Yufang Guo

Single Nucleotide Polymorphism-based Genetic Diversity in the Reference Set of Peanut (Arachis spp.) by Developing and Applying Cost-Effective Kompetitive Allele Specific Polymerase Chain Reaction Genotyping Assays

Kompetitive allele‐specific polymerase chain reaction (KASP) assays have emerged as cost‐effective marker assays especially for molecular breeding applications. Therefore, a set of 96 informative single nucleotide polymorphisms (SNPs) was used to develop KASP assays in groundnut or peanut (Arachis spp.). Developed assays were designated as groundnut KASP assay markers (GKAMs) and screened on 94 genotypes (validation set) that included parental lines of 27 mapping populations, seven synthetic autotetraploid and amphidiploid lines, and 19 wild species accessions. As a result, 90 GKAMs could be validated and 73 GKAMs showed polymorphism in the validation set. Validated GKAMs were screened on 280 diverse genotypes of the reference set for estimating diversity features and elucidating genetic relationships. Cluster analysis of marker allelic data grouped accessions according to their genome type, subspecies, and botanical variety. The subspecies Arachis hypogaea L. subsp. fastigiata Waldron and A. hypogaea subsp. hypogaea formed distinct cluster; however, some overlaps were found indicating their frequent intercrossing during the course of evolution. The wild species, having diploid genomes, were grouped into a single cluster. The average polymorphism information content value for polymorphic GKAMs was 0.32 in the validation set and 0.31 in the reference set. These validated and highly informative GKAMs may be useful for genetics and breeding applications in Arachis species.

A high-density genetic map of Arachis duranensis, a diploid ancestor of cultivated peanut

BackgroundCultivated peanut (Arachis hypogaea) is an allotetraploid species whose ancestral genomes are most likely derived from the A-genome species, A. duranensis, and the B-genome species, A. ipaensis. The very recent (several millennia) evolutionary origin of A. hypogaea has imposed a bottleneck for allelic and phenotypic diversity within the cultigen. However, wild diploid relatives are a rich source of alleles that could be used for crop improvement and their simpler genomes can be more easily analyzed while providing insight into the structure of the allotetraploid peanut genome. The objective of this research was to establish a high-density genetic map of the diploid species A. duranensis based on de novo generated EST databases. Arachis duranensis was chosen for mapping because it is the A-genome progenitor of cultivated peanut and also in order to circumvent the confounding effects of gene duplication associated with allopolyploidy in A. hypogaea.ResultsMore than one million expressed sequence tag (EST) sequences generated from normalized cDNA libraries of A. duranensis were assembled into 81,116 unique transcripts. Mining this dataset, 1236 EST-SNP markers were developed between two A. duranensis accessions, PI 475887 and Grif 15036. An additional 300 SNP markers also were developed from genomic sequences representing conserved legume orthologs. Of the 1536 SNP markers, 1054 were placed on a genetic map. In addition, 598 EST-SSR markers identified in A. hypogaea assemblies were included in the map along with 37 disease resistance gene candidate (RGC) and 35 other previously published markers. In total, 1724 markers spanning 1081.3 cM over 10 linkage groups were mapped. Gene sequences that provided mapped markers were annotated using similarity searches in three different databases, and gene ontology descriptions were determined using the Medicago Gene Atlas and TAIR databases. Synteny analysis between A. duranensis, Medicago and Glycine revealed significant stretches of conserved gene clusters spread across the peanut genome. A higher level of colinearity was detected between A. duranensis and Glycine than with Medicago.ConclusionsThe first high-density, gene-based linkage map for A. duranensis was generated that can serve as a reference map for both wild and cultivated Arachis species. The markers developed here are valuable resources for the peanut, and more broadly, to the legume research community. The A-genome map will have utility for fine mapping in other peanut species and has already had application for mapping a nematode resistance gene that was introgressed into A. hypogaea from A. cardenasii.

QTLs for node of first fruiting branch in a cross of an upland cotton, Gossypium hirsutum L., cultivar with primitive accession Texas 701

Primitive cottons (Gossypium spp.) represent resources for genetic improvement. Most primitive accessions are photoperiod sensitive; they do not flower under the long days of the U.S. cotton belt. Molecular markers were used to locate quantitative trait loci (QTLs) for node of first fruiting branch (NFB), a trait closely related to flowering time in cotton. An F2 population consisted of 251 plants from the cross of a day neutral cultivar Deltapine 61, and a photoperiod sensitive accession Texas 701, were used in this study. Segregation in the population revealed the complex characteristics of NFB. Interval mapping and multiple QTL mapping were used to determine QTLs contributing to NFB. Three significant QTLs were mapped to chromosome 16, 21, and 25; two suggestive QTLs were mapped to chromosome 15 and 16. Four markers associated with these QTLs accounted for 33% of the variation in NFB by single and multiple-marker regression analyses. Two pairs of epistasis interaction between markers were detected. Our results suggested that at least three chromosomes contain factors associated with flowering time for this population with epistasis interactions between chromosomes. This research represent the first flowering time QTL mapping in cotton. Makers associated with flowering time may have the potential to facilitate day neutral conversion of accessions.

Use of fiber and fuzz mutants to detect QTL for yield components, seed, and fiber traits of upland cotton

This research detected QTL or molecular markers associated with yield, fiber, and seed traits within multiple fuzz and fiber loci genetic backgrounds. Two F2 populations from crosses of MD17, a fuzzless-lintless line containing three fuzzless loci, N1, n2 and a postulated n3, with line 181, fuzzless-linted and with FM966, a fuzzy-linted cultivar, were used. QTL explaining 68.3 (population with FM966) to 87.1% (population with 181) of the phenotypic variation for lint percentage and 62.8% (population with 181) for lint index were detected in the vicinity of BNL3482-138 on chromosome 26. Single marker regression analyses indicated STV79-108, on the long arm of chromosome 12 had significant association with lint percentage (R2 26.7%), lint index (R2 30.6%), embryo protein percentage (R2 15.4%) and micronaire (R2 20.0%). Two-locus epistatic interactions were also observed. Results from this research will facilitate further understanding the complex mechanisms of yield, fiber, and seed traits of cotton.

Comparative mapping in intraspecific populations uncovers a high degree of macrosynteny between A- and B-genome diploid species of peanut

BackgroundCultivated peanut or groundnut (Arachis hypogaea L.) is an important oilseed crop with an allotetraploid genome (AABB, 2n = 4x = 40). Both the low level of genetic variation within the cultivated gene pool and its polyploid nature limit the utilization of molecular markers to explore genome structure and facilitate genetic improvement. Nevertheless, a wealth of genetic diversity exists in diploid Arachis species (2n = 2x = 20), which represent a valuable gene pool for cultivated peanut improvement. Interspecific populations have been used widely for genetic mapping in diploid species of Arachis. However, an intraspecific mapping strategy was essential to detect chromosomal rearrangements among species that could be obscured by mapping in interspecific populations. To develop intraspecific reference linkage maps and gain insights into karyotypic evolution within the genus, we comparatively mapped the A- and B-genome diploid species using intraspecific F2 populations. Exploring genome organization among diploid peanut species by comparative mapping will enhance our understanding of the cultivated tetraploid peanut genome. Moreover, new sources of molecular markers that are highly transferable between species and developed from expressed genes will be required to construct saturated genetic maps for peanut.ResultsA total of 2,138 EST-SSR (expressed sequence tag-simple sequence repeat) markers were developed by mining a tetraploid peanut EST assembly including 101,132 unigenes (37,916 contigs and 63,216 singletons) derived from 70,771 long-read (Sanger) and 270,957 short-read (454) sequences. A set of 97 SSR markers were also developed by mining 9,517 genomic survey sequences of Arachis. An SSR-based intraspecific linkage map was constructed using an F2 population derived from a cross between K 9484 (PI 298639) and GKBSPSc 30081 (PI 468327) in the B-genome species A. batizocoi. A high degree of macrosynteny was observed when comparing the homoeologous linkage groups between A (A. duranensis) and B (A. batizocoi) genomes. Comparison of the A- and B-genome genetic linkage maps also showed a total of five inversions and one major reciprocal translocation between two pairs of chromosomes under our current mapping resolution.ConclusionsOur findings will contribute to understanding tetraploid peanut genome origin and evolution and eventually promote its genetic improvement. The newly developed EST-SSR markers will enrich current molecular marker resources in peanut.

Genetic detection of node of first fruiting branch in crosses of a cultivar with two exotic accessions of upland cotton

Flowering time has biological and agricultural significance for crops. In Upland cotton (Gossypium hirsutum L.), photoperiodic sensitivity is a major obstacle in the utilization of primitive accessions in breeding programs. Quantitative trait loci (QTLs) analysis was conducted in two F2 populations from the crosses between a day-neutral cultivar Deltapine 61 (DPL61) and two photoperiod sensitive G. hirsutum accessions (T1107 and T1354). Node of first fruiting branch (NFB) was used to measure relative time of flowering. Different flowering time genetic patterns were observed in the two populations. Two QTLs were found across five scoring dates, accounting 28.5 (qNFB-c21-1) and 15.9% (qNFB-c25-1) of the phenotypic variation at the last scoring date in Pop. 1107 (DPL61 by T1107); whereas, one major QTL (qNFB-c25-1) can be detected across five scoring dates, explained 63.5% of the phenotypic variation at the last scoring date in Pop. 1354 (DPL61 by T1354). QTLs with minor effects appeared at various scoring date(s), indicating their roles in regulating flowering at a lower or higher node number. Genetic segregation analysis and QTL mapping results provide further information on the mechanisms of cotton photoperiodic sensitivity.

TILLING by sequencing to identify induced mutations in stress resistance genes of peanut ( Arachis hypogaea )

BackgroundTargeting Induced Local Lesions in Genomes (TILLING) is a powerful reverse genetics approach for functional genomics studies. We used high-throughput sequencing, combined with a two-dimensional pooling strategy, with either minimum read percentage with non-reference nucleotide or minimum variance multiplier as mutation prediction parameters, to detect genes related to abiotic and biotic stress resistances. In peanut, lipoxygenase genes were reported to be highly induced in mature seeds infected with Aspergillus spp., indicating their importance in plant-fungus interactions. Recent studies showed that phospholipase D (PLD) expression was elevated more quickly in drought sensitive lines than in drought tolerant lines of peanut. A newly discovered lipoxygenase (LOX) gene in peanut, along with two peanut PLD genes from previous publications were selected for TILLING. Additionally, two major allergen genes Ara h 1 and Ara h 2, and fatty acid desaturase AhFAD2, a gene which controls the ratio of oleic to linoleic acid in the seed, were also used in our study. The objectives of this research were to develop a suitable TILLING by sequencing method for this allotetraploid, and use this method to identify mutations induced in stress related genes.ResultsWe screened a peanut root cDNA library and identified three candidate LOX genes. The gene AhLOX7 was selected for TILLING due to its high expression in seeds and roots. By screening 768 M2 lines from the TILLING population, four missense mutations were identified for AhLOX7, three missense mutations were identified for AhPLD, one missense and two silent mutations were identified for Ara h 1.01, three silent and five missense mutations were identified for Ara h 1.02, one missense mutation was identified for AhFAD2B, and one silent mutation was identified for Ara h 2.02. The overall mutation frequency was 1 SNP/1,066 kb. The SNP detection frequency for single copy genes was 1 SNP/344 kb and 1 SNP/3,028 kb for multiple copy genes.ConclusionsOur TILLING by sequencing approach is efficient to identify mutations in single and multi-copy genes. The mutations identified in our study can be used to further study gene function and have potential usefulness in breeding programs.

A Cotton-Fiber-Associated Cyclin-Dependent Kinase A Gene: Characterization and Chromosomal Location

A cotton fiber cDNA and its genomic sequences encoding an A-type cyclin-dependent kinase (GhCDKA) were cloned and characterized. The encoded GhCDKA protein contains the conserved cyclin-binding, ATP binding, and catalytic domains. Northern blot and RT-PCR analysis revealed that the GhCDKA transcript was high in 5–10 DPA fibers, moderate in 15 and 20 DPA fibers and roots, and low in flowers and leaves. GhCDKA protein levels in fibers increased from 5–15 DPA, peaked at 15 DPA, and decreased from 15 t0 20 DPA. The differential expression of GhCDKA suggested that the gene might play an important role in fiber development. The GhCDKA sequence data was used to develop single nucleotide polymorphism (SNP) markers specific for the CDKA gene in cotton. A primer specific to one of the SNPs was used to locate the CDKA gene to chromosome 16 by deletion analysis using a series of hypoaneuploid interspecific hybrids.