Simple DNA marker‐assisted selection of endophyte (Epichloë uncinata)‐infected festulolium

In this study, to breed endophyte (Epichloe uncinata)‐infected festololium (x Festulolium Aschers.et Graebn.), we backcrossed endophyte‐infected Italian ryegrass (Lolium multiflorum Lam.) to festulolium. To prevent self‐pollinated plants being mixed with the progeny, we used polymerase chain reaction (PCR)‐based DNA marker sets distinguishing between Lolium and Festuca species. In the first backcrossing (F₁ development), 20 of the 21 progeny plants showed Festuca‐specific band patterns and were endophyte‐infected. We developed six maternal lines (BC₁ development) by backcrossing six selected F₁ plants. More than 97% of plants from five maternal lines had novel Festuca‐specific band patterns which their maternal parents lacked. However, only 54% plants of the maternal line F₁–₁₈ exhibited novel Festuca‐specific band patterns. F₁–₁₈ showed Festuca‐specific band patterns with almost all tested DNA marker sets, and its f ratio (the ratio of Festuca‐specific region to the whole genome) was 31.1%, indicating that F₁–₁₈ had an almost complete set of the Festuca genome. Because paternal backcrossing festulolium parents were amphidiploids, an f ratio of approximately 25% corresponds to a complete set of the Festuca genome. We found that it was difficult to evaluate BC₂ hybridity using the same DNA marker sets because very few DNA markers were distinctive. In total, we bred and selected at least 146 true BC₁ hybrids and endophyte‐infected plants using DNA markers.