Response of Bacterial Community in Soil of Banana Plantation to Combined Use of Organic and Inorganic Fertilizers
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摘要:目的 探明化肥减量配施有机肥对香蕉土壤细菌群落结构特征的影响。方法 设计5个不同施肥处理：不施肥处理（CK，T1）；25%化肥配施有机肥（T2）；50%化肥配施有机肥（T3）；100%化肥（100% CF，T4）和50%化肥（50% CF，T5）。采用Illumina MiSeq高通量测序平台，对土壤细菌16S rRNA基因保守区进行测序，并对不同施肥处理下土壤细菌群落数量、结构和多样性的差异进行生物信息学分析。结果 T1、T2、T3、T4和T5处理的OTUs数分别是30、33、31、34、31个。不同处理中，优势菌群所占的比例各不相同。T3处理提高了土壤中有益菌变形杆菌门（Proteobacteria）和酸杆菌门（Acidobacteria）的相对丰度。各处理土壤细菌的Shannon指数大小顺序为：T3＞T5＞T2＞T1＞T4。和单施50% CF（T5）化肥相比，50% CF配施有机肥处理（T3）提高了土壤细菌多样性。与T5处理相比，T2处理的多样性降低，说明化肥用量过低不利于土壤多样性的提高。T4处理的微生物多样性最低，表明过量的化肥施入降低了土壤微生物多样性。主坐标分析和热图分析结果表明，不同处理细菌群落结构、相对丰度和优势菌群发生了明显的变化。结论 与单施化肥相比，合理的有机无机配施能够提高土壤细菌多样性、改善土壤细菌群落结构。减氮50%配施有机肥（T3）处理不仅减少了氮肥施用量，而且增加了土壤有益细菌的相对丰度和土壤细菌多样性，有利于土壤生物肥力的提高。Abstract:Objective Effects of combined application of organic and chemical fertilizers on the bacterial community in soil of banana plantations were studied.Method Various proportions of chemical fertilizer combined with an organic fertilizer, as well as all or reduced chemical fertilizer, were mixed in the soil from a banana plantation to determine the effect of the applications on the bacterial community in soil. The treatments included the uses of no fertilizer as control (T1), 25% chemical fertilizer (T2), 50% chemical fertilizer (T3), 100% chemical fertilizer (T4), and 50% reduced chemical fertilizer without organic fertilizer (T5). 16S rRNA genes of the bacteria were analyzed by Illumina MiSeq high-throughput sequencing. A bioinformatic analysis was performed to determine the structure, abundance, and diversity of the bacterial communities in soil under treatments.Result The OTUs of the T1, T2, T3, T4, and T5 treatments were 30, 33, 31, 34, and 31, respectively. The proportion of beneficial bacteria in the soils varied by the treatments. The relative abundances of beneficial proteobacteria and acidobacteria were increased by the treatment of T3. The Shannon index of the bacteria in soil under different treatments ranked as T3＞T5＞T2＞T1＞T4. In comparison to T5, T3 improved and T2 reduced the soil bacterial diversity indicating a disadvantage of the reduced use of chemical fertilizer. However, the lowest diversity was observed under T4 which showed excessive chemical fertilization to be detrimental, nonetheless. The PCoA and heat map analyses revealed significant changes on the bacterial compositions, relative abundance, and beneficial bacteria in the soil by the treatments.Conclusion Comparing to the use of chemical fertilizer alone, appropriate combination of organic and inorganic fertilizers effectively improved the bacterial diversity and composition in soil. A 50% reduction of chemical fertilizer usage coupled with organic fertilizer (T3) could not only conserve the chemical fertilizer, but also enhance the relative abundances of beneficial bacteria and bacterial diversity resulting in an improved soil fertility.
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0. 引言
【研究意义】紫黑米作为一种重要的功能稻米，富含多种维生素、花色苷及微量元素，是天然的药、食兼用的功能性保健食品，深受消费者喜爱 [1]。紫黑米种皮内沉积的花色苷是花青素与糖结合的产物，其中花青素具有抗氧化清除自由基、降低血脂、抗癌、消炎等作用[2-3]，已被广泛应用于食品、化妆品、医药及保健品[4-5]。此外花青素作为天然染料应用于纺织品的研究也有所报道[6]。作为中国消费量最大的功能稻米，紫黑米具有广阔的市场前景和工业加工价值。分子标记辅助选择可以极大提升作物育种效率，加快育种进程。利用与目标基因紧密连锁的分子标记解析品种农艺性状遗传构成，可为品种的进一步应用和改良提供分子依据。【前人研究进展】我国紫黑米资源丰富，但多数代表品种均存在生育期长、产量低、抗性差等问题[7]。20世纪80年代特种稻种质资源的创新利用研究开始兴起[8]，迄今已培育出黑糯1号、晚籼紫宝等一系列新的紫黑米种质[9-15]。然而与普通稻米育种研究相比，特种稻育种应用分子辅助技术的还比较少。唐清杰等应用分子标记检测和分析了海丰黑糯2号的抗病虫基因[16]。王军等[17]对香糯龙晴4号的紫色和香味进行了基因型分析。许峰等[18]将稻瘟病抗性基因导入香血糯335，并与中间品系杂交，选育出了香血稻515。刘耀光课题组[19]通过开发的新一代高效多基因载体系统TGS II（TransGene Stacking II），把花青素合成相关的8个关键基因转入水稻，实现了花青素在水稻胚乳特异合成，创造出首例富含花青素的水稻新种质“紫晶米”。【本研究切入点】前人对紫黑米的研究着重于新种质的创制和应用，分子辅助育种也仅限于个别性状基因的转育，全面解析优异种质遗传特性的研究尚鲜见报道。【拟解决的关键问题】紫两优737是福建省农科院水稻研究所利用自育的紫糯两系不育系紫392S[20]与紫糯恢复系福恢737配组育成的杂交紫糯稻新品种，是国内首个通过省级审定的紫糯两系杂交稻，填补了国内外紫糯两系杂交稻品种空白。该品种在云南、福建、安徽、广西多地试种示范，产量高、品质好、适应性广、富含花青素，具有很好的应用前景。本研究以紫两优737及其亲本紫392S、福恢737为材料，采用重要农艺性状相关基因的特异性标记对其进行检测，分析紫两优737及其亲本携带的有利等位基因，为该品种的进一步利用提供科学依据。
1. 材料与方法
1.1 水稻材料
供试水稻材料为紫两优737，以及双亲紫392S和福恢737。所检测基因座位上的对照材料包含日本晴、9311、珍汕97和明恢63等，详见表1。
表 1 检测10个基因的相关标记信息Table 1. Markers’ information for 10 genes基因
Gene标记
Marker引物序列
Primer sequence (5′-3′)退火温度
Annealing temperature/℃等位基因参照
Allele referenceGn1a Gn1a-M1[21] CTCTTGCTTCATTATCAATC 55 明恢63 Minghui 63 AAACTACACAAGAATCTGCT GS3 GS3-PstⅠ[21]
（限制性内切酶 Restriction enzyme：PstⅠ）TATTTATTGGCTTGATTTCCTGTG 55 珍汕97 Zhenshan 97，明恢63 Minghui 63 GCTGGTTTTTTACTTTCATTTGCC Hd3a hd3afnp inner[22] AGCGGCAGGAGaGTCTACAA 62 日本晴 Nipponbare， Kasalath TCaGGATCATCGTTAGCTAGGG hd3afnp outer AAtCGAGGGGAGTATATTGCTAGT GCTaCATGAGAGACCTTAGCCTT Hd1 Si9337[23] AGATGTCCCTTCACTTCAGC 60 9311，日本晴 Nipponbare CGAAACGGCCCTTGATCC wx We 2-2[24] CACTACAAGACACACTTGCAC 55 荆糯6 Jingnuo 6, 9311 GTCATCTAGCCCACCACCTT Wx-t1[25] ATGTCGGCTCTCACCACG 55 荆糯6 Jingnuo 6, 9311 ACCGACCGCTGCTGCTTG 484/W2R[26]
（限制性内切酶 Restriction enzyme：AccⅠ）CTTTGTCTATCTCAAGACAC 55 9311，明恢63 Minghui 63 TTTCCAGCCCAACACCTTAC PCR- AccⅠ [27]
（限制性内切酶 Restriction enzyme：AccⅠ）GCTTCACTTCTCTGCTTGTG 55 日本晴Nipponbare, 明恢63 Minghui 63 ATGATTTAACGAGAGTTGAA Sbe1 Sbe1[28] GAGTTGAGTTGCGTCAGATC 57 9311，日本晴 Nipponbare AATGAGGTTGCTTGCTGCTG sbe3-rs RS/SpeⅠ[29]
（限制性内切酶 Restriction enzyme：SpeⅠ）ATGTGATGTGCTGGATTTGG 55 密阳 23 Miyang 23，宜优673 Yiyou 673 TGTGGTTTTCATACCGTTCTTA AGPlar AGPlar M1[30] CGTTCAGGTTCAGGCAATCA 58 珍汕97 Zhenshan 97, 9311 GGAAGGGTGGTGATGTGGAG PUL PUL M2[30] GACAACCGTCCGCTTTAGTTTC 58 9311, 宜优673 Yiyou 673 GCATTTGAGAGGGTTTGGATTC Pb CAPSPb [31]
（限制性内切酶 Restriction enzyme：BamHⅠ）AAATCAGTTGTCCCGTCCA 58 9311，日本晴 Nipponbare TTAGGGAGTTGGTGATGGG 1.2 分子标记
应用13个分子标记（表1）对紫392S、福恢737、紫两优737的重要农艺性状相关基因的基因型进行检测。其中产量性状相关基因2个，抽穗期相关基因2个，品质相关基因5个，紫色种皮基因1个。13个标记引物序列来自前人文献报道[21-31]。
1.3 标记检测
采用CTAB法提取水稻基因组DNA，DNA质量及浓度使用Thermo Scientific NanoDrop 2000检测。PCR反应体系（10 μl）：5 μl含染料2× Hieff® PCR Master mix（Yeasen Biotechnology （Shanghai） Co., Ltd.），引物1 μl （4引物标记为各引物等量混合，10 μmol·L−1），DNA模板2 μl（50~150 ng·μl−1），ddH2O 2 μL。
PCR反应程序：94 ℃预变性5 min； 94 ℃变性30 S，55～62 ℃退火30 s，72 ℃延伸1 min，35个循环；72 ℃延伸10 min。
根据扩增产物片段大小，分别采用6%非变性聚丙烯酰胺凝胶和1.5%琼脂糖凝胶电泳分离，Gelstain显色。
2. 结果与分析
2.1 产量性状相关基因Gn1a与GS3的标记检测
紫两优737在云南、福建和安徽等省区试的产量表现[1,20]（表2），三地区试中紫两优737的产量均超过7500 kg·hm−2，云南区试中，紫两优737比对照癸能紫米的增产幅度达到152.1%。
Table 2. Yield performance of Ziliangyou 737 in regional trails in Yunnan, Fujian and Anhui试验类型
Type of test品种
Varieties平均产量
Average Yield/（kg·hm−2）比对照增减产
Ratio compared with CK/%云南区试 Reginal trial in Yunnan 紫两优737 Ziliangyou 737 7566.90 152.1.0 癸能紫米 Guinengzimi（CK） 3002.10 福建区试 Reginal trial in Fujian 紫两优737 Ziliangyou 737 7505.48 −3.82 宜优673 Yiyou 673（CK） 7804.07 安徽区试 Reginal trial in Anhui 紫两优737 Ziliangyou 737 8441.63 −0.16 Ⅱ优838（CK） 8456.55 Gn1a与GS3均为水稻产量性状的主要构成因子，Gn1a基因定位于水稻第1染色体，是控制水稻每穗实粒数的主效QTL。采用YAN等[21]针对Gn1a基因非翻译区存在的16 bp碱基缺失而开发的STS标记，以明恢63为对照对供试材料进行检测。结果显示所有检测材料均扩增出大小约为113 bp的片段（图1-A），即紫两优737及双亲在Gn1a座位上均携带高产突变型Ha-Gn1a。
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图 1 水稻产量因子Gn1a、 GS3基因相关标记对紫两优737及其亲本的检测结果A：Gn1a-M1。M：20bp DNA Marker；1：明恢63；2：宜优673；3：紫392S；4：福恢737；5：紫两优737。B： GS3-PstⅠ，原始扩增产物（左）和酶切产物（右）。M：100 bp DNA Marker； 1：珍汕97，2：明恢63，3：紫392S，4：福恢737；5：紫两优737。Figure 1. Detection of Ziliangyou 737 and its parents with markers for rice yield components Gn1a and GS3A: Gn1a-M1. M: 20bp DNA Marker; 1: Minghui 63; 2: Yiyou 673; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737. B: GS3-PstⅠ, original amplified products(left) and enzyme digestion products (right). M: 100 bp DNA Marker; 1: Zhengshan 97; 2: Minghui 63; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737.GS3位于第3染色体，是控制水稻粒重和粒长的主效QTL，也是控制水稻粒宽和籽粒充实度的微效QTL。YAN等[21]根据GS3基因第2外显子的单核苷酸差异设计CAPS标记GS3-PstⅠ。短粒型在该变异位点为半胱氨酸密码子（TGC），长粒型为终止密码子（TGA），该变异位点能被限制性内切酶PstⅠ识别。以珍汕97和明恢63为对照，采用GS3-PstⅠ标记对供试材料进行检测，各品种均扩增出大小约为512 bp的片段，经酶切处理后，仅有珍汕97的扩增产物被酶切成大小约为294 bp和218 bp的两个片段（图1-B），表明紫两优737及其两个亲本在GS3座位上均携带长粒型等位基因MH- GS3。
2.2 抽穗期相关基因标记检测
抽穗期是决定水稻品种区域与季节适应性的重要因素，且对水稻抽穗期控制具有主效作用的基因往往对产量和株高也有重要作用[23]。Hd3a编码的成花素是调控水稻抽穗通路的关键因子，在短日照下促进抽穗，长日照下推迟抽穗。利用常远等[22]根据Hd3a第4外显子的碱基突变设计的共显性分子标记hd3afnp对供试材料进行检测。结果显示，紫392S、福恢737和紫两优737的基因型分别为Hd3aNip、hd3aKasa和Hd3aNip/hd3aKasa（图2-A）。
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图 2 水稻Hd3a、 Hd1基因相关标记对紫两优737及其亲本的检测结果A： hd3afnp。M：100 bp DNA Marker；1：日本晴；2：Kasalath；3：紫392S；4：福恢737；5：紫两优737。B：Si9337。M：20 bp DNA Marker；1：日本晴；2：9311；3：紫392S；4：福恢737；5：紫两优737。Figure 2. Detection of Ziliangyou 737 and its parents with markers for Hd3a and Hd1A: hd3afnp. M: 100 bp DNA Marker; 1: Nipponbare; 2: Kasalath; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737. B: Si9337. M: 20 bp DNA Marker; 1: Nipponbare; 2: 9311; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737.Hd1在短日照下激活Hd3a的表达、促进开花，长日照条件下抑制Hd3a的表达、延迟开花。陈俊宇等[23]根据珍汕97B和密阳46 Hd1基因内的序列差异设计了InDel标记Si9337，用该标记鉴定紫392S、福恢737、紫两优737的Hd1基因型，结果表明其基因型分别为Hd1jap、Hd1ind及Hd1jap /Hd1ind（图2-B）。
2.3 品质性状相关基因及紫色种皮基因Pb的标记检测
紫两优737在云南、福建区试的稻米品质测试结果显示紫两优737米质好，其直链淀粉含量分别为2.6%、2.1%、1.2%，胶稠度分别为90、97 、100 mm[1]（表3）。
表 3 紫两优737在云南、福建区域试验稻米品质[1]Table 3. Rice quality of Ziliangyou 737 in regional trails in Yunnan and Fujian类别 Types 糙米率
Brown rice percentage/%精米率
Head rice percentage/%整精米率
Head rice percentage/%粒长
Grain length/mm长宽比 Ratio of grain length to width 直链淀粉 Amylase content/% 碱消值 Alkali value 胶稠度
Gel consistency/mm云南区试Regional trial in Yunnan 78.4 69.4 55.3 6.3 2.7 2.6 7.0 90 福建区试 Reginal trial of in Fujian 79.3 68.5 65.1 6.4 2.9 2.1 7.0 97 采用基于糯稻基因组第2外显子上23 bp插入片段设计的共显性STS标记We2-2[24]和Wx-t1[25]对紫392S、福恢737及紫两优737的蜡质基因进行检测，三个品种均扩增出糯稻特征条带（图3-A、图3-B），表明三个品种均含糯稻蜡质基因wx。
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图 3 糯稻蜡质基因相关标记对紫两优737及其亲本的检测结果A：We 2-2。M：20 bp DNA Marker；1：荆糯6；2：9311；3：紫392S；4：福恢737；5：紫两优737。B：Wx-t1。M：20 bp DNA Marker；1：荆糯6；2：9311；3：紫392S；4：福恢737；5：紫两优737。C：484/W2R，原始扩增产物（左）和酶切产物（右）；M：100 bp DNA Marker；1：9311；2：明恢63；3：紫392S；4：福恢737；5：紫两优737。D：PCR-AccⅠ，原始扩增产物（左）和酶切产物（右）；M：100 bp DNA Marker；1：9311；2：明恢63；3：紫392S；4：福恢737；5：紫两优737。Figure 3. Detection of Ziliangyou 737 and its parents with markers for wxA: We 2-2. M: 20 bp DNA Marker; 1: Jingnuo 6; 2: 9311; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737. B: Wx-t1. M: 20 bp DNA Marker; 1: Jingnuo 6; 2: 9311; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737. C: 484/W2R, original amplified products (left) and enzyme digestion products (right); M: 100 bp DNA Marker; 1: 9311; 2: Minghui 63; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737. D: PCR-AccⅠ, original amplified products (left) and enzyme digestion products (right); M: 100 bp DNA Marker; 1: 9311; 2: Minghui 63; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737.水稻蜡质基因第1内含子+1的碱基类型与稻米中直链淀粉含量类型直接相关。当该位置的碱基为G时，能产生较多大小为2.3 kb的Wx基因成熟mRNA，从而积累更多GBSS蛋白，使得稻米中直链淀粉含量较高；当第1内含子碱基为T时，无法正常剪接，Wx翻译受阻，合成的GBSS蛋白较少，使得稻米中直链淀粉含量较低[32]。采用标记484/W2R和PCR-AccⅠ对供试材料进行检测。结果显示，所有供试材料均分别扩增出大小约为250 bp和460 bp的目标条带，但均不能被酶切（图3-C，图3-D），说明紫392S，福恢737及紫两优737 wx基因第1内含子+1位碱基均为T型，即表现为低直链淀粉含量。
采用严长杰等[27]根据籼粳差异开发的STS标记，对供试材料的Sbe1基因型进行检测，发现紫两优737及其亲本均扩增出与籼型对照一致的大小约为548 bp的条带（图4-A），说明待测材料均携带Sbe1i型等位基因。
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图 4 水稻淀粉合成相关基因、紫色种皮基因Pb相关标记对紫两优737及其亲本的检测结果A：Sbe1。M：100 bp DNA Marker。1：9311；2：明恢63；3：紫392S；4：福恢737；5：紫两优737。B：AGPlar M1。M：20 bp DNA Marker；1：珍汕97B；2：9311；3：紫392S；4：福恢737；5：紫两优737。C：PUL M2。M：20 bp DNA Marker；1：9311；2：宜优673；3：紫392S；4：福恢737；5：紫两优737。D：RS/SpeⅠ，原始扩增产物（左）和酶切产物（右）。M：100 bp DNA Marker；1：密阳23；2：宜优673；3：紫392S；4：福恢737；5：紫两优737。E：CAPSPb，原始扩增产物（左）和酶切产物（右）。M：100 bp DNA Marker；1：日本晴； 2：9311；3：紫392S；4：福恢737；5：紫两优737。Figure 4. Detection of Ziliangyou 737 and its parents with markers for starch synthesis-related genes and Pb geneA: Sbe1. M: 100 bp DNA Marker. 1: 9311; 2: Minghui 63; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737. B: AGPlar M1. M: 20 bp DNA Marker; 1: Zhenshan 97B; 2: 9311; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737. C: PUL M2. M: 20 bp DNA Marker; 1: 9311; 2: Yiyou 673; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737. D: RS/SpeⅠ, original amplified products (left) and enzyme digestion products (right). M: 100 bp DNA Marker; 1: Miyang 23; 2: Yiyou 673; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737. E: CAPSPb, original amplified products (left) and enzyme digestion products (right). M: 100 bp DNA Marker; 1: Nipponbare; 2: 9311; 3: Zi 392S; 4: Fuhui 737; 5: Ziliangyou 737.使用焦磷酸化酶大亚基基因AGPlar核心标记AGPlar M1[30]对供试材料进行鉴定，结果表明紫392S为Ⅰ型等位基因，福恢737为Ⅱ型等位基因，紫两优737为Ⅰ/Ⅱ杂合型（图4-B）。
极限糊精酶基因PUL为编码脱分支酶基因之一，研究表明PUL基因对稻米蒸煮食用品质的部分理化指标如胶稠度等有显著影响[33]。采用田志喜等[30]开发的核心分子标记PUL M2对供试品种进行鉴定，三个品种均扩增出与9311一致的条带，即Ⅱ型等位基因（图4-C）。
抗性淀粉（RS）具有降血糖血脂、促进肠道健康等重要的保健功能。采用基于控制水稻RS含量基因sbe3-rs的突变位点开发的RS/SpeⅠ功能标记[29]检测供试材料。所有材料均扩增出了大小约为571 bp的条带，且都被酶切成大小约为375 bp和196 bp两个条带（图4-D），表明与对照密阳23一样均为野生型SBE3。
用根据紫色果皮Pb与白色果皮pb等位基因在第7外显子的差异设计的CAPSPb标记[31]检测供试材料。紫两优737、紫392S、福恢737和对照日本晴、9311均扩增出大小约为1198bp的目标条带，经BamHⅠ酶切处理后，紫两优737及其亲本的扩增产物均被切开（图4-E），表明所有材料均携带紫色果皮等位基因型。
3. 讨论与结论
水稻杂种优势利用在普通稻米育种实践中已经被广泛使用。本课题组采用杂交育种方法，利用紫392S与福恢737配组选育出具有紫黑色种皮的紫糯两系特种稻新品种紫两优737[1]。紫两优737是国内首个通过省级审定的紫糯两系杂交稻，填补了国内外紫糯两系杂交稻品种空白。紫两优737的成功培育，是本课题组对杂种优势原理的创新性应用，成功开拓了紫糯稻两系法杂种优势利用的新途径。经云南、福建、安徽多地示范种植，紫两优737表现出适应好、产量高，直链淀粉含量低，糯性好，食用口感佳等优点[34]。
紫两优737在云南、福建、安徽种植的品种亩产量均超过500 kg，在提高紫糯米品种产量方面取得了重大突破。检测结果表明，紫两优737及其亲本在穗实粒数主效基因Gn1a座位上均携带高产等位基因Ha-Gn1a；在粒重和粒长主效基因GS3座位上均携带长粒型等位基因MH-GS3。这些基因的存在为紫两优737区别于其他常规紫糯稻的高产特性奠定了分子基础。
抽穗期是决定水稻品种适应地区和季节的关键性状，紫两优737对环境适应性好，已经通过云南省（滇审稻2019004）、福建省（闽审稻20200067）、安徽省（皖审稻20212002）品种审定和广西引种备案。抽穗期基因的检测结果为我们解释紫两优737适应性好这一特征提供了一些分子依据。Hd3a编码的成花素是水稻抽穗调控通路中的关键因子，来自aus稻品种Kasalath的ha3aKasa相对来自温带粳稻品种日本晴的Hd3aNip是高光效基因，但会导致水稻开花推迟，影响正常生产[22]。紫两优737在该基因位点的基因型则属于产量高和抽穗延迟适中的杂合态Hd3aNip/hd3aKasa。此外抽穗期基因Hd1在短日照下激活Hd3a的表达，促进开花；长日照下抑制Hd3a表达，延迟开花[23]，紫两优737在该位点也是杂合型Hd1jap /Hd1ind。
长期以来，糙米存在蒸煮难度大、食味欠佳的问题，阻碍了人们消费黑米的意愿，因而改善黑米的蒸煮食味品质是寻找开发稻米产能和营养富矿的“金钥匙”[35]。本课题培育的紫两优737具有直链淀粉含量低，糯性好，适口性好的优点。安徽区试中，紫两优737米质鉴定结果符合优质三等优质糯稻品种品质规定要求。品质相关基因检测结果表明，紫两优737的双亲均携带紫色种皮基因Pb，糯稻蜡质基因wx，淀粉分支酶基因Sbe1i和SBE3以及极限糊精酶基因PUL。在焦磷酸化酶大亚基基因AGPlar座位上，双亲分别为Ⅰ和Ⅱ型。严长杰等[28]的试验表明，蜡质基因对淀粉的理化特性具有决定性作用，在该位点不同等位基因的崩解值（Breakdown Value, BDV）、冷胶粘度（Cool Paste Viscosity, CPV）、回复值（Consistency Value, CSV）、直链淀粉含量（Amylose Content, AC）和胶稠度（Gel Consistency, GC）等都有显著或极显著差异。紫两优737含有糯稻蜡质基因wx，且其第1内含子+1的碱基类型均为T型，这一检测结果跟前人研究一致。韩月澎等[36]对籼、粳两个糯性突变品种的研究表明籼稻糯性突变品种的wx基因第1内含子剪切位点+1位的碱基由G突变为T，而粳稻糯性突变品种则与原品种相同（均为T）。进而推测wx基因第1内含子+1位碱基为T是糯稻品种的特征，该变异使得品种具有中等直链淀粉含量（AC）和较软的胶稠度（GC）[37]
除上述基因之外，紫两优737还携带一些其他性状的有利等位基因，如分蘖角基因TAC1、氮高效利用基因NRT1.1B等。前人研究表明硝酸盐转运蛋白NRT1.1B的自然变异是导致水稻籼粳间氮利用效率差异的重要原因，田间试验证明携带籼型等位基因NRT1.1B的粳稻品种在正常施肥条件下增产15%[38]。这些基因对紫两优737优异农艺性状形成的贡献还有待进一步探讨。
综上所述，本研究通过分析紫糯两系特种稻紫两优737重要农艺性状的遗传构成，初步阐释了紫两优737具有的高产、适应性好、优质等优异农艺性状的分子基础。研究结果为紫两优737及其亲本在育种和生产上的进一步应用提供了理论依据，也为后续的定向改良工作奠定了基础。
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图 1 不同处理土壤细菌维恩图
Figure 1. Venn diagram of bacterial community in soil under treatments
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图 2 不同处理下土壤细菌在门水平上的群落结构
Figure 2. Bacterial community composition at phylum level in soil under treatments
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图 3 不同处理土壤细菌群落差异分析
Figure 3. Differences in bacteria community structure of soil under treatments
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图 4 不同处理土壤细菌群落门水平热图
Figure 4. Heatmap of bacteria community at phylum level in soil under treatments
表 1 不同施肥处理下土壤主要细菌门的相对丰度
Table 1 Relative abundances of major bacterial phyla in soil under treatments
细菌种类Bacterial species 处理1 T1 处理2 T2 处理3 T3 处理4 T4 处理5 T5 变形杆菌门Proteobacteria 16.65 19.81 20.44 16.26 18.95 放线菌门Actinobacteria 15.78 18.75 16.44 19.09 18.17 绿弯菌门Chloroflexi 16.22 13.94 15.31 16.53 17.25 酸杆菌门Acidobacteria 11.72 12.73 16.09 15.03 13.23 厚壁菌门Firmicutes 14.61 12.14 12.09 12.39 12.64 浮霉菌门Planctomycetes 9.57 9.88 8.14 7.07 7.04 芽单胞菌门Gemmatimonadetes 2.05 2.52 1.90 1.78 2.00 拟杆菌门Bacteroidetes 2.62 2.31 1.23 1.08 1.14 疣微菌门Verrucomicrobia 3.94 1.35 1.03 0.74 0.54 糖细菌门Saccharibacteria 0.23 0.82 0.62 0.98 1.02 
下载: 导出CSV
表 2 不同处理土壤细菌群落多样性指数
Table 2 Diversity index of bacteria in soil under treatments
处理Treatment Shannon指数Shannon index Simpson指数Simpson index 测序深度指数Sequencing depth index OTU数量OTU number 处理1 T1 9.109 0.993 0.995 2559.000 处理2 T2 9.111 0.996 0.995 2341.333 处理3 T3 9.217 0.996 0.995 2467.000 处理4 T4 8.845 0.996 0.996 2181.667 处理5 T5 9.114 0.996 0.996 2379.333
下载: 导出CSV
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