Population Improvement of Resistance to Late Blight in Tetraploid Potato:A Case Study in Combination with AFLP Marker Assisted Background Selection

Cultivated potato with high level of horizontal resistance against late blight is one of the most important goals of potato breeding.The recurrent selection has been adopted to increase the level of potato horizontal resistance and a B3C1 population without R1-R11 dominant genes has been released by the International Potato Center at the short-day condition of Peru.The present research was carried out to further improve the resistance and the agronomic traits of B3C1 population under long-day condition of Hubei,China,with maximized retention of its genetic diversity.Twenty-seven individual clones of B3C1 were used to generate population B3C2 by in-population crossing with the bulk pollens aiming to elevate the frequency of late blight resistance genotypes and to improve the adaptation to local long-day conditions.The late blight resistance and the main agronomic traits including the maturity,the plant characters and the tuber traits were evaluated for the foreground selection in three years,by which 130 pedigrees were maintained as the basic population of B3C2 for further selection.A total of 312 polymorphic loci detected by 9 AFLP marker combinations were used to monitor the genetic diversity of the populations for the background selection.The B3C2 population of 51 clones was finally selected,of which the frequency of resistant genotypes increased by 23.8% points and the genetic diversity was maintained by about 96% as referred to B3C1.Our results strongly suggested that combination of the foreground selection for target traits and the background selection for the genetic diversity is an efficient strategy in the recurrent selection of tetraploid potato to improve quantitative traits.

Isolation and Characterization of Ell Gene Promoter from Tomato

A fragment of 2000 bp upstream sequence of Ell clone was amplified from genomic DNA of the tomato cultivar Zhongshu- 5. Sequence analysis showed that the upstream contains the regulatory elements： TATA box （-29 - -22）, CAAT box （-193 - -189）, wound, and drought response elements. Expression vectors of Ell promoter gus fusion were constructed with the promoters of 1 200 and 2 000 bp regions, respectively. Transgenic tomato plants were obtained through Agrobacteriummediated transformation. Histochemical analysis of GUS activity in various tissues showed that the two promoters were able to direct fruit-specific gene expression. The expression driven by promoter of 2 000 bp upstream fragment could increase GUS activity with the maturation of tomato fruits. The promoter of -1 200 bp fragment could direct gus gene expression in fruits with the inductions of drought and wounding. The regulatory region for fruit-specificity was probably located in the region of 1 200 bp of 5′-flanking sequence and some positive regulatory elements or enhancers may exist in the region from -1 200 to -2000 bp.

Interactions between ShPP2-1, an F-box family gene, and ACR11A regulate cold toleranee of tomato

There is a critical need to identify germplasm resources and genes that promote cold tolerance of tomato because global tomato production is threatened by cold stress.We found that the expression of an F-box gene family member named ShPP2-1 from Solanum habrochaites is cold inducible and studied its contribution to cold tolerance.Overexpression of ShPP2-1 in cultivated tomato(AC)reduced cold tolerance by intensifying damage to cell membranes.To explore the underlying molecular mechanism,we conducted a yeast two-hybrid library screen and found that a protein containing ACT domain repeats named ACR11A interacts with PP2-1.Overexpression of SIACR11A in AC enhanced the cold tolerance of seedlings and germinating seeds.Cold tolerance decreased in tomato plants that overexpressed both of these genes.Additionally,we performed seed germination experiments in the cold with 177 tomato accessions and identified two alleles of SlACR11A that differ in one single-nucleotide polymorphism.We found that one of these alleles,SlACR11A G,is significantly enriched in cold-tolerant tomato plants.Taken together,our fi ndings indicate that the combination of low expression levels of PP2-1 and high expression levels of ACR11A can promote cold tolerance.These genes may therefore serve as direct targets for both genetic engineering and improvement projects that aim to enhance the cold tolerance of tomato.

Alterations in Ascorbic Acid Levels by Transformation of the L-galactono-1,4-lactone Dehydrogenase Gene in Solanum tuberosum

L-galactono-1, 4-lactone dehydrogenase(GLDH) is an important enzyme that catalyzes the last step of the ascorbate biosynthetic pathways in plants. A full-length c DNA clone encoding GLDH was isolated from potato(Solanum tuberosum L. ‘Favorita') leaf and subcloned into a binary vector, p BI121, to construct sense and antisense recombinant plant expression vectors. The recombinants were introduced into potato via Agrobacterium-mediated transformation, and plants were confirmed as transgenic using PCR and quantitative real-time PCR. Two anti-sense potato lines(G1 and G2) and three sense lines(G3, G4 and G5) were obtained. The GLDH activity of G4 and G5 were increased in vivo. Moreover, the ascorbic acid(As A) and dehydroascorbate(DHA) contents were up-regulated in both leaves and tubers. However, the shoots of G1 were suppressed and its leaves were deformed. Additionally, the As A contents in G1 leaves and tubers decreased by 28.8% and 10.3%, respectively. The GLDH activity in leaves treated with L-galactono-1,4- lactone(L-Gal L) increased in all lines, as did the As A and DHA contents. These results indicate that GLDH activity plays an important role in regulating the As A level as well as the growth and development of potato plants.

Functional Characterization of Mi, a Root-knot Nematode Resistance Gene from Tomato （ Lycopersicon esculentum L.）

Root-knot nematodes （Meloidogyne spp.） cause major economic damage to numerous crop species around the world. Plant resistance is the most important attribute that is able to suppress invasion by the rootknot nematodes. In the present study, a candidate root-knot nematode resistance gene （Mi） was isolated from the resistant tomato （Lycopersicon esculentum L.） line RN-1. Expression profiling analysis revealed that this gene was expressed specifically in the roots, stems, and leaves, but not in the flowers or fruits. To verify the real function of this candidate gene, both sense and inteference RNA （RNAi） vectors were constructed. We obtained 31 transgenic plants with between one and seven copies of T-DNA inserts of sense Mi from two nematode-susceptible tomato cultivars as assayed by polymerase chain reaction （PCR） and Southern blotting analysis. Reverse transcription-PCR analysis revealed that expression levels of the Mi gene varied in different transgenic plants. Nematode assays showed that the resistance to root-knot nematodes was significantly improved in some transgenic lines compared with untransformed susceptible controls and that the resistance was heritable in selfed progeny. Loss of function via RNAi further confirmed the role of the Mi gene and the original resistant lines became susceptible to root-knot nematodes.