| Puccinelliachinampoensisand Madicago sativa are two important perennialgrasses in northeast China, and are classified as the main cultivated grass in west JilinProvince. It is important to do the basic research on the germplasm of this two kind ofgrass and is benefit for salt-resistant breeding, grassland degradation amelioration, thedevelopment of quality animal husbandry.In order to know more about the genetic diversity, genetic structure andgeneticdifferentiation of P. chinampoensispopulations, ISSR molecular markers wereused to analysis the genetic diversity of P. chinampoensis mainly from northeastChina. We cloned the CAT gene from P. chinampoensis, and expression analysis of itunder different stress treatments.In order to understand the potential production ofdifferent alfalfa hybrid combinations, we studied photosynthetic rate response to lightintensity and CO2concentration characteristics in order to identify the combination ofecological environmental conditions and ecological adaptability, seeking the bestphotosynthetic management.15alfalfa combinations hybrid were used as materials tostudy the yield and quality factors，and provides a theoretical basis and reference forthe establishment of high-quality, high yield cultivation technology system. Thereaults are as followed:（1） It is necessary to establish and optimize the ISSR-PCR reaction system forstudying the genetic diversity of P. chinampoensis. To obtain the best amplificationresult of P. chinampoensiswith clear, repeatable and rich polymorphism bands, theISSR reaction system including dNTPs, Mg2+, primer, template DNA and Taq DNApolymerase was optimized. Results showed that the optimum concentrations of fivereactants in20μl reaction mixture were as follows: genomic DNA40ng, dNTPs0.2mmol/L, primer0.8μmol/L, Taq DNA polymerase1U, MgCl22.5mmol/L and10×PCR Buffer（Mg2+free）2μL.10primers with stable amplification bands and richpolymorphism for ISSR-PCR were selected from100candidate primers, which theoptimal annealing temperature was also found. This optimized ISSR reaction systemwould provide reference for genetic diversity analysis, germplasm resourcesclassification and map construction.（2） ISSR was used to analyse the genetic diversity of5P.chinampoensispopulations.The results of ISSR analysis shows that the geneticdiversity among5Puccinellia populations.198（63.94%） bands are found to bepolymorphic,150wild materials are amplified by10ISSR Primers, the gene among populations (Nm) is0.6152. Higher genetic diversity is both observed at species leveland population level.The genetic differentiation（Gst） between populations is44.83%. Baseonthe clusteranalysisthat fivepopulations canbedividedinto twocategories.（3） In this study, the full length cDNAsequence of CAT gene was cloned fromAlkaligrass （Puccinellia chinampoensis） leaves using RT-PCR method, the primerswere designed according to the homologous CAT gene sequences of other plantspecies. Sequence analysis showed that the nucleotide sequence of this gene is1487bp, containing a complete open reading frame and encoding492amino acids,Genebank: HM230827. Nucleotide and amino acid sequence analysis revealed thatPuCAT shares high identity with the orthologs from Triticum aestivum and Hordeumvulgare. And its signal peptide, hydrophobicity/hydrophilic, trans-membrane domain,secondary structure and main functional domains were predicted. Semi-quantitativeRT-PCR analysis showed that PuCAT expressed in different tissues, but theexpression in root was lower, and in leaf much higher. Various elevated levels ofPuCAT expression have been detected when exposed to4different stressexperimental treatment, and the results was not the same.（4）9alfalfa varieties were used as materials to study the response ofphotosynthesis rate to light intensity and CO2concentration in budding period withLicor-6400instrument on the nature sunshine condition. The results indicated that9kinds of hybrid alfalfa were differed in response to light intensity and CO2concentration. Photosynthetic rate varies with each type increases with the increase oflight intensity and CO2concentration are available Non-rectangular hyperbola modelto simulate, and draw some photosynthetic response characteristic parameters:apparent quantum yield（AQY）, carboxylation efficiency（CE）, light compensationpoint（LCP） and light saturation point（Lk）, dark respiration rate（Rd） and photorespiration rate（Rp）, lower CO2compensation poin（tCCP）and CO2saturation point（CSP）. According to these parameters can be preliminarily concluded that thehybrid combination Ⅴ, Ⅱ, Ⅳ light use scope is bigger, good adaptability to light;Combination Ⅰ, Ⅷ, Ⅲ’s ability to use CO2is stronger; Combination Ⅰ, Ⅷ, Ⅱand Ⅴ are the combination of high yield potential; Resistance shade combination areⅢ, Ⅸ, Ⅰ, while Ⅷ and Ⅴ is more heliophilous resistance; Combination Ⅳ, Ⅴ,Ⅷ of light energy use efficiency is the largest, has the strong ability of weak lightutilization; Combination Ⅶ and Ⅲ has the high activity of RuBPCase: combinationⅧ, Ⅴ, Ⅳ leaf physiological activity is higher, can provide more energy for plant growth; Combination Ⅵ, Ⅲ, Ⅶ Pi deficiency to the restrictions of the process ofelectron transfer and photosynthetic, phosphorylation are relatively small.（5）15alfalfa combinations hybrid were used as materials to study the plantheight, branch number, fresh yield, hay yield of yield traits and CP,EE,NFE,NDF,ADF, stem/leaf of quality factors at the beginning of flowering of the firstcutting, and comprehensive evaluation on it by cluster analysis and correlationanalysis. The results showed that different alfalfa hybrid combinations are certaindifferences both production and quality, the combinations that male parentwas imported Russian resources is better in height, the hybrid combinations that maleparent was our country were far more than the other two countries in hay and freshyield performance, and the hybrid combination that male parent. resources were fromour country in and the United States have more excellent potential high quality.Comprehensive showed that alfalfa male sterile line MS-GN breeding with ourcountry alfalfa varieties had the potential that can get a combination of high qualityand high yield at least in the northeast, the second is the resources of the United States,but we can consider male parent resources of Russia, if the variety choice is for aspecific purpose.The study on genetic diversity of Puccinellia and cloning PuCAT gene can be usedfor protectionand utilization of Puccinelliawild resources. The alfalfa breeding workin China is still slower than some western countries. The “Medicago sativa L. No.1Gongnong” is still widely used in northeast China which was bred in1980’s. Atpresent, only the United States completed the alfalfa “three line” hybrids bred alfalfa.It’s yield has more than10%-15%then other alfalfa varieties Alfalfa hybrid breedingis a major breakthrough in this area, and benefit for the development of animalhusbandry and alfalfa industry. |
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