Research About Formation Mechanism Of Hay Heterosis Of Alfalfa Male Sterility

Study the formation mechanism of heterosis in alfalfa was important for the breeding of hybrid alfalfa. This study configured 32 hybridized combinations using incomplete diallel cross design (NCⅡ), and 8 alfalfa male sterile lines as female parents and 4 alfalfa varieties (or species) with superior characters as male parents. The research studied the character performance of alfalfa heterosis in agronomy and physiology, and investigated the relationship between heterosis and gene differential expression in gene regulation level, and further analyzed the formation mechanism of yield heterosis. The main results were as follows:(1) Combining ability effect of forage yield showed that the genotypic variance proportion of general combining ability of all parents was 71.18%,therefore,additive effect of genotype played a leading role on the formation of hybrid yield characters. General combining ability of cytoplasmic male sterility(CMS) had a significant impact on the formation of forage yield advantage of hybrid than specific combining ability. CMS 2, 10 and 12 had a higher general combining ability and utilization value. Strong heterotic combinations were 2×Ⅱ, 2×Ⅲ, 4×I and 12×I respectively,and all of their competitive advantage were more than 40%.(2) 32 hybrid combinations were divided into three kind of combination based on forage production and heterosis analysis, which were strong heterotic crosses, weak heterotic crosses and dominant heterotic crosses. The forage yield (43.02g/strain) and heterosis (41.88%) of strong heterotic crosses were significantly higher than the weak heterotic crosses (P<0.01). Correlation analysis showed that there was a remarkable positive correlation between plant height, branch number, main stem diameter and node number of 8 yield traits and yield heterosis respectively (P<0.01).(3) Water use efficiency was the main indices of photosynthetic physiology that affected forage yield heterosis formation, and followed by net photosynthetic rate. High water use efficiency and net photosynthetic rate leading to higher dry matter synthesis was to facilitate the formation of yield heterosis. But transpiration rate and chlorophyll content had no significant effect on heterosis.(4) Gene differential expression of leaves under different phenophase were analyzed using cDNA-AFLP technology. 8577 stable bands were detected using 14 primers, and proportion of differentially expressed genes was 14.77%. Differentially expressed genes in the squaring period were more than branching period, respectively 20.27% and 8.31%. There were 6 differential gene expression patterns between hybrids and their parents, bands observed only in hybrids (UNF1) and bands observed only in female (UNP1) played a major role on the formation of the yield heterosis. UNF1 was significant positive correlation with forage yield advantage at returning green, branching and squaring period (r=0.8726).(5) 12 gene fragments were randomly cloned that showed differentially expressed between hybrids and parents had homologous nucleotide sequence in the GenBank database, and their similarities were greater than 75%. 6 gene fragments had high homologous sequence with encoded protein sequences (similarity≥42%). The function of homologous proteins was further speculated and the results showed that alfalfa yield advantage had a relationship with a variety of metabolic pathway. They included basic metabolism, biological transport, transcription regulation, signal transduction, in which the basic metabolism was the main.