Liping Jin

Cloning and Functional Characterization of SAD Genes in Potato

Stearoyl-acyl carrier protein desaturase (SAD), locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD) were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD) was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.

Gene transcription analysis during interaction between potato and Ralstonia solanacearum

Bacterial wilt (BW) caused by Ralstonia solanacearum (Rs) is an important quarantine disease that spreads worldwide and infects hundreds of plant species. The BW defense response of potato is a complicated continuous process, which involves transcription of a battery of genes. The molecular mechanisms of potato-Rs interactions are poorly understood. In this study, we combined suppression subtractive hybridization and macroarray hybridization to identify genes that are differentially expressed during the incompatible interaction between Rs and potato. In total, 302 differentially expressed genes were identified and classified into 12 groups according to their putative biological functions. Of 302 genes, 81 were considered as Rs resistance-related genes based on the homology to genes of known function, and they have putative roles in pathogen recognition, signal transduction, transcription factor functioning, hypersensitive response, systemic acquired resistance, and cell rescue and protection. Additionally, 50 out of 302 genes had no match or low similarity in the NCBI databases, and they may represent novel genes. Of seven interesting genes analyzed via RNA gel blot and semi-quantitative RT-PCR, six were induced, one was suppressed, and all had different transcription patterns. The results demonstrate that the response of potato against Rs is rapid and involves the induction of numerous various genes. The genes identified in this study add to our knowledge of potato resistance to Rs.

Mapping and Analysis of QTL for Early Maturity Trait in Tetraploid Potato (Solanum tuberosum L.)

Maturity is one of the important traits of potato. In order to get the genetic segment of potato early maturity trait, a tetraploid potato maturity segregation population of Zhongshu 19 × Zhongshu 3 was used for genetic analysis through the combination of high throughput simplified genome sequencing (2b-RAD) and bulked segregation analysis (BSA). A genetic segment related to the early maturity trait at the 3.7~4.2 Mb locus on the short arm of chromosome 5 was obtained and eight markers were developed based on this segment, while five of them were closely linked to the early maturity trait loci. Moreover, 42 SSR markers were developed based on the reference sequence of DM. Finally, a genetic map of chromosome 5 contained 50 markers was constructed using the Tetraploidmap software. The total map length was 172 cM with an average genetic distance of 3.44 cM. Combining with phenotypic data of the segregation population, we mapped the early maturity trait QTL with the contribution of 33.55% on the short arm of chromosome 5, located at 84cM between the flanking markers SSR5-85-1 and SCAR5-8 with the physical interval of 471kb. Gene annotation showed that there exist 34 genes in this region, 12 of them are unknown function. Among the other 22 annotated genes, E3 ubiquitin ligase gene PUB14 may be related to maturity and regulate tuber formation. Our fine mapping of the early maturity QTL made a solid foundation for cloning of the early maturity controlled gene or genes. Key message Early maturity site was mapped using a tetraploid potato segregation population derived from cv. Zhongshu 19 and Zhongshu 3. One major QTL with 33.55% contribution to early maturity was fine mapped in physical interval of 471kb on chromosome 5.

Mapping and QTL Analysis of Early-Maturity Traits in Tetraploid Potato (Solanum tuberosum L.)

Early maturity is one of the most important agronomical traits in potato breeding. To identify the DNA segment that codes for early maturity, a tetraploid potato segregation population of “Zhongshu 19” × “Zhongshu 3” was genetically analyzed, using a combination of high throughput simplified genome sequencing (2b-RAD) and bulked segregant analysis (BSA). The DNA segment related to the early-maturity trait was identified at the 3.7~4.2 Mb locus on the short arm of chromosome 5. Eight molecular markers were developed, of which five were closely linked to the early-maturity trait loci. Additionally, 42 simple sequence repeats (SSR) markers were constructed based on the reference sequence of Solanum tuberosum group Phureja DM1-3 516 R44 (DM). Using the TetraploidMap software, the linkage map of chromosome 5 was constructed with 50 markers. The total map length was 172 centiMorgan (cM), with an average genetic distance of 3.44 cM. Correlating molecular and phenotypic data of the segregating population, the mapped Quantitative Trait Loci (QTL) on the short arm of chromosome 5 contributed to 33.55% of the early-maturity phenotype. The early-maturity QTL was located at 84 cM, flanked by the SSR5-85-1 and SCAR5-8 markers. The QTL was fine-mapped to 471 kb. Using DNA sequence annotation, 34 genes were identified in this region, 12 of them with unknown function. Among the other 22 annotated genes, E3 ubiquitin ligase gene PUB14 could be related to maturity and regulation of tuber formation. The constructed QTL map is a useful basic tool for the cloning of early-maturity related genes in tetraploid potatoes.

Pedigree-Based Deciphering of Genome-Wide Conserved Patterns in an Elite Potato Parental Line

Elite parental lines are more likely to breed fine varieties, but knowledge about elite parents and their genetic backgrounds is limited. In this paper, we investigated the pedigree relationships of potato varieties bred worldwide and in China. Several elite parents were identified, and these parents were more frequently used as parents in breeding programs across different time periods and countries. We next used 2b-RAD, a reduced-representation sequencing method, to genotype the elite parent Mira and 24 of its offspring. These cultivars span 5 generations, making this lineage the longest continuous pedigree among Chinese bred potatoes. A total of 47,314 tetraploid single nucleotide polymorphisms (SNPs) identified by FreeBayes were used to trace the conserved segments of the Mira genome. The conserved segments had identical or similar allele-specific SNPs across the analyzed genotypes. In Mira, 3,788 segments comprising over 10,000 bp, or 20.8% of the genome, were defined as conserved segments. These segments contain genes involved in crucial biological processes that are of special interest to breeders. These regions, which have been conserved across generations of highly selective breeding, may be helpful for further breeding and performing genome-wide breeding by design.

NBS Profiling Identifies Potential Novel Locus from Solanum demissum That Confers Broad-Spectrum Resistance to Phytophthora infestans

Abstract Potato late blight, caused by the oomycete pathogen Phytophthora infestans , is the most serious disease of potato worldwide. The adoption of varieties with resistance genes, especially broad-spectrum resistance genes, is the most efficient approach to control late blight. Solanum demissum is a well-known wild potato species from which 11 race-specific resistance genes have been identified, however, no broad-spectrum resistance genes like RB have been reported in this species. Here, we report a novel reisistance locus from S. demissum that potentially confer broad-spectrum resistance to late blight. A small segregating population of S. demissum were assessed for resistance to aggressive P. infestans isolates (race 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11). This coupled with nucleotide binding site (NBS) profiling analyses, led to the identification of three fragments that linked to the potential candidate resistance gene(s). Cloning and sequence analysis of these fragments suggested that the identified resistance gene locus is located in the region containing R2 resistance gene at chromosome 4. Based on the sequences of the cloned fragments, a co-segregating sequence characterized amplified region (SCAR) marker, RDSP, was developed. The newly identified marker RDSP will be useful for marker assisted breeding and further cloning of this potential resistance gene locus.