高油酸、中果型花生新材料的创制与鉴定

The Development and Identification of New Peanut Germplasm Materials with High Oleic Acid and Medium Pod

【目的】种质资源是作物育种的基础,创制具有多种表型特征的高油酸花生新材料,对高油酸花生育种具有重要意义。【方法】以常规品种白沙1016和高油酸花生品系CTWE为材料,通过对2个亲本ahFAD2A和ahFAD2B ORF的扩增与序列分析,确定其突变类型;利用等位基因特异性PCR(allele-specific PCR,AS-PCR)对两亲本油酸性状基因型进行确认;采用单粒近红外光谱技术(near infrared reflectance spectroscopy,NIRS)结合气相色谱(gas chromatographic,GC)分析对各世代单株的油酸及亚油酸含量进行测定;利用产量性状及油酸含量双重选择压力对F2:3家系及F4种子进行筛选,并对筛选得到的新材料进行表型鉴定及AS-PCR鉴定。【结果】ahFAD2A和ahFAD2B ORF区域扩增与序列测定结果表明,CTWE与突变体F435的突变位点完全一致,即ahFAD2A在448 bp处发生G-A碱基替换,同时ahFAD2B在442 bp处发生碱基A的插入。白沙1016在这2个位点保持野生型状态。借助AS-PCR反应确定两亲本油酸性状基因型,结果表明,CTWE为ol1ol1ol2ol2,白沙1016为OL1OL1OL2OL2,两亲本存在2对差异基因。该结果与测序结果相一致。通过设立产量性状和油酸性状双重选择压力对241个F2:3家系进行选择,单株荚果重高于对照冀花2号30%的家系共20个,对20个高产家系均随机抽取5个单粒进行NIRS检测,淘汰13个低油酸家系。对至少含有1粒高油酸类型的7个家系内的所有单株进行AS-PCR检测,共检测到4种基因型的个体。通过对F2:3家系和F4单株的室内外考种及GC值的测定,筛选出具有不同表型特征、产量高出对照冀花2号30%的纯合高油酸材料4个,百仁重介于54.00—75.29 g,均属中果类型,油酸GC值介于81.10%—82.71%,油亚比介于28.66—41.19。11-3和13-2的株型均为匍匐3型,均有轻微果嘴和轻微网纹。11-3为蜂腰形荚果,轻微果腰。13-2为普通形荚果,无果腰。15-2株型为直立型、普通形荚果、无果腰、无果嘴和中等明显程度网纹。16-1株型为匍匐2型、普通形荚果、无果腰、轻微果嘴和轻微网纹。【结论】借助AS-PCR辅助选择的方法创制了具有不同表型特征的中果型高油酸新材料4份。

【Objective】 Germplasm resources is the base for crop breeding. The development of new high oleate peanut materials with various phenotypic characteristics is great significant for high oleic peanut breeding program. 【Method】Conventional variety Baisha 1016 and high oleic peanut line CTWE were used and their mutation types were determined by amplification and sequencing results of partial ORF of ahFAD2 A and ahFAD2 B. The oleic trait genotypes of two parents were confirmed by allele-specific PCR（AS-PCR）. Oleic acid and linoleic acid content of each generation plants were detected by near infrared reflectance spectroscopy（NIRS） combined with gas chromatographic（GC） analysis. Double selection pressures of yield and oleic acid were used in 241 F2：3 family lines and F4 seeds, and the identification of phenotypic and AS-PCR were conducted on new selected materials.【Result】The amplification and sequencing results of partial ORF of ahFAD2 A and ahFAD2 B showed that the mutated sites of CTWE were the same as that of mutant F435 with a base substitution in（G448A） for ahFAD2 A, and a single base pair insertion（442insA） for ahFAD2 B while Baisha 1016 maintained wild type state on the same both sites. The AS-PCR analysis demonstrated that the parental lines differed in both genotypes and oleate content for ahFAD2 A and ahFAD2 B. The genotype of CTWE was o1lo1lol2ol2, and Baisha 1016 was OL1OL1OL2OL2, which was consistent with the sequencing results. Double selection pressures of yield and oleic acid were used in 241 F2：3 family lines and 20 high oleate lines were selected, which the pods weight per plant was 30% higher than that of control of Jihua 2. Thirteen low-oleic family lines were eliminated by NIRS single particle detection on randomly picked 5 kernels of 20 high yield family lines respectively. All plants containing at least 1 kernels of high oleic acid type were detected, and four different genotypes in 7 lines were confirmed by AS-PCR. Four homozygous high oleate germplasm materials with various phenotypic characteristics and the yield 30% higher than that of control Jihua 2 were ultimately selected through field investigation and indoor test and ascertained by gas chromatography. Their 100-seed weights were between 54.00-75.29 g and belonged to medium-pod type. O/L ratio ranged from 28.66-41.19. The plant types of 11-3 and 13-2 were creeping type 3. They were both slight pod beak and slight pod reticulation. But the pod trait of 11-3 was bee waist shape with slight pod constriction, that of 13-2 was common shape with none pod constriction. The plant type of 15-2 was upright type and pod trait is common shape, none pod constriction, none pod beak and medium pod reticulation. The plant type of 16-1 was creeping type 2, pod trait was common shape, none pod constriction, slight pod beak and slight pod reticulation. 【Conclusion】 Four new medium-pod high oleic germplasm materials with different phenotypic characteristics were selected by AS-PCR assisted selection.