CAJ | 학술논문

【目的】针对花生染色体较小，染色体细胞学标记少，细胞遗传研究相对滞后，染色体分类识别困难的问题，建立能够准确区分栽培花生（Arachis hypogaea L.，2n=4x=40，AABB）A、B染色体组的新核型，提高染色体识别准确率，以揭示栽培花生和野生供体亲本的染色体对应关系，鉴定栽培种花生染色体结构变异体。【方法】以花生栽培种（Arachis hypogaea L.，2n=4x=40，AABB）的2个可能供体亲本即花生野生种Arachis duranensis （2n=2x=20，BB）和Arachis ipa?nsis（2n=2x=20，AA）全基因组DNA及5S rDNA和45S rDNA为探针，利用顺序基因组荧光原位杂交（GISH）和多色荧光原位杂交（McFISH）技术（简称顺序GISH-FISH）结合DAPI染色，在准确区分花生栽培种A、B染色体组的基础上，对花生栽培品种Z5163及其供体亲本染色体进行分析，建立花生栽培种新核型，并利用该核型对其他栽培品种的染色体进行分析，以探讨该核型的应用潜力和栽培花生染色体组成特点。【结果】以A. ipa?nsis和A.duranensis全基因组DNA为探针的GISH分析表明，以A. ipa?nsis为探针在花生栽培种20条B组染色体上能够产生清晰稳定的杂交信号，在A组染色体上没有信号，而以A.duranensis为探针，只在18条A组染色体能产生信号，但1对A组的小染色体“A染色体”不易被区分，因此，以A. ipa?nsis为探针可以准确区分花生栽培种A、B染色体组；综合5S rDNA和45S rDNA Mc-FISH和DAPI染色分析，发现花生栽培种A、B染色体组DAPI带纹、5S rDNA和45S rDNA的分布分别与A.duranensis和A. ipa?nsis一致，此结果支持A.duranensis和A.ipa?nsis是花生栽培种的供体亲本。DAPI染色结果显示，A. ipa?nsis及花生栽培种的B组染色体均有14条染色体显示着丝粒带纹，明显多于前人报道，表明仅利用DAPI染色来区分花生栽培种A、B组染色体的方法具有局限性。综合DAPI染色、rDNA、A.duranensis和A. ipa?nsis基因组探针进行顺序GISH-FISH分析，建立了可以准确识别花生栽培种A、B染色体组新核型。然后利用该核型对3个栽培种品种的染色体组成进行了分析，首次发现一个自发的花生染色体代换系MS B1（A1），揭示了栽培花生染色体B1与A1之间存在部分同源关系。【结论】野生花生A. duranensis和A. ipa?nsis分别与栽培花生A和B基因组染色体间具有很好的对应关系；研究建立的基于GISH-FISH和DAPI染色的栽培花生新核型，不但可以准确区分大部分A、B组染色体，而且还能识别栽培花生在多倍体化和人工进化过程中可能存在的自发的染色体变异，揭示A、B组染色体间的部分同源性。【目的】針對花生染色體較小，染色體細胞學標記少，細胞遺傳研究相對滯後，染色體分類識彆睏難的問題，建立能夠準確區分栽培花生（Arachis hypogaea L.，2n=4x=40，AABB）A、B染色體組的新覈型，提高染色體識彆準確率，以揭示栽培花生和野生供體親本的染色體對應關繫，鑒定栽培種花生染色體結構變異體。【方法】以花生栽培種（Arachis hypogaea L.，2n=4x=40，AABB）的2箇可能供體親本即花生野生種Arachis duranensis （2n=2x=20，BB）和Arachis ipa?nsis（2n=2x=20，AA）全基因組DNA及5S rDNA和45S rDNA為探針，利用順序基因組熒光原位雜交（GISH）和多色熒光原位雜交（McFISH）技術（簡稱順序GISH-FISH）結閤DAPI染色，在準確區分花生栽培種A、B染色體組的基礎上，對花生栽培品種Z5163及其供體親本染色體進行分析，建立花生栽培種新覈型，併利用該覈型對其他栽培品種的染色體進行分析，以探討該覈型的應用潛力和栽培花生染色體組成特點。【結果】以A. ipa?nsis和A.duranensis全基因組DNA為探針的GISH分析錶明，以A. ipa?nsis為探針在花生栽培種20條B組染色體上能夠產生清晰穩定的雜交信號，在A組染色體上沒有信號，而以A.duranensis為探針，隻在18條A組染色體能產生信號，但1對A組的小染色體“A染色體”不易被區分，因此，以A. ipa?nsis為探針可以準確區分花生栽培種A、B染色體組；綜閤5S rDNA和45S rDNA Mc-FISH和DAPI染色分析，髮現花生栽培種A、B染色體組DAPI帶紋、5S rDNA和45S rDNA的分佈分彆與A.duranensis和A. ipa?nsis一緻，此結果支持A.duranensis和A.ipa?nsis是花生栽培種的供體親本。DAPI染色結果顯示，A. ipa?nsis及花生栽培種的B組染色體均有14條染色體顯示著絲粒帶紋，明顯多于前人報道，錶明僅利用DAPI染色來區分花生栽培種A、B組染色體的方法具有跼限性。綜閤DAPI染色、rDNA、A.duranensis和A. ipa?nsis基因組探針進行順序GISH-FISH分析，建立瞭可以準確識彆花生栽培種A、B染色體組新覈型。然後利用該覈型對3箇栽培種品種的染色體組成進行瞭分析，首次髮現一箇自髮的花生染色體代換繫MS B1（A1），揭示瞭栽培花生染色體B1與A1之間存在部分同源關繫。【結論】野生花生A. duranensis和A. ipa?nsis分彆與栽培花生A和B基因組染色體間具有很好的對應關繫；研究建立的基于GISH-FISH和DAPI染色的栽培花生新覈型，不但可以準確區分大部分A、B組染色體，而且還能識彆栽培花生在多倍體化和人工進化過程中可能存在的自髮的染色體變異，揭示A、B組染色體間的部分同源性。
【목적】침대화생염색체교소，염색체세포학표기소，세포유전연구상대체후，염색체분류식별곤난적문제，건립능구준학구분재배화생（Arachis hypogaea L.，2n=4x=40，AABB）A、B염색체조적신핵형，제고염색체식별준학솔，이게시재배화생화야생공체친본적염색체대응관계，감정재배충화생염색체결구변이체。【방법】이화생재배충（Arachis hypogaea L.，2n=4x=40，AABB）적2개가능공체친본즉화생야생충Arachis duranensis （2n=2x=20，BB）화Arachis ipa?nsis（2n=2x=20，AA）전기인조DNA급5S rDNA화45S rDNA위탐침，이용순서기인조형광원위잡교（GISH）화다색형광원위잡교（McFISH）기술（간칭순서GISH-FISH）결합DAPI염색，재준학구분화생재배충A、B염색체조적기출상，대화생재배품충Z5163급기공체친본염색체진행분석，건립화생재배충신핵형，병이용해핵형대기타재배품충적염색체진행분석，이탐토해핵형적응용잠력화재배화생염색체조성특점。【결과】이A. ipa?nsis화A.duranensis전기인조DNA위탐침적GISH분석표명，이A. ipa?nsis위탐침재화생재배충20조B조염색체상능구산생청석은정적잡교신호，재A조염색체상몰유신호，이이A.duranensis위탐침，지재18조A조염색체능산생신호，단1대A조적소염색체“A염색체”불역피구분，인차，이A. ipa?nsis위탐침가이준학구분화생재배충A、B염색체조；종합5S rDNA화45S rDNA Mc-FISH화DAPI염색분석，발현화생재배충A、B염색체조DAPI대문、5S rDNA화45S rDNA적분포분별여A.duranensis화A. ipa?nsis일치，차결과지지A.duranensis화A.ipa?nsis시화생재배충적공체친본。DAPI염색결과현시，A. ipa?nsis급화생재배충적B조염색체균유14조염색체현시착사립대문，명현다우전인보도，표명부이용DAPI염색래구분화생재배충A、B조염색체적방법구유국한성。종합DAPI염색、rDNA、A.duranensis화A. ipa?nsis기인조탐침진행순서GISH-FISH분석，건립료가이준학식별화생재배충A、B염색체조신핵형。연후이용해핵형대3개재배충품충적염색체조성진행료분석，수차발현일개자발적화생염색체대환계MS B1（A1），게시료재배화생염색체B1여A1지간존재부분동원관계。【결론】야생화생A. duranensis화A. ipa?nsis분별여재배화생A화B기인조염색체간구유흔호적대응관계；연구건립적기우GISH-FISH화DAPI염색적재배화생신핵형，불단가이준학구분대부분A、B조염색체，이차환능식별재배화생재다배체화화인공진화과정중가능존재적자발적염색체변이，게시A、B조염색체간적부분동원성。
Objective]The cytogenetic study of peanut has been hindered mainly by the small chromosomes and limited cytological markers of peanut. In order to reveal the correspondence between cultivated peanut (Arachis hypogaea L., 2n=4x=40, AABB) and wild donor parents chromosomes and to characterize the chromosome structure variants, the objective of this study is to establish a highly distinguishable karyotype of A and B genome chromosomes for improving accuracy of chromosome identification.[Method]Using the total genomic DNA of the two possible donors (A.duranensis, 2n=2x=20, BBandA.ipa?nsis, 2n=2x=20, AA) of cultivated peanut and 5S and 45S rDNA as probes, sequential GISH-FISH combined with DAPI staining were employed to develop the karyotypes of Z5163 and the two donors based on distinguishing A and B genome ofA. hypogaea clearly. The new karyotype was then used to analyze more peanut cultivars in order to reveal the characteristics of peanut chromosome constitution.[Result] GISH indicated that all 20 B genome chromosomes ofA. hypogaea produced clear and stable signals after hybridized with the total genomic DNA ofA.ipa?nsis, while only 18 chromosomes except “A chromosomes” of A genome produced signals usingA. duranensis as a probe. Sequential mc-FISH using 5S rDNA and 45S rDNA as probes and combined with DAPI staining revealed that all the signals distributed on A and B genome ofA. hypogaea were almost identical to its respective possible donor genome chromosomes ofA. duranensisand A.ipa?nsis, which suggested thatA. duranensisand A.ipa?nsis were the donors ofA. hypogaea. Furthermore, the present study also found that 14 B genome chromosomes showed centromeric bands after DAPI staining, which were quite more than the previous reports, indicating that it was difficult to distinguish the A and B genome chromosomes ofA. hypogaea only by DAPI staining as previous reports. Therefore, based on the above findings, a new GISH-FISH karyotype of the cultivated peanut was developed which could clearly distinguish all the chromosomes of A genome from those of B genome inA. hypogaea. And then the karyotype was used to characterize three peanut varieties, and a spontaneous monosomic substitution line ofA. hypogaea, MSB1(A1), was found, indicating the homoeologous relationship between chromosomes B1 and A1.[Conclusion]The two genomes of cultivated peanut were very separately correspondent to its possible donor parentsA. duranensis andA. ipa?nsis; The new karyotype of the cultivated peanut could not only distinguish most of A and B genome chromosomes but also identify spontaneous chromosome variations produced in the progress of human-selection and polyploidization of peanut, indicating homoeologous relationships between chromosomes of A and B genomes of peanut.