegetatively. Therefore, sufficient number of polymorphic, informative, and functional molecular markers are essential for studying a wide range of genetic parameters in Lilium species. We attempted to develop, characterize and design SSR (simple sequence repeat) markers using online genetic resources for analyzing genetic diversity and population structure of Lilium species. We found di-nucleotide repeat motif were more frequent (4684) within 0.14 gb (giga bases) transcriptome than other repeats, of which was two times higher than tetra-repeat motifs. Frequency of di-(AG/CT), tri-(AGG/CTT), tetra-(AAAT), penta-(AGAGG), and hexa-(AGAGGG) repeats was 34.9%, 7.0%, 0.4%, 0.3%, and 0.2%, respectively. A total of 3607 non-redundant SSR primer pairs was designed based on the sequences of CDS, 5'-UTR and 3'-UTR region covering 34%, 14%, 23%, respectively. Among them, a sub set of primers (245 SSR) was validated using polymerase chain reaction (PCR) amplification, of which 167 primers gave expected PCR amplicon and 101 primers showed polymorphism. Each locus contained 2 to 12 alleles on average 0.82 PIC (polymorphic information content) value. A total of 87 lily accessions was subjected to genetic diversity analysis using polymorphic SSRs and found to separate into seven groups with 0.73 to 0.79 heterozygosity. Our data on large scale SSR based genetic diversity and population structure analysis may help to accelerate the breeding programs of lily through utilizing different genomes, understanding genetics and characterizing germplasm with efficient manner.

The landscape of microsatellites in the enset (Ensete ventricosum) genome and web-based marker resource development.

Ensete ventricosum (Musaceae, enset) is an Ethiopian food security crop. To realize the potential of enset for rural livelihoods, further knowledge of enset diversity, genetics and genomics is required to support breeding programs and conservation. This study was conducted to explore the enset genome to develop molecular markers, genomics resources, and characterize enset landraces while giving insight into the organization of the genome. We identified 233 microsatellites (simple sequence repeats, SSRs) per Mbp in the enset genome, representing 0.28% of the genome. Mono- and di-nucleotide repeats motifs were found in a higher proportion than other classes of SSR-motifs. In total, 154,586 non-redundant enset microsatellite markers (EMM) were identified and 40 selected for primer development. Marker validation by PCR and low-cost agarose gel electrophoresis revealed that 92.5% were polymorphic, showing a high PIC (Polymorphism Information Content; 0.87) and expected heterozygosity (He = 0.79-0.82). In silico analysis of genomes of closely related species showed 46.86% of the markers were transferable among enset species and 1.90% were transferable to Musa. The SSRs are robust (with basic PCR methods and agarose gel electrophoresis), informative, and applicable in measuring enset diversity, genotyping, selection and potentially breeding. Enset SSRs are available in a web-based database at https://enset-project.org/EnMom@base.html (or https://enset.aau.edu.et/index.html , downloadable from Figshare).

Transcriptome Analysis by RNA-Seq Reveals Genes Related to Plant Height in Two Sets of Parent-hybrid Combinations in Easter lily (Lilium longiflorum).

In this study, two different hybrids of Easter lily (Lilium longiflorum), obtained from two cross combinations, along with their four parents were sequenced by high-throughput RNA-sequencing (RNA-Seq) to find out differentially expressed gene in parent-hybrid combinations. The leaf mRNA profiles of two hybrids and their four parents were RNA-sequenced with a view to identify the potential candidate genes related to plant height heterosis. In both cross combinations, based to morphological traits mid-parent heterosis (MPH) was higher than high-parent heterosis (HPH) for plant height, leaf length, and number of flowers whereas HPH was higher than MPH for flowering time. A total of 4,327 differentially expressed genes (DEGs) were identified through RNA-Seq between the hybrids and their parents based on fold changes (FC) ≥ 2 for up- and ≤ -2 for down-regulation. Venn diagram analysis revealed that there were 703 common DEGs in two hybrid combinations, those were either up- or down-regulated. Most of the commonly expressed DEGs exhibited higher non-additive effects especially overdominance (75.9%) rather than additive (19.4%) and dominance (4.76%) effects. Among the 384 functionally annotated DEGs identified through Blast2GO tool, 12 DEGs were up-regulated and 16 of them were down-regulated in a similar fashion in both hybrids as revealed by heat map analysis. These 28 universally expressed DEGs were found to encode different types of proteins and enzymes those might regulate heterosis by modulating growth, development and stress-related functions in lily. In addition, gene ontology (GO) analysis of 260 annotated DEGs revealed that biological process might play dominant role in heterotic expression. In this first report of transcriptome sequencing in Easter lily, the notable universally up-regulated DEGs annotated ABC transporter A family member-like, B3 domain-containing, disease resistance RPP13/1, auxin-responsive SAUR68-like, and vicilin-like antimicrobial peptides 2-2 proteins those were perhaps associated with plant height heterosis. The genes expressed universally due to their overdominace function perhaps influenced MPH for greater plant height- largely by modulating biological processes involved therein. The genes identified in this study might be exploited in heterosis breeding for plant height of L. longiflorum.

Transcriptional regulation of anthocyanin biosynthesis in a high-anthocyanin resynthesized Brassica napus cultivar.

BACKGROUND: Anthocyanins are plant secondary metabolites with key roles in attracting insect pollinators and protecting against biotic and abiotic stresses. They have potential health-promoting effects as part of the human diet. Anthocyanin biosynthesis has been elucidated in many species, enabling the development of anthocyanin-enriched fruits, vegetables, and grains; however, few studies have investigated Brassica napus anthocyanin biosynthesis. RESULTS: We developed a high-anthocyanin resynthesized B. napus line, Rs035, by crossing anthocyanin-rich B. rapa (A genome) and B. oleracea (C genome) lines, followed by chromosome doubling. We identified and characterized 73 and 58 anthocyanin biosynthesis genes in silico in the A and C genomes, respectively; these genes showed syntenic relationships with 41 genes in Arabidopsis thaliana and B. napus. Among the syntenic genes, twelve biosynthetic and six regulatory genes showed transgressively higher expression in Rs035, and eight structural genes and one regulatory gene showed additive expression. We identified three early-, four late-biosynthesis pathways, three transcriptional regulator genes, and one transporter as putative candidates enhancing anthocyanin accumulation in Rs035. Principal component analysis and Pearson's correlation coefficients corroborated the contribution of these genes to anthocyanin accumulation. CONCLUSIONS: Our study lays the foundation for producing high-anthocyanin B. napus cultivars. The resynthesized lines and the differentially expressed genes we have identified could be used to transfer the anthocyanin traits to other commercial rapeseed lines using molecular and conventional breeding.

SNP discovery of Korean short day onion inbred lines using double digest restriction site-associated DNA sequencing.

Onion (Allium cepa L.) is an economically important vegetable crop around the world. Genetic and genomic research into various onion accessions will provide insights into the onion genome to enhance breeding strategies and improve crops. However, the onion's large genome size means that studies of molecular markers are limited in onion. This study aimed to discover high quality single nucleotide polymorphisms (SNPs) from 192 onion inbred lines relating to short-day cultivation in Korea. Paired-end (PE) double digested restriction site-associated DNA sequencing (ddRAD-seq) was used to discover SNPs in onion. A total of 538,973,706 reads (25.9 GB), with an average of 2,658,491 high-quality reads, were generated using ddRAD-seq. With stringent filtering, 1904 SNPs were discovered based on onion reference scaffolds. Further, population structure and genetic relationship studies suggested that two well-differentiated sub-populations exist in onion lines. SNP-associated flanking sequences were also compared with a public non-redundant database for gene ontology and pathway analysis. To our knowledge, this is the first report to identify high-quality SNPs in onion based on reference sequences using the ddRAD-seq platform. The SNP markers identified will be useful for breeders and the research community to deepen their understanding, enhance breeding programs, and support the management of onion genomic resources.

LSAT: Liliaceae Simple Sequences Analysis Tool, a web server.

LSAT is a web-based microsatellite SSR marker designer tool specific for the Liliaceae family. It is developed using HTML, CSS, PHP, Perl and Java scripts. It works without extra add-ons on standard browsers. LSAT provides SSR primer designing service using the web interface. It helps in SSR mining and primer design. LSAT is user friendly with customizable search parameters producing visual output having download options. The current version of LSAT is backed by two data sets, namely, lily EST (Expressed Sequence Tag) from NCBI and lily nr (non redundant) with 4,099 and 216,768 unigenes, respectively. LSAT will be updated regularly upon availability of additional data (either EST and/or transcriptome) on Liliaceae. AVAILABILITY: LSAT is available for free at http://210.110.86.160/Lsat/Lsat.html.

Whole-genome sequencing of Brassica oleracea var. capitata reveals new diversity of the mitogenome.

Plant mitochondrial genomes (mtDNAs) vary in sequence structure. We assembled the Brassica oleracea var. capitata mtDNA using a mean coverage depth of 25X whole genome sequencing (WGS) and confirmed the presence of eight contigs/fragments by BLASTZ using the previously reported KJ820683 and AP012988 mtDNA as reference. Assembly of the mtDNA sequence reads resulted in a circular structure of 219,975 bp. Our assembled mtDNA, NCBI acc. no. KU831325, contained 34 protein-coding genes, 3 rRNA genes, and 19 tRNA genes with similarity to the KJ820683 and AP012988 reference mtDNA. No large repeats were found in the KU831325 assembly. However, KU831325 showed differences in the arrangement of bases at different regions compared to the previously reported mtDNAs. In the reference mtDNAs KJ820683 and AP012988, contig/fragment number 4 is partitioned into two contigs/fragments, 4a and 4b. However, contig/fragment number 4 was a single contig/fragment with 29,661 bp in KU831325. PCR and qRT-PCR using flanking markers from separate parts of contig/fragment number 4 confirmed it to be a single contig/fragment. In addition, genome re-alignment of the plastid genome and mtDNAs supported the presence of heteroplasmy and reverse arrangement of the heteroplasmic blocks within the other mtDNAs compared to KU831325 that might be one of the causal factors for its diversity. Our results thus confirm the existence of different mtDNAs in diverse B. oleracea subspecies.

Genome-Wide Identification, Characterization, and Expression Profiling of Glutathione S-Transferase (GST) Family in Pumpkin Reveals Likely Role in Cold-Stress Tolerance.

Plant growth and development can be adversely affected by cold stress, limiting productivity. The glutathione S-transferase (GST) family comprises important detoxifying enzymes, which play major roles in biotic and abiotic stress responses by reducing the oxidative damage caused by reactive oxygen species. Pumpkins (Cucurbitamaxima) are widely grown, economically important, and nutritious; however, their yield can be severely affected by cold stress. The identification of putative candidate genes responsible for cold-stress tolerance, including the GST family genes, is therefore vital. For the first time, we identified 32 C. maxima GST (CmaGST) genes using a combination of bioinformatics approaches and characterized them by expression profiling. These CmaGST genes represent seven of the 14 known classes of plant GSTs, with 18 CmaGSTs categorized into the tau class. The CmaGSTs were distributed across 13 of pumpkin's 20 chromosomes, with the highest numbers found on chromosomes 4 and 6. The large number of CmaGST genes resulted from gene duplication; 11 and 5 pairs of CmaGST genes were segmental- and tandem-duplicated, respectively. In addition, all CmaGST genes showed organ-specific expression. The expression of the putative GST genes in pumpkin was examined under cold stress in two lines with contrasting cold tolerance: cold-tolerant CP-1 (C. maxima) and cold-susceptible EP-1 (Cucurbita moschata). Seven genes (CmaGSTU3, CmaGSTU7, CmaGSTU8, CmaGSTU9, CmaGSTU11, CmaGSTU12, and CmaGSTU14) were highly expressed in the cold-tolerant line and are putative candidates for use in breeding cold-tolerant crop varieties. These results increase our understanding of the cold-stress-related functions of the GST family, as well as potentially enhancing pumpkin breeding programs.

Exploration and Exploitation of Novel SSR Markers for Candidate Transcription Factor Genes in Lilium Species.

Lilies (Lilium sp.) are commercially important horticultural crops widely cultivated for their flowers and bulbs. Here, we conducted large-scale data mining of the lily transcriptome to develop transcription factor (TF)-associated microsatellite markers (TFSSRs). Among 216,768 unigenes extracted from our sequence data, 6966 unigenes harbored simple sequence repeats (SSRs). Seventy-one SSRs were associated with TF genes, and these were used to design primers and validate their potential as markers. These 71 SSRs were accomplished with 31 transcription factor families; including bHLH, MYB, C2H2, ERF, C3H, NAC, bZIP, and so on. Fourteen highly polymorphic SSRs were selected based on Polymorphic Information Content (PIC) values and used to study genetic diversity and population structure in lily accessions. Higher genetic diversity was observed in Longiflorum compared to Oriental and Asiatic populations. Lily accessions were divided into three sub-populations based in our structure analysis, and an un-rooted neighbor-joining tree effectively separated the accessions according to Asiatic, Oriental, and Longiflorum subgroups. Finally, we showed that 46 of the SSR-associated genes were differentially expressed in response to Botrytiselliptica infection. Thus, our newly developed TFSSR markers represent a powerful tool for large-scale genotyping, high-density and comparative mapping, marker-aided backcrossing, and molecular diversity analysis of Lilium sp.

Genome-wide characterization and expression profiling of PDI family gene reveals function as abiotic and biotic stress tolerance in Chinese cabbage (Brassica rapa ssp. pekinensis).

BACKGROUND: Protein disulfide isomerase (PDI) and PDI-like proteins contain thioredoxin domains that catalyze protein disulfide bond, inhibit aggregation of misfolded proteins, and function in isomerization during protein folding in endoplasmic reticulum and responses during abiotic stresses.Chinese cabbage is widely recognized as an economically important, nutritious vegetable, but its yield is severely hampered by various biotic and abiotic stresses. Because of, it is prime need to identify those genes whose are responsible for biotic and abiotic stress tolerance. PDI family genes are among of them. RESULTS: We have identified 32 PDI genes from the Br135K microarray dataset, NCBI and BRAD database, and in silico characterized their sequences. Expression profiling of those genes was performed using cDNA of plant samples imposed to abiotic stresses; cold, salt, drought and ABA (Abscisic Acid) and biotic stress; Fusarium oxysporum f. sp. conglutinans infection. The Chinese cabbage PDI genes were clustered in eleven groups in phylogeny. Among them, 15 PDI genes were ubiquitously expressed in various organs, while 24 PDI genes were up-regulated under salt and drought stress. By contrast, cold and ABA stress responsive gene number were ten and nine, respectively. In case of F. oxysporum f. sp. conglutinans infection 14 BrPDI genes were highly up-regulated. Interestingly, BrPDI1-1 gene was identified as putative candidate against abiotic (salt and drought) and biotic stresses, BrPDI5-2 gene for ABA stress, and BrPDI1-4, 6-1 and 9-2 were putative candidate genes for both cold and chilling injury stresses. CONCLUSIONS: Our findings help to elucidate the involvement of PDI genes in stress responses, and they lay the foundation for functional genomics in future studies and molecular breeding of Brassica rapa crops. The stress-responsive PDI genes could be potential resources for molecular breeding of Brassica crops resistant to biotic and abiotic stresses.

Genome-wide expression profiling of aquaporin genes confer responses to abiotic and biotic stresses in Brassica rapa.

BACKGROUND: Plants contain a range of aquaporin (AQP) proteins, which act as transporter of water and nutrient molecules through living membranes. AQPs also participate in water uptake through the roots and contribute to water homeostasis in leaves. RESULTS: In this study, we identified 59 AQP genes in the B. rapa database and Br135K microarray dataset. Phylogenetic analysis revealed four distinct subfamilies of AQP genes: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like intrinsic proteins (NIPs) and small basic intrinsic proteins (SIPs). Microarray analysis showed that the majority of PIP subfamily genes had differential transcript abundance between two B. rapa inbred lines Chiifu and Kenshin that differ in their susceptibility to cold. In addition, all BrPIP genes showed organ-specific expression. Out of 22 genes, 12, 7 and 17 were up-regulated in response to cold, drought and salt stresses, respectively. In addition, 18 BrPIP genes were up-regulated under ABA treatment and 4 BrPIP genes were up-regulated upon F. oxysporum f. sp. conglutinans infection. Moreover, all BrPIP genes showed down-regulation under waterlogging stress, reflecting likely the inactivation of AQPs controlling symplastic water movement. CONCLUSIONS: This study provides a comprehensive analysis of AQPs in B. rapa and details the expression of 22 members of the BrPIP subfamily. These results provide insight into stress-related biological functions of each PIP gene of the AQP family, which will promote B. rapa breeding programs.

Genome-Wide Computational Analysis of Musa Microsatellites: Classification, Cross-Taxon Transferability, Functional Annotation, Association with Transposons & miRNAs, and Genetic Marker Potential.

The development of organized, informative, robust, user-friendly, and freely accessible molecular markers is imperative to the Musa marker assisted breeding program. Although several hundred SSR markers have already been developed, the number of informative, robust, and freely accessible Musa markers remains inadequate for some breeding applications. In view of this issue, we surveyed SSRs in four different data sets, developed large-scale non-redundant highly informative therapeutic SSR markers, and classified them according to their attributes, as well as analyzed their cross-taxon transferability and utility for the genetic study of Musa and its relatives. A high SSR frequency (177 per Mbp) was found in the Musa genome. AT-rich dinucleotide repeats are predominant, and trinucleotide repeats are the most abundant in transcribed regions. A significant number of Musa SSRs are associated with pre-miRNAs, and 83% of these SSRs are promising candidates for the development of therapeutic SSR markers. Overall, 74% of the SSR markers were polymorphic, and 94% were transferable to at least one Musa spp. Two hundred forty-three markers generated a total of 1047 alleles, with 2-8 alleles each and an average of 4.38 alleles per locus. The PIC values ranged from 0.31 to 0.89 and averaged 0.71. We report the largest set of non-redundant, polymorphic, new SSR markers to be developed in Musa. These additional markers could be a valuable resource for marker-assisted breeding, genetic diversity and genomic studies of Musa and related species.

Genome wide characterization of short tandem repeat markers in sweet orange (Citrus sinensis).

Sweet orange (Citrus sinensis) is one of the major cultivated and most-consumed citrus species. With the goal of enhancing the genomic resources in citrus, we surveyed, developed and characterized microsatellite markers in the ≈347 Mb sequence assembly of the sweet orange genome. A total of 50,846 SSRs were identified with a frequency of 146.4 SSRs/Mbp. Dinucleotide repeats are the most frequent repeat class and the highest density of SSRs was found in chromosome 4. SSRs are non-randomly distributed in the genome and most of the SSRs (62.02%) are located in the intergenic regions. We found that AT-rich SSRs are more frequent than GC-rich SSRs. A total number of 21,248 SSR primers were successfully developed, which represents 89 SSR markers per Mb of the genome. A subset of 950 developed SSR primer pairs were synthesized and tested by wet lab experiments on a set of 16 citrus accessions. In total we identified 534 (56.21%) polymorphic SSR markers that will be useful in citrus improvement. The number of amplified alleles ranges from 2 to 12 with an average of 4 alleles per marker and an average PIC value of 0.75. The newly developed sweet orange primer sequences, their in silico PCR products, exact position in the genome assembly and putative function are made publicly available. We present the largest number of SSR markers ever developed for a citrus species. Almost two thirds of the markers are transferable to 16 citrus relatives and may be used for constructing a high density linkage map. In addition, they are valuable for marker-assisted selection studies, population structure analyses and comparative genomic studies of C. sinensis with other citrus related species. Altogether, these markers provide a significant contribution to the citrus research community.

2n megagametophyte formed via SDR contributes to tetraploidization in polyembryonic 'Nadorcott' tangor crossed by citrus allotetraploids.

KEY MESSAGE: 2 n megagametophyte formation plays an important role in polyploidization in polyembryonic citrus and is valuable for plant improvement. Tetraploid plants are frequently observed in the seedlings of diploid polyembryonic citrus genotypes. However, the mechanisms underlying the formation of tetraploids are still indistinct when apomictic citrus genotypes are used as female parent to cross with tetraploids. Herein, 54 tetraploid progenies, which were unexpectedly obtained previously from four 2x × 4x crosses using polyembryonic 'Nadorcott' tangor as seed parent, were analyzed by 22 simple sequence repeat (SSR) markers, aiming to reveal their genetic origin and the mechanism underlying 2n megagametophyte formation. The results showed that 13 tetraploids from all these four crosses were doubled diploids as indicated by their identical SSR allelic profile with their female parent; while the remaining 41 tetraploids apparently exhibited paternally derived alleles, which confirmed their zygotic origin. Furthermore, the genotyping of all hybrids indicated that all of them arose from 2n megagametophytes. Based on the genotypes of 2n megagametophytes, the analysis of maternal heterozygosity restitution (HR) for each marker showed that it varied from 0.00 to 87.80 % with a mean value of 40.89 %. In addition, it was observed that 13 markers displayed a lower rate than 50 %. On the basis of the above results, it can be speculated that the second division restitution (SDR) is the mechanism underlying the 2n megagametophyte formation in 'Nadorcott' tangor. The elucidation of the mechanism of 2n megagametophyte formation will be of great help to optimize further sexual hybridization for polyploids in citrus.