Implementation and optimization of the doubled haploid technology for tropical maize (Zea mays L.) breeding programs

摘要

Doubled haploid (DH) technology is currently the fastest way to achieve homozygosity in maize and it offers numerous quantitative genetic, operational, and economic advantages. Hybrid maize breeding with DH lines is common in temperate areas, yet adoption of this technology is still to be realized in tropical areas. Therefore, the main goal of my thesis project was to establish and validate the DH technology for tropical maize breeding programs at the International Maize and Wheat Improvement Center (CIMMYT) in Mexico.In vivo production of maternal haploids and DH lines involves four steps: (i) inducing haploidy by pollinating source germplasm with pollen of a haploid inducer; (ii) identifying seeds with haploid embryos based on a visually scorable marker; (iii) duplicating chromosomes of putative haploids by treating the seedlings with a mitotic inhibitor; and (iv) self-pollinating DH plants to multiply their seed. To impart knowledge on each of the above steps, we compiled a detailed protocol and produced a publicly available video which will be very useful for capacity building.Lack of reliable information on the performance of temperate inducers under nontemperate conditions is one reason for the slow adoption of DH technology in tropical maize breeding programs. Therefore, we assessed haploid induction rates (HIR) and agronomic performance of three temperate inducers in tropical lowland environments in Mexico. HIR obtained under tropical conditions were similar to those previously reported from evaluations under temperate conditions, indicating that temperate inducers can be used for initiation of DH breeding programs in the tropics. However, the inducers showed poor pollen production, poor seed set, and strong susceptibility to tropical leaf diseases. Hence, better adapted inducers would be advantageous for large-scale induction of haploidy in tropical DH programs.To develop better adapted haploid inducers, segregating populations were generated from crosses between temperate inducers and eight tropical CIMMYT maize lines (CML) from Mexico and Zimbabwe. Mass selection of individual F2 plants was conducted for visually scorable and highly heritable traits, followed by family-based selection for HIR and agronomic traits. Several tropical inducer candidates (TIC) were identified with HIR of up to 10% and notably improved agronomic performance under tropical lowland conditions. Compared to backcrosses to the inducers, backcrosses to the CML showed similar HIR combined with a significantly later anthesis date and improved plant vigor. Hence, backcrossing to the adapted parent may be a suitable approach to improve adaptation of new inducers while maintaining high HIR levels. Furthermore, we screened randomly chosen South American maize accessions and observed HIR of up to 3%, suggesting that novel sources of haploid induction ability may be present in CIMMYT?s vast germplasm collection. Although extensively exploited in DH line production, the genetic mechanisms underlying in vivo induction of maternal haploids in maize are still largely unknown. We conducted comparative quantitative trait locus (QTL) mapping for HIR to explore the genetic architecture of this phenomenon. Segregating populations were generated from four crosses composed of two temperate haploid inducer lines and three non-inducer lines. One major QTL on chromosome 1 (qhir1; bin 1.04) explaining up to 66% of the genotypic variance was detected in the three populations involving non-inducer lines. Hence, bin 1.04 represents an interesting region for map-based cloning. Further, qhir1 was affected by strong segregation distortion against the inducer allele, indicating that natural selection disfavors haploid induction ability. Seven QTL with smaller effects were detected in the CAUHOI×UH400 population. Further, we proposed a conceptual genetic framework for inheritance of in vivo haploid induction ability in maize. Common methods for artificial duplication of haploid chromosome sets mostly involve toxic and costly reagents and are extremely labor-intensive. This leads to serious bottlenecks during DH line development. When screening haploid populations derived from 260 diverse temperate and tropical source germplasm, we observed significant genetic variation for fertility-related traits, suggesting that haploid fertility can be effectively improved by recurrent selection. This may facilitate abolishment of artificial chromosome doubling during DH production, which seems particularly relevant for enabling small national maize breeding programs and seed companies in developing countries to adopt the DH technology.To study the suitability of different population types for DH line extraction, we developed 131 DH lines from five tropical elite single crosses (SC) and five tropical open-pollinated populations (OP) and evaluated them for testcross performance in Mexico. While testcross grain yield means of the two population types did not differ significantly, significant genetic variance was only revealed for OP-derived DH lines. Several DH lines from OP excelled in testcross performance and may be useful for tropical hybrid breeding programs. In addition, tropical OP may harbor valuable untapped genetic variation that can effectively be exploited with DH technology.This thesis work demonstrated that established protocols for in vivo DH line development can be readily applied to tropical maize breeding programs. Adoption of the DH technology promises to greatly increase the efficiency of breeding programs and DH lines are also an exciting tool to (i) immortalize genetic resources, (ii) conduct high-resolution genetic analyses of important traits, and (iii) accelerate the arrival of improved varieties to farmers? fields.