No abstract

Commercial maize hybrid production has corroborated the usefulness of producing inbred lines; however, the delivery of new lines has always been a major time constraint in breeding programs. Traditional methods for developing inbred lines typically require 6 to 10 generations of self-pollination to obtain sufficient homozygosity. To bypass the time and costs associated with the development of inbred lines, doubled haploid (DH) systems have been widely adopted in the commercial production of maize. Within just two generations, DH systems can create completely homozygous and homogeneous lines. A typical maize DH system, utilizing anthocyanin markers R1-nj or Pl1 for haploid selection, is described in this protocol. ABSTRACTCommercial maize hybrid production has corroborated the usefulness of producing inbred lines, however, the delivery of new lines has always been a major time constraint in breeding programs. Traditional methods for developing inbred lines typically require 6-10 generations of self-pollination to obtain sufficient homozygosity. To bypass the time and costs associated with the development of inbred lines, doubled haploid (DH) systems have been widely adopted in the commercial production of maize. Within just two generations, DH systems can create completely homozygous and homogeneous lines. A typical maize DH system, utilizing anthocyanin markers R1-nj or Pl1 for haploid selection, is described in this protocol.

Doubled haploid technology is highly successful in maize breeding programs and is contingent on the ability of maize inducers to efficiently produce haploids. Knowledge of the genes involved in haploid induction is important for not only developing better maize inducers, but also to create inducers in other crops. The main quantitative trait loci involved in maize haploid induction are qhir1 and qhir8. The gene underlying qhir1 has been discovered and validated by independent research groups. Prior to initiation of this study, the gene associated with qhir8 had yet to be recognized. Therefore, this research focused on characterizing positional candidate genes underlying qhir8. Pursuing this goal, a strong candidate for qhir8, GRMZM2G435294 (MYO), was silenced by RNAi. Analysis of crosses with these heterozygous RNAi-transgenic lines for haploid induction rate revealed that the silencing of MYO significantly enhanced haploid induction rate by an average of 0.6% in the presence of qhir1. Recently, GRMZM2G465053 (ZmDMP) was identified by map-based gene isolation and shown to be responsible for qhir8. While our results suggest that MYO may contribute to haploid induction rate, results were inconsistent and only showing minor increases in haploid induction rate compared to ZmDMP. Instead, reciprocal crosses clearly revealed that the silencing of MYO causes male sterility.

Core Ideas There are many potential benefits of incorporating embryo culture into a doubled haploid program. There is no known non‐transgenic method of selecting haploid embryos following embryo culture. Our goal was to establish a non‐transgenic haploid selection method following embryo culture. These methods allow early haploid selection based on differential root growth features. Potential benefits of incorporating embryo culture (EC) into a doubled haploid (DH) program, including shortening the breeding cycle and increasing chromosome doubling rates, make the laborious and tedious task of excising embryos worth the effort. Difficulties arise during embryo selection considering the marker gene R1‐nj, which is typically used in DH programs, is not expressed in early stages after pollination. Although transgenic approaches have been implemented to bypass this issue, there is so far no known non‐transgenic method of selecting haploid embryos. The findings of this study reveal methods of selecting haploid embryos that allow the possibility of incorporating EC into a DH program without using transgenic inducers. The best performing method involves a machine‐learning classifier, specifically a support vector machine, which uses primary root lengths and daily growth rates as traits for classification. Selection by this method can be achieved on the third day after germination. By this method, an average false negative rate of 2% and false positive rate of 9% was achieved. Therefore, the methods presented in this research allow efficient and non‐transgenic selection of haploid embryos that is simple and effective.