Xinyou Zhang

Differential gene expression in leaf tissues between mutant and wild-type genotypes response to late leaf spot in peanut (Arachis hypogaea L.)

Late leaf spot (LLS) is a major foliar disease in peanut (A. hypogaea L.) worldwide, causing significant losses of potential yield in the absence of fungicide applications. Mutants are important materials to study the function of disease-related genes. In this study, the mutant line M14 was derived from cultivar Yuanza 9102 treated with EMS. Yuanza 9102 was selected from an interspecific cross of cultivar Baisha 1016 with A. diogoi, and is resistant to several fungal diseases. By contrast, the M14 was highly susceptible to late leaf spot. RNA-Seq analysis in the leaf tissues of the M14 and its wild type Yuanza 9102 under pathogen challenge showed 2219 differentially expressed genes including1317 up-regulated genes and 902 down-regulated genes. Of these genes, 1541, 1988, 1344, 643 and 533 unigenes were obtained and annotated by public protein databases of SwissPort, TrEMBL, gene ontology (GO), KEGG and clusters of orthologous groups (COG), respectively. Differentially expressed genes (DEGs) showed that expression of inducible pathogenesis-related (PR) proteins was significantly up-regulated; in the meantime DEGs related to photosynthesis were down-regulated in the susceptible M14 in comparison to the resistant WT. Moreover, the up-regulated WRKY transcription factors and down-regulated plant hormones related to plant growth were detected in the M14. The results suggest that down-regulated chloroplast genes, up-regulated WRKY transcription factors, and depressed plant hormones related to plant growth in the M14 might coordinately render the susceptibility though there was a significant high level of PRs. Those negative effectors might be triggered in the susceptible plant by fungal infection and resulted in reduction of photosynthesis and phytohormones and led to symptom formation.

Chromosome painting of telomeric repeats reveals new evidence for genome evolution in peanut

Abstract Interspecific hybridization is an important approach to improve cultivated peanut varieties. Cytological markers such as tandem repeats will facilitate alien gene introgression in peanut. Telomeric repeats have also been frequently used in chromosome research. Most plant telomeric repeats are (TTTAGGG)n that are mainly distributed at the chromosome ends, although interstitial telomeric repeats (ITRs) are also commonly identified. In this study, the telomeric repeat was chromosomally localized in 10 Arachis species through sequential GISH (genomic in situ hybridization) and FISH (fluorescence in situ hybridization) combined with 4’,6-diamidino-2-phenylindole (DAPI) staining. Six ITRs were identified such as in the centromeric region of chromosome Bi5 in Arachis ipaensis, pericentromeric regions of chromosomes As5 in A. stenosperma, Bho7 in A. hoehnei and Av5 in A. villosa, nucleolar organizer regions of chromosomes As3 in A. stenosperma and Adi3 in A. diogoi, subtelomeric regions of chromosomes Bho9 in A. hoehnei and Adu7 in A. duranensis, and telomeric region of chromosome Es7 in A. stenophylla. The distributions of the telomeric repeat, 5S rDNA, 45S rDNA and DAPI staining pattern provided not only ways of distinguishing different chromosomes, but also karyotypes with a higher resolution that could be used in evolutionary genome research. The distribution of telomeric repeats, 5S rDNA and 45S rDNA sites in this study, along with inversions detected on the long arms of chromosomes Kb10 and Bho10, indicated frequent chromosomal rearrangements during evolution of Arachis species.

Genetic Diversity, Population Structure, and Botanical Variety of 320 Global Peanut Accessions Revealed Through Tunable Genotyping-by-Sequencing

Cultivated peanut (Arachis hypogaea L.) were classified into six botanical varieties according to the morphological characteristics. However, their genetic evolutionary relationships at the genome-wide level were still unclear. A total of 320 peanut accessions, including four of the six botanical varieties, and 37,128 high-quality single nucleotide polymorphisms (SNPs) detected by tunable genotyping-by-sequencing (tGBS) were used to reveal the evolutionary relationships among different botanical varieties and verify the phenotypic classification. A phylogenetic tree indicated that the tested accessions were grouped into three clusters. Almost all of the peanut accessions in cluster C1 belong to var. fastigiata, and clusters C2 and C3 mainly consisted of accessions from var. vulgaris and subsp. hypogaea, respectively. The results of a principal component analysis were consistent with relationships revealed in the phylogenetic tree. Population structure analysis showed that var. fastigiata and var. vulgaris were not separated when K = 2 (subgroup number), whereas they were clearly divided when K = 3. However, var. hypogaea and var. hirsuta could not be distinguished from each other all the way. The nucleotide diversity (π) value implied that var. vulgaris exhibited the highest genetic diversity (0.048), followed by var. fastigiata (0.035) and subsp. hypogaea (0.012), which is consistent with the result of phylogenetic tree. Moreover, the fixation index (FST) value confirmed that var. fastigiata and var. vulgaris were closely related to each other (FST = 0.284), while both of them were clearly distinct from var. hypogaea (FST > 0.4). The present study confirmed the traditional botanical classifications of cultivated peanut at the genome-wide level. Furthermore, the reliable SNPs identified in this study may be a valuable resource for peanut breeders.

High-resolution chromosome painting with repetitive and single-copy oligonucleotides in Arachis species identifies structural rearrangements and genome differentiation

BackgroundArachis contains 80 species that carry many beneficial genes that can be utilized in the genetic improvement of peanut (Arachis hypogaea L. 2n = 4x = 40, genome AABB). Chromosome engineering is a powerful technique by which these genes can be transferred and utilized in cultivated peanut. However, their small chromosomes and insufficient cytological markers have made chromosome identification and studies relating to genome evolution quite difficult. The development of efficient cytological markers or probes is very necessary for both chromosome engineering and genome discrimination in cultivated peanut.ResultsA simple and efficient oligonucleotide multiplex probe to distinguish genomes, chromosomes, and chromosomal aberrations of peanut was developed based on eight single-stranded oligonucleotides (SSONs) derived from repetitive sequences. High-resolution karyotypes of 16 Arachis species, two interspecific F1 hybrids, and one radiation-induced M1 plant were then developed by fluorescence in situ hybridization (FISH) using oligonucleotide multiplex, 45S and 5S rDNAs, and genomic in situ hybridization (GISH) using total genomic DNA of A. duranensis (2n = 2x = 20, AA) and A. ipaënsis (2n = 2x = 20, BB) as probes. Genomes, chromosomes, and aberrations were clearly identifiable in the established karyotypes. All eight cultivars had similar karyotypes, whereas the eight wild species exhibited various chromosomal variations. In addition, a chromosome-specific SSON library was developed based on the single-copy sequence of chromosome 6A of A. duranensis. In combination with repetitive SSONs and rDNA FISH, the single-copy SSON library was applied to identify the corresponding A3 chromosome in the A. duranensis karyotype.ConclusionsThe development of repetitive and single-copy SSON probes for FISH and GISH provides useful tools for the differentiation of chromosomes and identification of structural chromosomal rearrangement. It facilitates the development of high-resolution karyotypes and detection of chromosomal variations in Arachis species. To our knowledge, the methodology presented in this study demonstrates for the first time the correlation between a sequenced chromosome region and a cytologically identified chromosome in peanut.

Allelic Expression Variation of ahFAD2A and Its Relationship with Oleic Acid Accumulation in Peanut: Allelic Expression Variation of ahFAD2A and Its Relationship with Oleic Acid Accumulation in Peanut

花生 ahFAD2A 是控制种子油酸、亚油酸含量和油亚比的关键基因。利用 ahFAD2A 基因特异引物检测远杂9102, 豫花9416等52个花生品种的 ahFAD2A 基因等位变异, 并比较其中13个品种的 ahFAD2A 基因序列。结果表明, 花生 ahFAD2A 基因存在G-A两种单核苷酸等位变异(野生型 ahFAD2A-wt 和突变体 ahFAD2A-m ), DNA序列比对结果证实, 豫花9416等10个品种(突变体)与远杂9102、延津花籽和开农白2号(野生型)相比, 在 ahFAD2A 基因的448 bp处存在核苷酸G-A突变。应用real-time PCR检测 ahFAD2A 等位基因在种子不同发育时期的表达动态显示突变体豫花9416等位基因( ahFAD2A-m )在种子发育中期表达量稍高, 种子发育后期表达量下降速度较野生型远杂9102( ahFAD2A-wt )更快。进一步测定豫花9416和远杂9102在种子不同发育时期的油酸、亚油酸积累和油亚比动态, 发现两品种间存在明显差异, 豫花9416在籽粒发育前期油酸相对含量已超过亚油酸, 油亚比大于１并逐渐增加, 而远杂9102到籽粒发育中后期油酸相对含量才高于亚油酸, 油亚比逐渐接近于１左右。

Embryonic Development and Changes of Endogenous Hormones in Interspecific Hybrids between Peanut (A. hypogaea L.) and Wild Arachis Species

The peanut cultivar Yuhua 15 was used as female parent to cross with wild species A. correntina and A. macedoi, re- spectively, with intervarietal cross Yuhua 15×Baisha 1016 as check. There were no significant differences observed in pegging percentage among all the crosses. It indicated normal fertilization in the interspecific crosses. Embryo development was observed through paraffin sections, and contents of endogenous hormones including IAA, GA 3 , ZR+DHZR, and ABA in the developing embryos of hybrids were determined with ELISA. In comparison with normal seed development of Yuhua 15×Baisha 1016 and Yuhua 15×A. correntina, Yuhua 15×A. macedoi was incompatible due to a deterred embryo development at approximately 12–17 days after pollination followed by the hyperplasia of integument into the embryo sac which suppressed further development of the embryo and finally resulted in abortion. Compared with two compatible crosses, the ABA content and ratios of ABA to total of other auxin in developing embryos of cross (Yuhua 15×A. macedoi) were relatively higher at the early development stage, while the contents of IAA, GA 3 , and ZR+DHZR remained at a relatively lower level, which speculates on that abnormal hormone ac- tivities may be an important reason leading to the abortion of hybrid embryos. Peg bases of Yuhua 15×A. macedoi were treated with IAA, KT, or mixture of the two, showing that the exogenous hormones can increase the length of ovule.