Surprisingly frequent chromosomal instability in cultivated peanut

A major gene controls flowering pattern in peanut, possibly encoding a TFL1-like. It was subjected to gain/loss events of a deletion and changes in mRNA expression levels, partly explaining the evolution of flowering pattern in Arachis. Flowering pattern (FP) is a major characteristic differentiating the two subspecies of cultivated peanut (Arachis hypogaea L.). Subsp. fastigiata possessing flowers on the mainstem (MSF) and a sequential FP, whereas subsp. hypogaealacks MSF and exhibits an alternate FP. FP is considered the main contributor to plant adaptability, and evidence indicates that its diversification occurred during the several thousand years of domestication. However, the genetic mechanism that controls FP in peanut is unknown. We investigated the genetics of FP in a recombinant inbred population, derivatives of an A. hypogaea by A. fastigiata cross. Lines segregated 1:1 for FP, indicating a single gene effect. Using Axiom_Arachis2 SNP-array, FP was mapped to a small segment in chromosome B02, wherein a Terminal Flowering 1-like (AhTFL1) gene with a 1492bp deletion was found in the fastigiata line, leading to a truncated protein. Remapping FP in the RIL population with the AhTFL1 indel as a marker increased the LOD score from 53.3 to 158.8 with no recombination in the RIL population. The same indel was found co-segregating with the phenotype in two independent EMS-mutagenized M2 families, suggesting a hotspot for gene conversion. Also, AhTFL1 was significantly less expressed in the fastigiata line compared to hypogaea and in flowering than non-flowering branches. Sequence analysis of the AhTFL1 in peanut world collections indicated significant conservation, supporting the putative role of AhTFL1 in peanut speciation during domestication and modern cultivation.

BackgroundGenomic research of cultivated peanut has lagged behind other crop species because of the paucity of polymorphic DNA markers found in this crop. It is necessary to identify additional DNA markers for further genetic research in peanut.ResultsMicrosatellite markers in cultivated peanut were developed using the SSR enrichment procedure. The results showed that the GA/CT repeat was the most frequently dispersed microsatellite in peanut. The primer pairs were designed for fifty-six different microsatellites, 19 of which showed a polymorphism among the genotypes studied. The average number of alleles per locus was 4.25, and up to 14 alleles were found at one locus. This suggests that microsatellite DNA markers produce a higher level of DNA polymorphism than other DNA markers in cultivated peanut.ConclusionsIt is desirable to isolate and characterize more DNA markers in cultivated peanut for more productive genomic studies, such as genetic mapping, marker-assisted selection, and gene discovery. The development of microsatellite markers holds a promise for such studies.

F-box proteins are a large gene family in plants, and play crucial roles in plant growth, development, and stress response. To date, a comprehensive investigation of F-box family genes in peanuts, and their expression pattern in lateral branch development has not been performed. In this study, a total of 95 F-box protein family members on 18 chromosomes, named AhFBX1-AhFBX95, were identified in cultivated peanut (Arachis hypogaea L.), which were classified into four groups (Group I–IV). The gene structures and protein motifs of these peanut FBX genes were highly conserved among most FBXs. We found that significant segmental duplication events occurred between wild diploid species and the allotetraploid of peanut FBXs, and observed that AhFBXs underwent strong purifying selection throughout evolution. Cis-acting elements related to development, hormones, and stresses were identified in the promoters of AhFBX genes. In silico analysis of AhFBX genes revealed expression patterns across 22 different tissues. A total of 32 genes were predominantly expressed in leaves, pistils, and the aerial gynophore tip. Additionally, 37 genes displayed tissue-specific expression specifically at the apex of both vegetative and reproductive shoots. During our analysis of transcriptome data for lateral branch development in spreading and erect varieties, namely M130 and JH5, we identified nine deferentially expressed genes (DEGs). Quantitative real-time PCR (qRT-PCR) results further confirmed the expression patterns of these DEGs. These DEGs exhibited significant differences in their expression levels at different stages between M130 and JH5, suggesting their potential involvement in the regulation of lateral branch development. This systematic research offers valuable insights into the functional dissection of AhFBX genes in regulating plant growth habit in peanut.

BackgroundPeanut (Arachis hypogaea) is an autogamous allotetraploid legume (2n = 4x = 40) that is widely cultivated as a food and oil crop. More than 6,000 DNA markers have been developed in Arachis spp., but high-density linkage maps useful for genetics, genomics, and breeding have not been constructed due to extremely low genetic diversity. Polymorphic marker loci are useful for the construction of such high-density linkage maps. The present study used in silico analysis to develop simple sequence repeat-based and transposon-based markers.ResultsThe use of in silico analysis increased the efficiency of polymorphic marker development by more than 3-fold. In total, 926 (34.2%) of 2,702 markers showed polymorphisms between parental lines of the mapping population. Linkage analysis of the 926 markers along with 253 polymorphic markers selected from 4,449 published markers generated 21 linkage groups covering 2,166.4 cM with 1,114 loci. Based on the map thus produced, 23 quantitative trait loci (QTLs) for 15 agronomical traits were detected. Another linkage map with 326 loci was also constructed and revealed a relationship between the genotypes of the FAD2 genes and the ratio of oleic/linoleic acid in peanut seed.ConclusionsIn silico analysis of polymorphisms increased the efficiency of polymorphic marker development, and contributed to the construction of high-density linkage maps in cultivated peanut. The resultant maps were applicable to QTL analysis. Marker subsets and linkage maps developed in this study should be useful for genetics, genomics, and breeding in Arachis. The data are available at the Kazusa DNA Marker Database (http://marker.kazusa.or.jp).

The concentration degree of floret flowering date (CDFFD) is an important selection index for earliness breeding of peanut, while the genetic basis of CDFFD-related traits in peanut is poorly understood. The aim of this study was to develop a high-density genetic linkage map of cultivated peanut by combining SLAF-seq (specific locus amplified fragment sequencing) with SSR (simple sequence repeat) techniques and to identify QTL (quantitative trait loci) controlling CDFFD. A RIL population derived from a cross between Silihong and Jinonghei 3 was sequenced by the HiSeq 2500 platform. A total of 1262.02 M high-quality paired-end reads were obtained, and 1,328,966 SNPs (single nucleotide polymorphisms) were developed from parents and progenies, of which 754,499 SNPs were successfully encoded and genotyping. The resulting map consisted of 3326 genetic markers (2996 SNPs and 330 SSRs) distributed in 20 linkage groups (LGs), spanning 1822.83 cM with an average distance of 0.55 cM between adjacent markers. These assigned SNP markers had average sequencing depths of 16.94-, 15.66-, and 20.94-fold in the male parent, female parent, and their progenies, respectively. Based on phenotyping in 4 environments, 15 significant QTL for the CDFFD were identified. And one major QTL for days to accumulation of 25 flowers (DA25F), qDA25F6.2, located on chromosome 6 was obtained, with a phenotypic variance explanation (PVE) of 12.49%. This map exhibited higher resolution and accuracy, which will facilitate more QTL discovery for interested agronomic and quality traits in cultivated peanut.

Genome-wide identified 116 ERF members in cultivated peanut. Comprehensive analysis shows that AhERF28 is the key factor for peanut in its response to salt and drought stresses. Ethylene response factor (ERF) belongs to the AP2/ERF (APETALA2/ERF) superfamily, which is widely involved in plant responses to various abiotic stresses, including drought and salt stresses. Although members of the ERF family have been reported in multiple plant species, the members and functions of the peanut ERF family are largely unknown. In this study, a total of 116 ERFs were identified in peanut (Arachis hypogaea L.) via genome-wide identification. The peanut ERFs were classified into ERF and DREB subfamilies. Through phylogenetic tree analysis, the 116 ERFs were divided into 11 different subgroups, and the genes in the same group had conserved motifs, exon coding sequences, and domains. The ERF family genes demonstrated differential expression across 22 tissues. Further analysis revealed that AhERFs contain a variety of cis-acting elements in their promoters. Among them, there are 6, 11, and 15 elements related to development, hormones, and abiotic/biotic stress, respectively. In addition, by analyzing the transcriptome data under salt and drought treatments and qRT-PCR verification of AhERFs, it was found that the expression level of AhERF28 increased after both salt and drought treatments. Further research indicates that silencing AhERF28 enhanced the tolerance of peanut to salt and drought stress, likely due to an increased capacity of ROS scavenging. This study identified the ERF members in the cultivated peanut and revealed the potential role of AhERF28 in salt and drought stresses, which provides a new perspective for understanding the functions of ERFs in peanut response to abiotic stress processes and a theoretical basis for crop improvement.

Abstract: Twenty-one random and 29 SSR primers were used to assess genetic variation and interrelationships among subspecies and botanical varieties of cultivated peanut, Arachis hypogaea (2n = 4x = 40), and phylogenetic relationships among cultivated peanut and wild species of the genus Arachis. In contrast with the previous generalization that peanut accessions lack genetic variation, both random and SSR primers revealed 42.7 and 54.4% polymorphism, respectively, among 220 and 124 genetic loci amplified from 13 accessions. Moreover, the dendrograms based on RAPD, ISSR, and RAPD + ISSR data precisely organized the five botanical varieties of the two subspecies into five clusters. One SSR primer was identified that could distinguish all the accessions analysed within a variety. Although the polymorphic index content varied from 0.1 to 0.5 for both ISSR and RAPD markers, primer index values were substantially higher for RAPD primers (0.35-4.65) than for SSR primers (0.35-1.73). It was possible to identify accessions, particularly those of divergent origins, by RAPD and (or) ISSR fingerprints. Based on these results, marker-based genetic improvement in A. hypogaea appears possible. None of the 486 RAPD and 330 ISSR amplification products were found to be commonly shared among 13 species of section Arachis and one species each of sections Heteranthae, Rhizomatosae, and Procumbentes. Dendrograms constructed from RAPD, ISSR, and RAPD + ISSR data showed overall similar topologies. They could be resolved into four groups corresponding to the species grouped in four taxonomic sections. The present results strongly support the view that Arachis monticola (2n = 4x = 40) and A. hypogaea are very closely related, and indicate that A. villosa and A. ipaensis are the diploid wild progenitors of these tetraploid species.

Peanut (Arachis hypogaea L.) is one of the valuable oilseed crops grown in drought-prone areas worldwide. Drought severely limits peanut production and productivity significantly. In order to decipher the drought tolerance mechanism in peanut under drought stress, RNA sequencing was performed in TAG - 24 (drought tolerant genotype) and JL-24 (drought susceptible genotype). Approximately 51million raw reads were generated from four different libraries of two genotypes subjected to drought stress exerted by 20% PEG 6000 stress and control conditions, of which ~ 41million (80.87%) filtered reads were mapped to the Arachis hypogaea L. reference genome. The transcriptome analysis detected 1,629 differentially expressed genes (DEGs), 186 genes encoding transcription factors (TFs) and 30,199 SSR among the identified DEGs. Among the differentially expressed TF encoding genes, the highest number of genes were WRKY followed by bZIP, C2H2, and MYB during drought stress. The comparative analysis between the two genotypes revealed that TAG-24 exhibits activation of certain key genes and transcriptional factors that are involved in essential biological processes. Specifically, TAG-24 showed activation of genes involved in the plant hormone signaling pathway such as PYL9, Auxin response receptor gene, and ABA. Additionally, genes related to water deprivation such as LEA protein and those involved in combating oxidative damage such as Glutathione reductase were also found to be activated in TAG-24. This genome-wide transcription map, therefore, provides a valuable tool for future transcript profiling under drought stress and enriches the genetic resources available for this important oilseed crop.

Germin-like proteins (GLPs) play diversified roles in plant development and defense response. Here, we identified 36 expressed sequence tags (ESTs) encoding GLPs from peanut (Arachis hypogaea L.). After assembly, these ESTs were integrated into eight unigenes named AhGLP1 to AhGLP8, of which, three (AhGLP1-3) were comprised 14, ten, and seven EST clones, respectively, whereas the remaining ones were associated with one single clone. The length of the deduced amino acid (AA) residues ranged from 208 to 223 AAs except for AhGLP6 and AhGLP8, which were incomplete at the carboxyl terminus. All of the AhGLPs contained a possible N-terminal signal peptide that was 17 to 24 residues in length excluding AhGLP7, where there is likely a non-cleavable amino terminus. Phylogenetic analysis showed that these AhGLPs were classified into three subfamilies. Southern blot analysis indicated that AhGLP1 and AhGLP2 likely have multiple copies in the peanut genome. The recombinant mature AhGLP1 and AhGLP2 proteins were successfully expressed in Escherichia coli. The purified AhGLP2 has superoxide dismutase (SOD) activity in enzymatic assay, but not oxalate oxidase activity. The SOD activity of AhGLP2 was stable up to 70°C and resistant to hydrogen peroxide, suggesting that AhGLP2 might be a manganese-containing SOD. Furthermore, AhGLP2 could confer E. coli resistance to oxidative damage caused by paraquat, suggesting that the AhGLP2 likely protects peanut plants from reactive oxygen metabolites. Thus, information provided in this study indicates the diverse nature of the peanut GLP family and suggests that some of AhGLPs might be involved in plant defense response.

Genome-wide analysis of the laccase gene family in Arachis hypogaea and functional characterization of AhLAC63 involved in lignin biosynthesis and abiotic stress.

SummaryCultivated peanut (Arachis hypogaea L.) is an important grain legume providing high‐quality cooking oil, rich proteins and other nutrients. Shelling percentage (SP) is the 2nd most important agronomic trait after pod yield and this trait significantly affects the economic value of peanut in the market. Deployment of diagnostic markers through genomics‐assisted breeding (GAB) can accelerate the process of developing improved varieties with enhanced SP. In this context, we deployed the QTL‐seq approach to identify genomic regions and candidate genes controlling SP in a recombinant inbred line population (Yuanza 9102 × Xuzhou 68‐4). Four libraries (two parents and two extreme bulks) were constructed and sequenced, generating 456.89–790.32 million reads and achieving 91.85%–93.18% genome coverage and 14.04–21.37 mean read depth. Comprehensive analysis of two sets of data (Yuanza 9102/two bulks and Xuzhou 68‐4/two bulks) using the QTL‐seq pipeline resulted in discovery of two overlapped genomic regions (2.75 Mb on A09 and 1.1 Mb on B02). Nine candidate genes affected by 10 SNPs with non‐synonymous effects or in UTRs were identified in these regions for SP. Cost‐effective KASP (Kompetitive Allele‐Specific PCR) markers were developed for one SNP from A09 and three SNPs from B02 chromosome. Genotyping of the mapping population with these newly developed KASP markers confirmed the major control and stable expressions of these genomic regions across five environments. The identified candidate genomic regions and genes for SP further provide opportunity for gene cloning and deployment of diagnostic markers in molecular breeding for achieving high SP in improved varieties.

Colorectal carcinoma (CRC) can be divided into two nonoverlapping groups: those that are chromosomally unstable but microsatellite stable (MSS CIN+) and those that are chromosomally stable but microsatellite unstable (MSI CIN-). However, a third group with neither chromosome nor microsatellite instability (MSS CIN-) makes a substantial contribution to the total CRC burden. The clinicopathologic features of MSS CIN- CRC are not well delineated. We assessed the relationship between age and chromosomal instability (CIN) status as measured by ploidy and allelic imbalance in a series of MSS tumors. We studied a prospectively collected series of CRC patients at Mayo Clinic Rochester. A total of 84 samples of MSS CRC in patients <or=50 years old were identified between 1994 and 1997. A consecutive series of 90 MSS CRC in patients >or=65 years old served as a comparison group. CIN status was assessed using two techniques: ploidy analysis by flow cytometry and small chromosome changes as measured by genomewide fractional allelic imbalance. CRC in the young-onset group was more likely to involve the rectum and to be high stage. MSS tumors in the young-onset group were more often diploid (46%) than those in older patients (26%; P = 0.006). This difference was maintained in the subset of MSS CRC that were high stage (42% versus 18%; P = 0.02) and in rectal cancers (50% versus 23%; P = 0.04). A greater proportion of young patients with MSS CRC has diploid tumors than patients who develop MSS CRC over age 65.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally. Adenomas, precursors to CRC, can be diagnosed early, but the genetic events leading to adenoma–adenocarcinoma conversion remain unclear. This study explored the role of chromosomal instabilities (CINs) in this conversion. Over a 17-year follow-up period, 119 adenomas were analyzed using low-coverage whole-genome sequencing (LC-WGS) and Ultrasensitive Chromosomal Aneuploidy Detector. Risk factors for adenocarcinoma development were identified through logistic regression analysis, and survival was assessed using Kaplan–Meier curves. CIN was found in 32% of adenomas, with a higher incidence in high-grade adenomas (P = 0.0359). Common chromosomal changes included loss of 18q, 1p, and 17p and gain of 8q (MYC), 20q, and 7p (EGFR). During the 17-year follow-up, 88 patients experienced recurrence, including 40 cases of adenomas and 48 cases of progression to adenocarcinoma. CIN was identified in 40% of progression cases, 33.6% of adenoma recurrence cases, and 26% of nonrecurrent cases. A strong genetic linkage was observed before and after tumor transformation, with a high match between the tumors and matched prior adenomas. CIN was significantly associated with disease progression (HR: 2.5, 95% CI: 1.4–4.5, P = 0.00162) and was an independent risk factor. Additionally, MFN2 gene copy number deletion was linked to recurrence and/or progression after resection, with reduced expression in tumor tissues. In conclusion, CIN is a key risk factor for adenoma recurrence and progression, and MFN2 gene copy number deletion is associated with adverse outcomes, providing insights for more accurate clinical prognostication of adenoma-to-adenocarcinoma transformation.

Nonrandom chromosome changes in Kaposi sarcoma: cytogenetic and FISH results in a new cell line (KS-IMM) and literature review

Cultivated peanut (Arachis hypogaea L.) consists of six botanical varieties. Identification of DNA markers associated with botanical varieties would be useful in plant genotyping, germplasm management, and evolutionary studies. We have developed 130 simple sequence repeat (SSR) markers in peanut, 38 of which were used in this study because of their ability in detecting genetic polymorphism among 24 peanut accessions. Eight SSR markers were found useful to classify botanical varieties. Among them, six SSR markers were specific to botanical varieties fastigiata and vulgaris, one to botanical varieties hypogaea and hirsuta, and one to botanical varieties peruviana, and aequatoriana. Also, three of them derived from peanut expressed sequence tags (ESTs) were associated with putative genes. As botanical varieties have different morphological traits and belong to different subspecies in A. hypogaea, these markers might be associated with genes involved in the expression of morphological traits. The results also suggested that SSRs (also called microsatellites) might play a role in shaping evolution of cultivated peanut. Multiplex PCR of botanical variety-specific markers could be applied to facilitate efficient genotyping of the peanut lines.