Introgression of heat shock protein (Hsp70 and sHsp) genes into the Malaysian elite chilli variety Kulai (Capsicum annuum L.) through the application of marker-assisted backcrossing (MAB)

Abstract

Backcrossing together with simple sequence repeat marker strategy was adopted to improve popular Malaysian chilli Kulai (Capsicum annuum L.) for heat tolerance. The use of molecular markers in backcross breeding and selection contributes significantly to overcoming the main drawbacks such as increase linkage drag and time consumption, in the ancient manual breeding approach (conventional), and speeds up the genome recovery of the recurrent parent. The strategy was adopted to introgress heat shock protein gene(s) from AVPP0702 (C. annuum L.), which are heat-tolerant, into the genetic profile of Kulai, a popular high-yielding chilli but which is heat sensitive. The parents were grown on seed trays, and parental screening was carried out with 252 simple sequence repeat markers. The selected parents were crossed and backcrossed to generate F₁ hybrids and backcross generations. Sixty-eight markers appeared to be polymorphic and were used to assess the backcross generation; BC₁F₁, BC₂F₁ and BC₃F₁. The average recipient allele of the selected four BC₁F₁ plants was 80.75% which were used to produce the BC₂F₁ generation. BC₁-P₇ was the best BC₁F₁ plant because it had the highest recovery at 83.40% and was positive to Hsp-linked markers (Hsp70-u2 and AGi42). After three successive generations of backcrossing, the average genome recovery of the recurrent parent in the selected plants in BC₃F₁ was 95.37%. Hsp gene expression analysis was carried out on BC₁F₁, BC₂F₁ and BC₃F₁ selected lines. The Hsp genes were found to be up-regulated when exposed to heat treatment. The pattern of Hsp expression in the backcross generations was similar to that of the donor parent. This confirms the successful introgression of a stress-responsive gene (Hsp) into a Kulai chilli pepper variety. Furthermore, the yield performance viz. plant height, number of fruits, fruit length and weight and total yield of the improved plant were similar with the recurrent parent except that the plant height was significantly lower than the Kulai (recurrent) parent.

Keywords: Backcrossing; Chilli; Heat shock protein 70; Heat stress; Marker-assisted selection; Simple sequence repeat (SSR) markers.

Fig. 1

Screening of parental lines (A AVPP0702 and K Kulai) for polymorphism using some of the SSR marker. Running on 2.5% metaphor agarose gel stained with midori green. M 50 bp ladder

Fig. 2

Genotyping F₁ (derived from K Kulai × A AVPP0702) using foreground markers Hsp70-u2 (14 individuals) and AGi42 (26 individuals) plus the two parents. H indicates heterozygous individuals. Running on 2.5% metaphor agarose gel stained with midori green. M 50 bp ladder

Fig. 3

Genotyping with markers Hsp70-u2 and AGi42 linked to Hsp genes in BC₁F₁ population of chilli derived from K Kulai × A AVPP0702. H indicates heterozygous individuals. Running on 2.5% metaphor agarose gel stained with midori green, only 14 samples plus the two parents for each marker are shown (M 50 bp ladder)

Fig. 4

BC₃F₁ confirmation using foreground markers Hsp70-u2 (9 individuals) and AGi42 (8 individuals). A AVPP0702 and K Kulai; H heterozygous region. Only eight individual samples were shown plus the two parents. M 50 bp ladder

Fig. 5

Standard curve with the C _q plotted against the log of the starting quantity of cDNA template for Hsp70-u2

Fig. 6

Amplification levels of candidate target Hsp70-u2 gene in the different parents and backcross generations under differential heat treatment and exposure time (A 2 h and B 4 h), C _q threshold cycle (triplicate) indicates the abundance of the target nucleic acid; the lower the C _q level, the higher the nucleic acid (in this case, Hsp70-u2)

Fig. 7

Accumulation of candidate target OsHsp24 gene in the different parents and backcross generations under differential heat treatment and exposure time (A 2 h and B 4 h), C _q threshold cycle (triplicate) indicates the abundance of the target nucleic acid; the lower the C _q level, the higher the nucleic acid (in this case, OsHsp24)

Fig. 8

Changes in the expression level of Hsp70-u2 gene in AVPP0702 (tolerant), “Kulai” (sensitive) BC₁F₁, BC₂F₁ and BC₃F₁ Capsicum annuum genotypes under heat shock treatment of 25, 35 and 45 °C for 2 and 4 h. UBI-3 and EF1-α were used as endogenous control. Error bars indicate SE (n = 3)

Fig. 9

Changes in the expression level of OsHsp24 gene in AVPP0702 (tolerant), “Kulai” (sensitive), BC₁F₁, BC₂F₁ and BC₃F₁ Capsicum annuum genotypes under heat shock treatment of 25, 35 and 45 °C for 2 and 4 h. UBI-3 and EF1-α were used as endogenous control. Error bars indicate SE (n = 3)