Development and Application of a PCR-Based Molecular Marker for the Identification of High Temperature Tolerant Cabbage (Brassica oleracea var. capitata) Genotypes

Global warming accelerates the development of high temperature (HT)- and high humidity (HH)-tolerant varieties. This is further facilitated by the identification of HTHH-tolerant genes and the development of molecular markers based on these genes. To identify genes involved in HTHH tolerance in cabbage (Brassica oleracea var. capitata), we performed RNA-seq analysis of two inbred lines, BN1 (HTHH-tolerant) and BN2 (HTHH-susceptible), and selected trehalose 6- phosphate phosphatase I-2 (BoTPPI-2) as one of the HTHH-tolerant-associated genes. We also developed a segregating F2 population from a cross between BN1 and BN2. RNA-seq results showed that BoTPPI-2 transcript levels were high in the HTHH-tolerant inbred line BN1, but not detectable in the HTHH-susceptible inbred line BN2. The expression pattern of BoTPPI-2 was not related to the expression of heat shock-related genes. Soft rot resistance, used as an indicator of HTHH tolerance, was higher in BN1 than in BN2. F2 individuals similar to BN1 with respect to phenotype appeared to be HTHH-tolerant, whereas BN2-types were susceptible to HTHH. Analysis of the genomic DNA revealed the presence of a long terminal repeat (LTR; ca. 4.6 kb) in the ninth intron of the BoTPPI-2_BN2 allele, thereby suppressing its transcription and exhibiting HTHH phenotype. Except for the LTR insertion, the sequence of BoTPPI-2_BN2 was almost identical to that of BoTPPI-2_BN1. On the basis of the LTR and BoTPPI-2 sequences, we developed a molecular marker to identify HTHH-tolerant genotypes and validated its efficiency using F2 individuals, inbred lines, and cultivars from diverse sources. The marker explained the genetic basis of HTHH tolerance in at least 80%, but not 100%, of the cabbage genotypes. Thus, additional markers associated with HTHH tolerance are needed for perfect selection.

PCR-based molecular marker for the Bdv2 Thinopyrum intermedium source of barley yellow dwarf virus resistance in wheat

Because of the importance of BYDV in wheat production worldwide, and given the difficulties of bioassaying for resistance, a molecular marker was developed for the resistance known as Bdv2 that originates on the long arm of chromosome 7Ai1 of Thinopyrum intermedium. This resistance was identified in a partial amphiploid line TAF46, a disomic addition line to wheat (L1), a telosomic addition line (7Ai1 L), and a series of recombinants and translocations. A RAPD (random amplified polymeric DNA) marker for the resistant germplasm was cloned and sequenced, and primers were designed against that sequence to produce a sequence characterised amplified region (SCAR) marker. A single PCR product is produced only with genotypes carrying the resistance from any of the available recombinants. The cloned sequence, recommended primers, and PCR protocols are described. The usefulness of the marker has been demonstrated for following Bdv2 in segregating wheat breeding germplasm, with the imminent release of a BYDV-resistant cultivar.