对湿加松SSR反应体系中模板DNA的用量、Mg2+浓度、dNTPs及Taq酶的用量、变性温度、退火温度、反应循环次数共7个影响SSR扩增效果的因素,逐一设置单因素多水平进行试验,以目测比较确定谱带条数、亮度、清晰度、背景透明度以及结果的重复性等为判定反应条件适合程度的指标,其反应体系为25μL,扩增程序经优化后确定为:10 ng的模板、1×PCR缓冲液、2.5 mM的Mg2+、0.2 mM dNTPs、0.6 UTaq酶、正反向引物各0.2μM;最优反应程序为94℃预变性2 m in之后进入循环,每个循环94℃变性45 s,退火45 s,72℃延伸45 s。退火温度从Tm开始,每隔2个循环降低1℃,直至(Tm-10℃),维持40个循环。循环结束,72℃延伸5 m in。
Quantitative trait locus(QTL)detection was carried out for growth traits in 122 F1 progenies of Pinus elliottii var.elliottii(PEE)×P.caribaea var.hondurensis(PCH)hybrid.The PCH male parent linkage map contained 108 markers in 16 linkage groups,while the PEE female parent contained 93 markers in 19 linkage groups.Sequence-related amplified polymorphism(SRAP),microsatellite(SSR),expressed sequence tag polymorphism(ESTP)and inter-simple sequence repeat(ISSR)were selected from an existing linkage map.Growth traits investigated were height at age five(HT05)and six years(HT06),diameter at breast height at age five(DBH05)and six years(DBH06)and annual growth from age five to six years of height(AGHT)and diameter at breast height(AGDBH).Kruskal-Wallis and interval mapping approaches were used to estimate levels of significance,the number of QTLs,the percentage of the phenotypic variation explained by each of QTLs and their positions on the genetic linkage maps.Twenty six QTLs with significance levels p<0.05 were detected on the parental maps for the six growth traits,which explained more than 15%of the phenotypic variation,suggesting an existence of major-effect genes.Several QTLs had the pleiotropy controlling more than one single growth trait.Overall,the proportion of phenotypic variation explained by QTLs ranged from 5.9%to 40.6%for HT05and HT06,from 6.6%to 42.0%for DBH05 and DBH06 and from 5.8%to 22.3%for AGHT and AGDBH.The results from this study provide a basis for marker-aided selection(MAS)in PEE×PCH hybrids.
