WO2020151417A1 - 一种构建甘蔗抗褐锈病基因定位遗传分离群体的方法 - Google Patents

一种构建甘蔗抗褐锈病基因定位遗传分离群体的方法 Download PDF

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WO2020151417A1
WO2020151417A1 PCT/CN2019/126509 CN2019126509W WO2020151417A1 WO 2020151417 A1 WO2020151417 A1 WO 2020151417A1 CN 2019126509 W CN2019126509 W CN 2019126509W WO 2020151417 A1 WO2020151417 A1 WO 2020151417A1
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brown rust
hybrid
primer
resistance
sugarcane
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PCT/CN2019/126509
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English (en)
French (fr)
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黄应昆
李文凤
王晓燕
单红丽
张荣跃
李婕
仓晓燕
尹炯
罗志明
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云南省农业科学院甘蔗研究所
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Publication of WO2020151417A1 publication Critical patent/WO2020151417A1/zh
Priority to ZA2021/03562A priority Critical patent/ZA202103562B/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

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  • the invention belongs to the technical field of crop disease resistance gene positioning, and specifically relates to a method for constructing a sugarcane brown rust resistance gene positioning genetic separation population.
  • Sugarcane brown rust is an important disease caused by Puccina.melanocephala H.Sydow&P.Sydow. It often occurs in the main sugarcane producing areas in China, causing huge economic losses and seriously affecting the performance of the sugarcane industry. Continuous development. Sugarcane brown rust is mainly manifested by the appearance of a large number of reddish brown spots on the sugarcane leaves, and the spots merge to form a large indefinite necrotic area. As a result, the sugarcane leaves die before they are ripe, even the young leaves.
  • the discovery and location of resistance genes can lay the foundation for the isolation and cloning of new brown rust resistance genes and the development of molecular breeding. It is of great significance for the effective prevention and control of sugarcane brown rust and ensuring the sustainable and stable development of my country's sucrose industry.
  • the foundation and key to the discovery and location of brown rust resistance genes requires the development and establishment of a simple and efficient method for the construction of genetic segregation populations, and the construction of genetic segregation populations for the location of brown rust resistance genes.
  • the present invention provides a simple and efficient method for constructing sugarcane brown rust resistance genes to locate genetically isolated populations.
  • a method for constructing a genetic segregation population of sugarcane brown rust resistance gene mapping including the following steps:
  • the brown rust resistance is evaluated according to the 1-9 grading standard.
  • the leaves are asymptomatic as the first grade high resistance; the diseased spots of the leaves account for less than 10% of the leaf area It is grade 2 disease resistance; leaf disease spots account for 11%-25% of leaf area, and it is grade 3 medium resistance; leaf disease spots account for 26%-35% of leaf area and it is grade 4 moderately susceptible; leaf disease spots account for 36%-50% of leaf area % Is grade 5 susceptible 1; leaf lesions account for 51%-60% of the leaf area as grade 6 susceptible 2; leaf lesions account for 61%-75% of the leaf area as grade 7 susceptible 3; leaf lesions account for leaf area 76%-90% are grade 8 high susceptibility 1; leaf lesions occupy 91%-100% of the leaf area, which means grade 9 high susceptibility 2;
  • the resistance and susceptibility segregation ratio of the genetically segregated populations of hybrid offspring (total number of disease-resistant single plants of grade 1-3): (total number of diseased single plants of grade 4-9),
  • the true hybrid F1 generation population with the resistance to susceptibility segregation ratio of the hybrid offspring genetic segregation population ⁇ 3:1 was selected as the sugarcane brown rust resistance gene mapping genetic segregation population.
  • the brown rust pathogen spore suspension described in step (5) is to collect brown rust spore-carrying sugarcane leaves to extract the spores for direct inoculation, specifically: collecting brown rust spore-carrying sugarcane leaves and soaking them in a plastic basin filled with clear water For 1h, rub the diseased leaves by hand to release the brown rust spores into clean water, and after filtering through a 24-mesh sieve, prepare a suspension of 40-50 spores/10 ⁇ 10 times the brown rust spores.
  • the sugarcane varieties with high brown rust resistance and no Bru1 gene from different sources mentioned in step (1) are Yuetang 00-236 or ROC24, and the sugarcane varieties with high brown rust resistance are Mex105 or Yuetang 03-393;
  • the hybrid combination prepared in step (2) is: Mex105 ⁇ Yuetang 00-236, Yuetang 03-393 ⁇ ROC24 or Mex105 ⁇ ROC24;
  • the SSR primers described in step (3) are: the SSR primers of Mex105 ⁇ Yuetang 00-236 are mSSCIR38 primers and mSSCIR48 primers, and the SSR primers of Yuetang 03-393 ⁇ ROC24 are mSSCIR67 primers and 66-SCC05 primers, Mex105 ⁇
  • the SSR primers of ROC24 are mSSCIR27 primer and mSSESTA17 primer;
  • the mSSCIR38 primer is composed of an upstream primer of mSSCIR38 and a lower primer of mSSCIR38, the base sequence of the upstream primer of mSSCIR38 is shown in SEQ ID NO:1, and the base sequence of the downstream primer of mSSCIR38 is shown in SEQ ID NO: 2;
  • the mSSCIR48 primer is composed of an upstream primer of mSSCIR48 and a downstream primer of mSSCIR48.
  • the base sequence of the upstream primer of mSSCIR48 is shown in SEQ ID NO: 3
  • the base sequence of the downstream primer of mSSCIR48 is shown in SEQ ID NO: 4;
  • the mSSCIR67 primer is composed of an upstream primer of mSSCIR67 and a downstream primer of mSSCIR67, the base sequence of the upstream primer of mSSCIR67 is shown in SEQ ID NO: 5, and the base sequence of the downstream primer of mSSCIR67 is shown in SEQ ID NO: 6;
  • the 66-SCC05 primer is composed of 66-SCC05 upstream primer and 66-SCC05 downstream primer.
  • the base sequence of the 66-SCC05 upstream primer is shown in SEQ ID NO: 7, and the base of the 66-SCC05 downstream primer The sequence is shown in SEQ ID NO: 8;
  • the mSSCIR27 primer is composed of an upstream primer of mSSCIR27 and a downstream primer of mSSCIR27, the base sequence of the upstream primer of mSSCIR27 is shown in SEQ ID NO: 9, and the base sequence of the downstream primer of mSSCIR27 is shown in SEQ ID NO: 10;
  • the mSSESTA17 primer is composed of an upstream primer of mSSESTA17 and a downstream primer of mSSESTA17, the base sequence of the upstream primer of mSSESTA17 is shown in SEQ ID NO: 11, and the base sequence of the downstream primer of mSSESTA17 is shown in SEQ ID NO: 12.
  • sugarcane brown rust resistance gene mapping genetic segregation population constructed by the method of the present invention is the isolation and cloning of new brown rust resistance genes.
  • Carrying out molecular breeding has laid a good foundation and provided technical support, which is of great significance and practical effect for the effective prevention and control of sugarcane brown rust using its disease resistance gene and ensuring the sustainable and stable development of China's sucrose industry.
  • the method of the present invention has simplified procedures and strong operability.
  • the three cross combinations of "Mex105 ⁇ Yuetang 00-236", “Yuetang 03-393 ⁇ ROC24”, and “Mex105 ⁇ ROC24” were constructed, and the segregation ratios of resistance to susceptibility of the hybrid offspring of the true hybrid F1 generation population were all ⁇ 3: 1. It shows that its disease resistance gene is controlled by a dominant single gene, and the three authentic hybrid F1 populations can be used for brown rust resistance gene mapping.
  • SEQ ID NO: 1 in the sequence table shows the base sequence of the upstream primer of mSSCIR38.
  • SEQ ID NO: 2 in the sequence table shows the base sequence of the downstream primer of mSSCIR38.
  • SEQ ID NO: 3 in the sequence table shows the base sequence of the upstream primer of mSSCIR48.
  • SEQ ID NO: 4 in the sequence table shows the base sequence of the downstream primer of mSSCIR48.
  • SEQ ID NO: 5 in the sequence table shows the base sequence of the upstream primer of mSSCIR67.
  • SEQ ID NO: 6 in the sequence table shows the base sequence of the downstream primer of mSSCIR67.
  • SEQ ID NO: 7 in the sequence table shows the base sequence of the 66-SCC05 upstream primer.
  • SEQ ID NO: 8 in the sequence table shows the base sequence of the 66-SCC05 downstream primer.
  • SEQ ID NO: 9 in the sequence table shows the base sequence of the upstream primer of mSSCIR27.
  • SEQ ID NO: 10 in the sequence table shows the base sequence of the downstream primer of mSSCIR27.
  • SEQ ID NO: 11 in the sequence table shows the base sequence of the upstream primer of mSSESTA17.
  • SEQ ID NO: 12 in the sequence table shows the base sequence of the downstream primer of mSSESTA17.
  • SEQ ID NO: 13 in the sequence table shows the base sequence of the upstream primer of mSSCIR9*.
  • SEQ ID NO: 14 in the sequence table shows the base sequence of the downstream primer of mSSCIR9*.
  • SEQ ID NO: 15 in the sequence table shows the base sequence of the upstream primer of mSSCIR36.
  • SEQ ID NO: 16 in the sequence table shows the base sequence of the downstream primer of mSSCIR36.
  • SEQ ID NO: 17 in the sequence list shows the base sequence of the upstream primer of SMC1490CL.
  • SEQ ID NO: 18 in the sequence table shows the base sequence of the downstream primer of SMC1490CL.
  • Sugarcane varieties with high brown rust resistance and no Bru1 gene from different sources and sugarcane varieties with high susceptibility to brown rust as parents refer to sugarcane varieties with different blood relatives from different geographical sources.
  • four sugarcane varieties with high brown rust resistance and no Bru1 gene from different sources were selected by conventional artificial inoculation resistance to brown rust identification and molecular detection of the conventional brown rust resistance gene Bru1 among many sugarcane varieties.
  • the polymorphic primers between the parents of "Mex105 ⁇ Yuetang 00-236" were selected as mSSCIR38 primer and mSSCIR48 primer, "ROC26 ⁇
  • the polymorphic primers between parents of "Yuetang 93-159" are mSSCIR67 primers and mSSCIR9* primers
  • the polymorphic primers between parents of "ROC28 ⁇ Decane 93-88” are mSSCIR36 primers and mSSCIR27 primers
  • “Yuetang 03-393 ⁇ ROC24 The polymorphic primers between parents are mSSCIR67 primer and 66-SCC05 primer
  • the polymorphic primers between parents of "Mex105 ⁇ ROC24” are mSSCIR27 primer and mSSESTA17 primer
  • the mSSCIR38 primer is composed of mSSCIR38 upstream primer and mSSCIR38 lower primer.
  • the mSSCIR48 primer is composed of mSSCIR48 upstream primer and mSSCIR48 downstream primer.
  • the mSSCIR67 primer is composed of mSSCIR67 upstream primer and mSSCIR67 downstream primer.
  • the mSSCIR9* primer is composed of mSSCIR9* upstream primer and mSSCIR9* downstream primer.
  • the mSSCIR36 primer is composed of mSSCIR36 upstream primer and mSSCIR36 downstream primer.
  • the mSSCIR27 primer is composed of mSSCIR27 upstream primer and mSSCIR27 downstream primer.
  • the 66-SCC05 primer is composed of 66-SCC05 upstream primer and 66-SCC05 downstream primer.
  • the mSSESTA17 primer is composed of mSSESTA17 upstream primer and mSSESTA17 downstream primer.
  • the SMC1490CL primer consists of the SMC1490CL upstream primer and the SMC1490CL downstream primer.
  • the reaction system and the reaction procedure of each primer for PCR are: 20 ⁇ L reaction system containing 8.5 ⁇ L ddH 2 O, 2 ⁇ Easy Taq PCR SuperMix for PAGE 9.0 ⁇ L, DNA template 0.5 ⁇ L, and upstream and downstream primers each 1.0 ⁇ L (10 ⁇ g/ ⁇ L) .
  • the reaction procedure is: 94°C pre-denaturation 4min; 94°C denaturation 15s, 54°C annealing 15s, 72°C extension 1min, 35 cycles; finally 72°C extension 5min.
  • the amplified bands of the offspring are all from the parents and are judged as true hybrids; the amplified bands of the offspring contain hybrid bands other than the parents, and they are judged as false hybrids; the amplified bands of the offspring contain only the female parent Band, judged as self-bred species.
  • the diseased leaves of sugarcane with brown rust spores were collected from the diseased sugarcane field in Maitreya, Yunnan, moisturized and brought back to the laboratory to extract the spores of the disease for direct inoculation, specifically: collecting brown rust spores of sugarcane
  • the diseased leaves were soaked in a plastic basin filled with clear water for 1 hour, and the diseased leaves were rubbed by hand to release the brown rust pathogen spores into the clear water. After filtering through a 24-mesh sieve, 40-50 spores/10 ⁇ 10 times A suspension of brown rust pathogen spores in the field of view.
  • asymptomatic leaves are level 1 high resistance; leaf diseased spots account for less than 10% of the leaf area as level 2 disease resistance; leaf diseased spots account for 11%-25% of the leaf area as level 3 medium resistance; leaf diseased spots account for 26 %-35% is grade 4 moderately susceptible; leaf disease spots account for 36%-50% of the leaf area and are grade 5 susceptible1; leaf disease spots account for 51%-60% of the leaf area are grade 6 susceptible2; leaf disease spots account for 61%-75% of leaf area is 7th grade susceptible 3; leaf diseased spots account for 76%-90% of leaf area, 8th grade high susceptibility 1; leaf diseased spots account for 91%-100% of leaf area, 9th grade high susceptibility 2.
  • the resistance and susceptibility segregation ratio of the hybrid offspring of the true cross F1 generation population of each cross combination is calculated according to the following formula:
  • the resistance and susceptibility segregation ratio of the genetically segregated populations of hybrid offspring (total number of disease-resistant single plants of grade 1-3): (total number of diseased single plants of grade 4-9),
  • the true hybrid F1 generation population with the resistance to susceptibility segregation ratio of the hybrid offspring genetic segregation population ⁇ 3:1 was selected as the sugarcane brown rust resistance gene mapping genetic segregation population.
  • the segregation ratio of resistance to susceptibility in the F1 generation of "Mex105 ⁇ ROC24" authentic cross is 294R:106S ⁇ 3:1.
  • the segregation ratio of resistance to susceptibility in the F1 generation population of "ROC26 ⁇ Dezhe 93-88" authentic cross is 18R:382S ⁇ 1:21.
  • R represents disease resistance
  • S represents susceptible disease

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Abstract

一种构建甘蔗抗褐锈病基因定位遗传分离群体的方法，包括：以来源不同的高感褐锈病甘蔗品种作母本×高抗褐锈病不含Bru1基因的甘蔗品种作父本配制组合杂交得到F1代种子，经播种、筛选真实性杂交F1代、种植真实性杂交F1代单株、接种褐锈病病菌、调查发病情况、按9级标准评价抗性、分析真实性杂交F1代群体抗病遗传，选取杂交后代遗传分离群体抗感分离比≈3:1的真实性杂交F1代群体为甘蔗抗褐锈病基因定位遗传分离群体。本发明方法程序简化，可操作性强，构建的真实性杂交F1群体抗感分离比≈3:1，其抗病基因由显性单基因控制，可用于抗褐锈病基因定位。

Description

一种构建甘蔗抗褐锈病基因定位遗传分离群体的方法 技术领域
本发明属于作物抗病基因定位技术领域，具体涉及一种构建甘蔗抗褐锈病基因定位遗传分离群体的方法。
背景技术
甘蔗褐锈病是由黑顶柄锈菌(Puccina.melanocephala H.Sydow&P.Sydow)引起的一种重要病害，在我国甘蔗主产区常发生流行，造成巨大的经济损失，严重影响着蔗糖产业的可持续发展。甘蔗褐锈病主要表现为蔗叶出现大量红褐色病斑，病斑合并形成大幅不定形的坏死区域，结果蔗叶未熟先死，甚至嫩叶也是这样。
甘蔗褐锈病的发生流行与品种抗病性密切相关，大面积种植感病品种是病害流行的重要原因。生产上，大面积施药防治甘蔗褐锈病非常困难，且收效甚微，选育和种植抗病品种是防治甘蔗褐锈病最经济有效的措施，前期研究发现有些甘蔗品种其具有高抗褐锈病特性，经分子标记检测分析未检测到已发现的褐锈病抗性基因Bru1，预示这些甘蔗品种可能含有新的抗褐锈病基因，发掘利用这些抗病种质资源是有效开展甘蔗抗褐锈病育种的基础和关键，利用其高抗褐锈病种质资源，研究明确其抗性遗传规律，发掘抗性基因资源十分必要。抗性基因的发掘与定位，可为抗褐锈病新基因的分离克隆及开展分子育种奠定基础，对有效防控甘蔗褐锈病和确保我国蔗糖产业持续稳定发展具有重要意义。而抗褐锈病基因发掘与定位的的基础和关键，需要开发建立简便、高效的遗传分离群体构建方法，构建用于抗褐锈病基因定位的遗传分离群体。
发明内容
针对甘蔗抗褐锈病基因发掘与定位的基础和关键，本发明提供一种简便、高效的构建甘蔗抗褐锈病基因定位遗传分离群体的方法。
本发明的技术方案以下：
一种构建甘蔗抗褐锈病基因定位遗传分离群体的方法，包括以下步骤：
(1)亲本选择:选取来源不同的高抗褐锈病不含Bru1基因的甘蔗品种和高感褐锈病甘蔗品种作为亲本；
(2)配制杂交组合杂交：以高感褐锈病甘蔗品种作母本×高抗褐锈病不含Bru1基因的甘蔗品种作父本配制杂交组合，通过杂交得到F1代种子；
(3)杂交组合亲本间多态性引物筛选：筛选出杂交组合在感病和抗病亲本间条带清晰、多态性明显、重复性好的SSR引物；
(4)筛选真实性杂交F1代群体：每个杂交组合F1代种子播种至少1000粒，利用步骤(3)所选的SSR引物对相应的杂交组合F1代种子苗进行杂交后代真实性鉴定，每个杂交组合筛选出真实性杂交F1代单株至少500苗；
(5)田间人工接种褐锈病病菌：田间种植真实性杂交F1代确保成活至少400苗，用褐锈病病菌孢子悬浮液于傍晚喷雾田间种植的真实性杂交F1代蔗叶接种；首次接种后，第二天按前述接种方式再接种1次；
(6)病情调查：接种4-5周后，调查记录各真实性杂交F1代单株发病情况，逐份材料进行调查，目测每份材料叶片褐锈病侵染面积百分率；
(7)抗病性评价：根据杂交F1代单株发病情况按1-9级分级标准进行褐锈病抗性评价，其中,叶片无症状为1级高抗；叶片病斑占叶面积10％以下为2级抗病；叶片病斑占叶面积11％-25％为3级中抗；叶片病斑占叶面积26％-35％为4级中感；叶片病斑占叶面积36％-50％为5级感病1；叶片病斑占叶面积51％-60％为6级感病2；叶片病斑占叶面积61％-75％为7级感病3；叶片病斑占叶面积76％-90％为8级高感1；叶片病斑占叶面积91％-100％为9级高感2；
(8)抗性遗传分析：根据每个杂交组合中真实性杂交F1代群体中褐锈病抗性及其单株数量，按如下公式计算每个杂交组合的真实性杂交F1代群体的杂交后代遗传分离群体抗感分离比：
杂交后代遗传分离群体抗感分离比＝(1-3级抗病总单株数):(4-9级感病总单株数)，
选取杂交后代遗传分离群体抗感分离比≈3:1的真实性杂交F1代群体为甘蔗抗褐锈病基因定位遗传分离群体。
步骤(5)中所述的褐锈病病菌孢子悬浮液为采集带褐锈病孢子蔗叶提取其孢子直接用于接种，具体是：采集带褐锈病孢子甘蔗病叶浸泡于盛有清水的塑料盆中1h，用手搓揉其病叶将褐锈病孢子释放至清水中，经24目分样筛过滤后配成40-50个孢子/10×10倍视野的褐锈病病菌孢子悬浮液。
步骤(1)中所述的来源不同的高抗褐锈病不含Bru1基因的甘蔗品种为粤糖00-236或ROC24，高感褐锈病甘蔗品种为Mex105或粤糖03-393；
步骤(2)中所配制的杂交组合为：Mex105×粤糖00-236，粤糖03-393×ROC24或Mex105×ROC24；
步骤(3)中所述的SSR引物为：Mex105×粤糖00-236的SSR引物为mSSCIR38引物和mSSCIR48引物，粤糖03-393×ROC24的SSR引物为mSSCIR67引物和66-SCC05引物，Mex105×ROC24的SSR引物为mSSCIR27引物和mSSESTA17引物；
所述mSSCIR38引物由mSSCIR38上游引物和mSSCIR38下引物组成，所述mSSCIR38上游引物的碱基序列如SEQ ID NO：1所示，mSSCIR38下游引物的碱基序列如SEQ ID NO：2所示；所述mSSCIR48引物由mSSCIR48上游引物和mSSCIR48下游引物组成，所述mSSCIR48上游引物的碱基序列如SEQ ID NO：3所示，mSSCIR48下游引物的碱基序列如SEQ ID NO：4所示；
所述mSSCIR67引物由mSSCIR67上游引物和mSSCIR67下游引物组成，所述mSSCIR67上游引物的碱基序列如SEQ ID NO：5所示，所述mSSCIR67下游引物的碱基序列如SEQ ID NO：6所示；所述66-SCC05引物由66-SCC05上游引物和66-SCC05下游引物组成，所述66-SCC05上游引物的碱基序列如SEQ ID NO：7所示，所述66-SCC05下游引物的碱基序列如SEQ ID NO：8所示；
所述mSSCIR27引物由mSSCIR27上游引物和mSSCIR27下游引物组成，所述mSSCIR27上游引物的碱基序列如SEQ ID NO：9所示，所述mSSCIR27下游引物的碱基序列如SEQ ID NO：10所示；所述mSSESTA17引物由mSSESTA17上游引物和mSSESTA17下游引物组成，所述mSSESTA17上游引物的碱基序列如SEQ ID NO：11所示，所述mSSESTA17下游引物的碱基序列如SEQ ID NO：12所示。
本发明的有益效果：
1、首次建立了一套简便、系统性构建甘蔗抗褐锈病基因定位遗传分离群体的方法，利用本发明方法构建的甘蔗抗褐锈病基因定位遗传分离群体，为抗褐锈病新基因的分离克隆及开展分子育种奠定了良好基础、提供了技术支撑，对利用其抗病基因有效防控甘蔗褐锈病和确保我国蔗糖产业持续稳定发展具有重要意义和现实作用。
2、本发明方法程序简化，可操作性强。构建的“Mex105×粤糖00-236”、“粤糖03-393×ROC24”、“Mex105×ROC24”3个杂交组合的真实性杂交F1代群体的杂交后代群体抗感分离比均≈3:1，表明其抗病基因由显性单基因控制，该3个真实性杂交F1代群体均可用于抗褐锈病基因定位。
序列表中SEQ ID NO：1所示的是mSSCIR38上游引物的碱基序列。
序列表中SEQ ID NO：2所示的是mSSCIR38下游引物的碱基序列。
序列表中SEQ ID NO：3所示的是mSSCIR48上游引物的碱基序列。
序列表中SEQ ID NO：4所示的是mSSCIR48下游引物的碱基序列。
序列表中SEQ ID NO：5所示的是mSSCIR67上游引物的碱基序列。
序列表中SEQ ID NO：6所示的是mSSCIR67下游引物的碱基序列。
序列表中SEQ ID NO：7所示的是66-SCC05上游引物的碱基序列。
序列表中SEQ ID NO：8所示的是66-SCC05下游引物的碱基序列。
序列表中SEQ ID NO：9所示的是mSSCIR27上游引物的碱基序列。
序列表中SEQ ID NO：10所示的是mSSCIR27下游引物的碱基序列。
序列表中SEQ ID NO：11所示的是mSSESTA17上游引物的碱基序列。
序列表中SEQ ID NO：12所示的是mSSESTA17下游引物的碱基序列。
序列表中SEQ ID NO：13所示的是mSSCIR9*上游引物的碱基序列。
序列表中SEQ ID NO：14所示的是mSSCIR9*下游引物的碱基序列。
序列表中SEQ ID NO：15所示的是mSSCIR36上游引物的碱基序列。
序列表中SEQ ID NO：16所示的是mSSCIR36下游引物的碱基序列。
序列表中SEQ ID NO：17所示的是SMC1490CL上游引物的碱基序列。
序列表中SEQ ID NO：18所示的是SMC1490CL下游引物的碱基序列。
具体实施方式
以下结合实施例对本发明作一步的说明，实施例中无特殊说明的为常规方法。
术语：
来源不同的高抗褐锈病不含Bru1基因的甘蔗品种和高感褐锈病甘蔗品种作为亲本是指不同地理来源含有不同血缘的甘蔗品种。
1、亲本选择
本发明在众多甘蔗品种中经常规的人工接种抗褐锈病鉴定和常规的抗褐锈病基因Bru1的分子检测，分别选取来源不同的4个高抗褐锈病不含Bru1基因的甘蔗品种粤糖00-236、粤糖93-159、德蔗93-88、ROC24和4个高感褐锈病甘蔗品种Mex105、ROC26、ROC28、粤糖03-393作为亲本(各材料均可通过商业渠道购买)。
2、配制杂交组合杂交
分别以高感褐锈病甘蔗品种作母本×高抗褐锈病不含Bru1基因的甘蔗品种作父本，配制“Mex105×粤糖00-236”、“ROC26×粤糖93-159”、“ROC28×德蔗93-88”、“粤糖03-393×ROC24”、“Mex105×ROC24”、“ROC26×德蔗93-88”、“ROC28×粤糖00-236”7个杂交组合，通过杂交得到7个杂交组合F1代种子。
3、杂交组合亲本间多态性引物筛选
选取84对甘蔗SSR引物，经过对杂交组合亲本间多态性SSR引物大量筛选实验，从中筛选出“Mex105×粤糖00-236”亲本间多态性引物为mSSCIR38引物和mSSCIR48引物，“ROC26×粤糖93-159”亲本间多态性引物为mSSCIR67引物和mSSCIR9*引物，“ROC28×德蔗93-88”亲本间多态性引物为mSSCIR36引物和mSSCIR27引物，“粤糖03-393×ROC24”亲本间多态性引物为mSSCIR67引物和66-SCC05引物，“Mex105×ROC24”亲本间多态性引物为mSSCIR27引物和mSSESTA17引物，“ROC26×德蔗93-88”亲本间多态性引物为mSSCIR36引物和SMC1490CL引物，“ROC28×粤糖00-236”亲本间多态性引物为mSSCIR38引物和mSSCIR67引物。
所述mSSCIR38引物由mSSCIR38上游引物和mSSCIR38下引物组成。
所述mSSCIR48引物由mSSCIR48上游引物和mSSCIR48下游引物组成。
所述mSSCIR67引物由mSSCIR67上游引物和mSSCIR67下游引物组成。
所述mSSCIR9*引物由mSSCIR9*上游引物和mSSCIR9*下游引物组成。
所述mSSCIR36引物由mSSCIR36上游引物和mSSCIR36下游引物组成。
所述mSSCIR27引物由mSSCIR27上游引物和mSSCIR27下游引物组成。
所述66-SCC05引物由66-SCC05上游引物和66-SCC05下游引物组成。
所述mSSESTA17引物由mSSESTA17上游引物和mSSESTA17下游引物组成。
所述SMC1490CL引物由SMC1490CL上游引物和SMC1490CL下游引物组成。
4、筛选真实性杂交F1代群体
“Mex105×粤糖00-236”、“ROC26×粤糖93-159”、“ROC28×德蔗93-88”、“粤糖03-393×ROC24”、“Mex105×ROC24”、“ROC26×德蔗93-88”、“ROC28×粤糖00-236”7个杂交组合F1代种子各播种至少1000粒，利用步骤3各杂交组合亲本间多态性引物对相应的杂交组合F1代种子苗进行杂交后代真实性鉴定，每个杂交组合筛选出真实性杂交F1代单株至少500苗。
各引物进行PCR的反应体系和反应程序是：20μL反应体系中含ddH 2O 8.5μL、2×Easy Taq PCR SuperMix for PAGE 9.0μL、DNA模板0.5μL、上下游引物各1.0μL(10μg/μL)。反应程序为：94℃预变性4min；94℃变性15s，54℃退火15s，72℃延伸1min，35个循环；最后72℃延伸5min。
真实性杂交F1代判别：后代扩增条带均来自父母本，判定为真杂种；后代扩增条带中含有父母本以外的杂带，判定为假杂种；后代扩增条带仅含有母本条带，判定为自交种。
5、田间人工接种褐锈病病菌：
7月分别将“Mex105×粤糖00-236”、“ROC26×粤糖93-159”、“ROC28×德蔗93-88”、“粤糖03-393×ROC24”、“Mex105×ROC24”、“ROC26×德蔗93-88”、“ROC28×粤糖00-236”7个杂交组合真实性杂交F1代单株移植田间，每个杂交组合种植至少5行区、每行80苗、7天后补苗1次、确保成活至少400苗，常规管理。
10月从云南弥勒发病蔗田采集带褐锈病病菌孢子甘蔗病叶(发褐锈病的甘蔗病叶)保湿带回实验室提取其病菌孢子直接用于接种，具体是：采集带褐锈病病菌孢子甘蔗病叶浸泡于盛有清水的塑料盆中1h，用手搓揉其病叶将褐锈病病菌孢子释放至清水中，经24目分样筛过滤后配成40-50个孢子/10×10倍视野的褐锈病病菌孢子悬浮液。于傍晚用高压机动喷雾器喷雾田间种植的真实性杂交F1代蔗叶接种。接种量控制在褐锈病病菌孢子悬浮液在蔗叶上不流淌为宜。首次接种后，第二天按前述接种方式再接种1次。
6、病情调查
接种4-5周后，调查记录各真实性杂交F1代单株发病情况，逐份材料进行调查，目测每份材料叶片褐锈病侵染面积百分率。
7、抗病性评价
根据各真实性杂交F1代单株发病情况按1-9级分级标准进行褐锈病抗性评价。其中,叶片无症状为1级高抗；叶片病斑占叶面积10％以下为2级抗病；叶片病斑占叶面积11％-25％为3级中抗；叶片病斑占叶面积26％-35％为4级中感；叶片病斑占叶面积36％-50％为5级感病1；叶片病斑占叶面积51％-60％为6级感病2；叶片病斑占叶面积61％-75％为7级感病3；叶片病斑占叶面积76％-90％为8级高感1；叶片病斑占叶面积91％-100％为9级高感2。
上述各组合中，“Mex105×粤糖00-236”真实性杂交F1代群体中307个单株表现1-3级抗病，93个单株表现4-9级感病；
“ROC26×粤糖93-159”真实性杂交F1代群体中38个单株表现1-3级抗病，362个单株表现4-9级感病；
“ROC28×德蔗93-88”真实性杂交F1代群体中56个单株表现1-3级抗病，344个单株表现4-9级感病；
“粤糖03-393×ROC24”真实性杂交F1代群体中298个单株表现1-3级抗病，102个单株表现4-9级感病；
“Mex105×ROC24”真实性杂交F1代群体中294个单株表现1-3级抗病，106个单株表现4-9级感病；
“ROC26×德蔗93-88”真实性杂交F1代群体中18个单株表现1-3级抗病，382个单株表现4-9级感病；
“ROC28×粤糖00-236”真实性杂交F1代群体中30个单株表现1-3级抗病，370个单株表现4-9级感病。
8、抗性遗传分析
根据每个杂交组合的真实性杂交F1代群体中褐锈病抗性及其单株数量，按如下公式计算每个杂交组合的真实性杂交F1代群体的杂交后代遗传分离群体抗感分离比：
杂交后代遗传分离群体抗感分离比＝(1-3级抗病总单株数):(4-9级感病总单株数)，
选取杂交后代遗传分离群体抗感分离比≈3:1的真实性杂交F1代群体为甘蔗抗褐锈病基因定位遗传分离群体。
如表1所示，根据各杂交组合的真实性杂交F1代群体褐锈病抗性及其单株数量，计算的杂交后代遗传分离群体抗感分离比(简称：抗感分离比)，分析各杂交组合的真实性杂交F1代群体褐锈病抗性遗传。
“Mex105×粤糖00-236”真实性杂交F1代群体抗感分离比307R:93S≈3:1。
“ROC26×粤糖93-159”真实性杂交F1代群体抗感分离比38R:362S≈1:10。
“ROC28×德蔗93-88”真实性杂交F1代群体抗感分离比56R:344S≈1:6。
“粤糖03-393×ROC24”真实性杂交F1代群体抗感分离比298R:102S≈3:1。
“Mex105×ROC24”真实性杂交F1代群体抗感分离比294R:106S≈3:1。
“ROC26×德蔗93-88”真实性杂交F1代群体抗感分离比18R:382S≈1:21。
“ROC28×粤糖00-236”真实性杂交F1代群体抗感分离比30R:370S≈1:12。
其中，“Mex105×粤糖00-236”、“粤糖03-393×ROC24”、“Mex105×ROC24”3个组合真实性杂交F1代群体抗感分离比均≈3:1，表明其抗病基因由显性单基因控制，该3个真实性杂交F1代群体均可用于甘蔗抗褐锈病基因定位遗传分离群体。
表1各杂交组合的真实性杂交F1代群体褐锈病抗性遗传分析结果
杂交组合 抗感分离比 抗性遗传类型
Mex105×粤糖00-236 307R:93S≈3:1 抗病基因为单基因显性遗传
ROC26×粤糖93-159 38R:382S≈1:10 抗病基因为隐性遗传
ROC28×德蔗93-88 56R:344S≈1:6 抗病基因为隐性遗传
粤糖03-393×ROC24 298R:102S≈3:1 抗病基因为单基因显性遗传
Mex105×ROC24 294R:106S≈3:1 抗病基因为单基因显性遗传
ROC26×德蔗93-88 18R:382S≈1:21 抗病基因为隐性遗传
ROC28×粤糖00-236 30R:370S≈1:12 抗病基因隐为性遗传
注：R表示抗病，S表示感病。

Claims (2)

  1. 一种构建甘蔗抗褐锈病基因定位遗传分离群体的方法，其特征在于，包括以下步骤：
    (1)亲本选择:选取来源不同的高抗褐锈病不含Bru1基因的甘蔗品种和高感褐锈病甘蔗品种作为亲本；所述的来源不同的高抗褐锈病不含Bru1基因的甘蔗品种为粤糖00-236、ROC24，高感褐锈病甘蔗品种为Mex105、粤糖03-393；
    (2)配制杂交组合杂交：以高感褐锈病甘蔗品种作母本×高抗褐锈病不含Bru1基因的甘蔗品种作父本配制杂交组合，通过杂交得到F1代种子；所配制的杂交组合为：Mex105×粤糖00-236，粤糖03-393×ROC24，Mex105×ROC24；
    (3)杂交组合亲本间多态性引物筛选：筛选出杂交组合在感病和抗病亲本间条带清晰、多态性明显、重复性好的SSR引物；
    Mex105×粤糖00-236的SSR引物为mSSCIR38引物和mSSCIR48引物，粤糖03-393×ROC24的SSR引物为mSSCIR67引物和66-SCC05引物，Mex105×ROC24的SSR引物为mSSCIR27引物和mSSESTA17引物；
    所述mSSCIR38引物由mSSCIR38上游引物和mSSCIR38下引物组成，所述mSSCIR38上游引物的碱基序列如SEQ ID NO：1所示，mSSCIR38下游引物的碱基序列如SEQ ID NO：2所示；所述mSSCIR48引物由mSSCIR48上游引物和mSSCIR48下游引物组成，所述mSSCIR48上游引物的碱基序列如SEQ ID NO：3所示，mSSCIR48下游引物组成的碱基序列如SEQ ID NO：4所示；
    所述mSSCIR67引物由mSSCIR67上游引物和mSSCIR67下游引物组成，所述mSSCIR67上游引物的碱基序列如SEQ ID NO：5所示，所述mSSCIR67下游引物的碱基序列如SEQ ID NO：6所示；所述66-SCC05引物由66-SCC05上游引物和66-SCC05下游引物组成，所述66-SCC05上游引物的碱基序列如SEQ ID NO：7所示，所述66-SCC05下游引物的碱基序列如SEQ ID NO：8所示；
    所述mSSCIR27引物由mSSCIR27上游引物和mSSCIR27下游引物组成，所述mSSCIR27上游引物的碱基序列如SEQ ID NO：9所示，所述mSSCIR27下游引物的碱基序列如SEQ ID NO：10所示；所述mSSESTA17引物由mSSESTA17上游引物和mSSESTA17下游引物组成，所述mSSESTA17上游引物的碱基序列如SEQ ID NO：11所示，所述mSSESTA17下游引物的碱基序列如SEQ ID NO：12所示；
    (4)筛选真实性杂交F1代群体：每个杂交组合F1代种子播种至少1000粒，利用步骤(3)所选的SSR引物对杂交组合F1代种子苗进行杂交后代真实性鉴定，每个杂交组合筛选出真实性杂交F1代单株至少500苗；
    (5)田间人工接种褐锈病病菌：田间种植真实性杂交F1代确保成活至少400苗，用褐锈病病菌孢子悬浮液于傍晚喷雾田间种植的真实性杂交F1代蔗叶接种；首次接种后，第二天按前述接种方式再接种1次；
    (6)病情调查：接种4-5周后，调查记录各真实性杂交F1代单株发病情况，逐份材料进行调查，目测每份材料叶片褐锈病侵染面积百分率；
    (7)抗病性评价：根据杂交F1代单株发病情况按1-9级分级标准进行褐锈病抗性评价，其中,叶片无症状为1级高抗；叶片病斑占叶面积10％以下为2级抗病；叶片病斑占叶面积11％-25％为3级中抗；叶片病斑占叶面积26％-35％为4级中感；叶片病斑占叶面积36％-50％为5级感病1；叶片病斑占叶面积51％-60％为6级感病2；叶片病斑占叶面积61％-75％为7级感病3；叶片病斑占叶面积76％-90％为8级高感1；叶片病斑占叶面积91％-100％为9级高感2；
    (8)抗性遗传分析：根据杂交组合中真实性杂交F1代群体中褐锈病抗性及其单株数量，按如下公式计算杂交组合的真实性杂交F1代群体的杂交后代遗传分离群体抗感分离比：
    杂交后代遗传分离群体抗感分离比＝(1-3级抗病总单株数):(4-9级感病总单株数)，
    选取杂交后代遗传分离群体抗感分离比≈3:1的真实性杂交F1代群体为甘蔗抗褐锈病基因定位遗传分离群体。
  2. 根据权利要求1所述的构建甘蔗抗褐锈病基因定位遗传分离群体的方法，其特征在于：步骤(5)中所述的褐锈病病菌孢子悬浮液为采集带褐锈病孢子蔗叶提取病菌孢子直接用于接种，具体是：采集带褐锈病孢子甘蔗病叶浸泡于盛有清水的塑料盆中1h，用手搓揉病叶将孢子释放至清水中，经24目分样筛过滤后配成40-50个孢子/10×10倍视野的褐锈病病菌孢子悬浮液。
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